bluuebunny/crossref_metadata_2025_split huggingface dataset:
Baskets for In Situ Hybridization and Immunohistochemistry: Figure 1.
Authors: Sive Hazel L. , Grainger Robert M. , Harland Richard M. 
Published: August 2007

INTRODUCTIONFor large in situ hybridization and immunohistochemistry experiments, changing solutions in vials becomes tedious, and baskets should be used. Commercially available baskets, such as 15-mm Netwell baskets (Corning), fit into 12-well tissue culture plates. Although they are expensive, they can, with extensive washing, be reused. However, these baskets are not readily adaptable to large-scale use, and their relatively shallow wells make cross-contamination between wells a real danger. This protocol describes a procedure for making homemade baskets with plastic microcentrifuge tubes and nylon mesh that are more adaptable to large-scale experiments. The baskets are narrow and deep and thus can be placed in a rack at high density. To change solutions, individual tubes or whole racks of tubes can be moved from one bath of solution to another. Because many tubes can be manipulated simultaneously, there is an enormous saving in work. It is also quite difficult to lose embryos in baskets; embryos in vials stand a good chance of being sucked into the aspirator during solution changes and lost.
Fetal Thymus Organ Culture: Figure 1.
Authors: Anderson Graham , Jenkinson Eric J. 
Published: August 2007

INTRODUCTIONThe generation of functionally competent T-cells from their progenitors involves a series of developmental events including proliferation, differentiation, and survival. T-cell development is a non-cell-autonomous event, and requires interactions with thymic stromal cells. Fetal thymus organ cultures provide an in vitro system in which isolated embryonic thymus lobes can be maintained in culture, allowing the study of T-cell development as well as thymic stromal cell function. This system remains the only in vitro system that supports a complete program of T-cell development, including positive and negative selection of the developing T-cell receptor repertoire. Modifications of the basic fetal thymus organ culture system, such as hanging drop cultures and reaggregate thymus organ cultures, provide useful systems to analyze thymus colonization and thymic stromal cell function, respectively.
A Rapid Protocol for Whole-Mount In Situ Hybridization on Xenopus Embryos
Authors: Monsoro-Burq Anne H. 
Published: August 2007

INTRODUCTIONThis in situ hybridization (ISH) protocol describes a simplified method using a digoxigenin-labeled antisense RNA probe on whole Xenopus embryos, suitable for both X. laevis and X. tropicalis. The protocol includes fixation, β-galactosidase staining (when lineage tracing is needed), and storage of the embryos prior to ISH. This method shortens the steps before hybridization, which limits RNA degradation in the sample, and preserves superficial structures. Hence, it is particularly suited for the analysis of ectoderm, neural, and mesodermal structures from blastula to early tadpole stages. Additional permeabilization steps are included to process later tadpole stages.
In Vivo Time-Lapse Imaging of Zebrafish Embryonic Development
Authors: Distel Martin , Köster Reinhard W. 
Published: August 2007

INTRODUCTIONIntravital time-lapse imaging is a powerful technique for investigating continuous developmental processes without missing crucial events. Because of the rapid embryogenesis, external development, and transparency of zebrafish embryos, their developmental processes can be visualized in time-lapse studies in the context of the living organism. The following protocol describes a method for performing intravital time-lapse imaging of zebrafish embryos over several days using confocal or compound stereomicroscopes.
Preparing DNA from Cell Pellets for Genotyping
Authors: Sahota Amrik , Brooks Andrew I. , Tischfield Jay A. 
Published: August 2007

INTRODUCTIONThis protocol describes large-scale DNA extraction from cell pellets using either in-house or commercial (PUREGENE) reagents. The cell pellets are prepared from human lymphoblast cell lines (LCLs) that have been established using the Epstein-Barr virus (EBV).
Preparing DNA from Blood for Genotyping
Authors: Sahota Amrik , Brooks Andrew I. , Tischfield Jay A. , King Irena B. 
Published: August 2007

INTRODUCTIONThis protocol describes the extraction of genomic DNA from whole blood samples (fresh or frozen) and buffy coats. It provides methods for (1) large-scale extraction of DNA using either in-house or commercial (PUREGENE) reagents; (2) mid-scale extraction of DNA using the QIAamp DNA Blood Midi Kit (for 0.5-mL samples); and (3) small-scale extraction of DNA using the QIAamp DNA Blood Mini Kits (for 200-μL samples). Also included are methods for extracting DNA from blood samples that are compromised or clotted. Compromised blood samples include samples that spent more than 3 days in transit, samples that have been stored for more than 1 wk at 4°C, samples that have been stored at -20°C or -70°C, and samples that were collected incorrectly (e.g., not mixed properly). The procedures described here are not high throughput. Potential drawbacks of making them high-throughput are lower recoveries of DNA and possible variability in data quality, thus requiring additional quality assurance and control checks. DNA yields for these methods vary with the number of nucleated cells in the blood sample and the quality of the sample.
Growth allometry of immature insects: larvae do not grow exponentially
Authors: TAMMARU TOOMAS , ESPERK TOOMAS 
Published: July 2007

SummaryThe allometric relationship between growth rate and body mass has received considerable attention but different taxa have not been equally studied. In particular, a limited amount of information is available on growth allometry of insect larvae.In life‐history studies, it is often assumed that insect larvae grow exponentially. This leads one to expect that potential rewards of extending growth periods are high in terms of increased adult body masses and fecundities. Therefore, it has been a challenge to find costs of large size that counterbalance the fecundity advantage of attaining larger sizes.This study examines the intraspecific growth allometry of lepidopteran larvae. Original methodology is proposed to address problems arising from the complexity of the insect growth curve, and the high sensitivity of growth rates to environmental conditions. To facilitate generalizations, larvae of 11 unrelated lepidopteran species were subjected to an identical study design.Instantaneous absolute growth rates of larvae were related to body size by an intraspecific allometric exponent in the range between 0·41 and 0·88. There were significant differences between the species but values of the exponent as high as 1 (exponential growth), and as low as 0 (linear growth) could safely be excluded.Instantaneous relative growth rates of larvae were typically 35% lower in their last instar when compared to the penultimate one. Using the exponential growth curve (i.e. assuming the constancy of relative growth rates) in modelling insect life‐histories may therefore lead to substantially biased conclusions.
Combined effects of fisheries and climate on a migratory long‐lived marine predator
Authors: Rolland V. , Barbraud C. , Weimerskirch H. 
Published: July 2007

Summary

The impact of climate on marine ecosystems is now well documented, but remains complex. Climate change may interact with human activities to effect population dynamics. In addition, in migratory species conditions are different between the breeding and wintering grounds, resulting in more complex dynamics. All these possible effects should be considered to predict the future of endangered species, but very few studies have investigated such combined interactions.
As a case study, we assessed the relative impact of fisheries and of oceanographic conditions in breeding and wintering sites on adult survival and breeding success of a population of the endangered black‐browed albatross Thalassarche melanophrys in the Kerguelen Islands, Southern Indian Ocean. This study was based on long‐term monitoring of individually marked individuals (1979–2005) and identification by tracking studies and band recoveries of the oceanic feeding zones used during breeding and non‐breeding seasons.
Breeding success was variable until 1997 and then declined gradually, from 0·88 to 0·48 chicks per egg laid. It was favoured by positive sea‐surface temperature anomalies (SSTA) and trawl fishery during the breeding period, whereas it was negatively affected by positive SSTA around Tasmania, where the species winters. Adult survival was 0·918 ± 0·004 on average and increased with SSTA during incubation, but decreased significantly with high tuna longlining effort in the wintering zone.
Our analyses show that demographic parameters were influenced by both climate and fisheries in both breeding and wintering grounds, but with different effect size. Black‐browed albatross breeding success was more favoured by trawlers’ offal and discards than by any of the seasonally/spatially oceanographic conditions, whereas their survival was equally affected by tuna longline fishery through incidental by‐catch and spring SSTA.
Synthesis and applications. Our work underlines that a comprehensive knowledge of the life history of a species in all the habitats used is important to disentangle the respective roles of environmental conditions and human factors on population dynamics. Identification of these effects is required when proposing effective conservation measures, because the conservation of threatened species may depend on their wintering country's exclusive economic zones.
The Arabidopsis AtPNG1 gene encodes a peptide: N‐glycanase
Authors: Diepold Andreas , Li Guangtao , Lennarz William J. , Nürnberger Thorsten , Brunner Frédéric 
Published: July 2007

SummaryDeglycosylation of misfolded proteins by the endoplasmic reticulum‐associated degradation (ERAD) pathway is catalyzed by peptide:N‐glycanases (PNGases) that are highly conserved among mammals and yeast. The catalytic mechanism of PNGases employs a catalytic triad consisting of Cys, His and Asp residues, which is shared by other enzyme families such as cysteine proteases and protein cross‐linking transglutaminases (TGases). In contrast to the yeast and mammalian systems, very little is known about ERAD in plants and the enzymes responsible for proper clearance of misfolded plant proteins. We have used a computer‐based modeling approach to identify an Arabidopsis thaliana PNGase (AtPNG1). AtPNG1 is encoded by a single‐copy gene and displays high structural homology with known PNGases. Importantly, heterologous expression of AtPNG1 restored N‐glycanase activity in a PNGase‐deficient Saccharomyces cerevisiae mutant. The AtPNG1 gene is uniformly and constitutively expressed at low levels throughout all developmental stages of the plant, and its expression does not appear to be subject to substantial regulation by external stimuli. Recently, recombinant AtPNG1 produced in Escherichia coli was reported to display TGase activity (Della Mea et al., Plant Physiol. 135, 2046–54, 2004). However, inactivation of the AtPNG1 gene did not result in decreased TGase activity in the mutant plant, and recombinant AtPNG1 produced in S. cerevisiae exhibited only residual TGase activity. We propose that the AtPNG1 gene encodes a bona fide peptide:N‐glycanase that contributes to ERAD‐related protein quality control in plants.
Low Temperature Effects during Seed Filling on Chickpea Genotypes (Cicer arietinum L.): Probing Mechanisms affecting Seed Reserves and Yield
Authors: Nayyar H. , Kaur G. , Kumar S. , Upadhyaya H. D. 
Published: July 2007

AbstractChickpea is sensitive to cold conditions (<15 °C), particularly at its reproductive phase and consequently it experiences significant decrease in the seed yield. The information about the effects of cold stress on chickpea during the seed filling phase is lacking. Moreover, the underlying metabolic reasons associated with the low temperature injury are largely unknown in the crop. Hence, the present study was undertaken with the objectives: (i) to find out the possible mechanisms leading to low temperature damage during the seed filling and (ii) to investigate the relative response of the microcarpa (Desi) and the macrocarpa (Kabuli) chickpea types along with elucidation of the possible mechanisms governing the differential cold sensitivity at this stage. At the time of initiation of the seed filling (pod size ∼1 cm), a set of plants growing under warm conditions of the glasshouse (temperature: 17/28 ± 2 °C as average night and day temperature) was subjected to cold conditions of the field (2.3/11.7 ± 2 °C as average night and day temperature), while another set was maintained under warm conditions (control). The chilling conditions resulted in the increase in electrolyte leakage, the loss of chlorophyll, the decrease in sucrose content and the reduction in water status in leaves, which occurred to a greater extent in the macrocarpa type than in the microcarpa type. The total plant weight decreased to the same level in both the chickpea types, whereas the rate and duration of the seed filling, seed size, seed weight, pods per plant and harvest index decreased greatly in the macrocarpa type. The stressed seeds of both the chickpea types experienced marked reduction in the accumulation of starch, proteins, fats, crude fibre, protein fractions (albumins, globulins, prolamins and glutelins) with a larger decrease in the macrocarpa type. The accumulation of sucrose and the activity levels of the enzymes like starch synthase, sucrose synthase and invertase decreased significantly in the seeds because of the chilling, indicating impairment in sucrose import. Minerals such as calcium, phosphorous and iron as well as several amino acids (phenylalanine, tyrosine, threonine, tryptophan, valine and histidine) were lowered significantly in the stressed seeds. These components were limited to a higher extent in the macrocarpa type indicating higher cold sensitivity of this type.
Effects of Cadmium on Carbon and Nitrogen Assimilation in Shoots of Mungbean [Vigna radiata (L.) Wilczek] Seedlings
Authors: Wahid A. , Ghani A. , Ali I. , Ashraf M. Y. 
Published: July 2007

AbstractIncreased cadmium (Cd) uptake from contaminated soils damages plant metabolism. The purpose of this study was to determine Cd‐induced time‐related changes in some shoot growth and physiological attributes, and their interrelationships in Cd‐tolerant (NM‐98) and sensitive (NM‐28) mungbean varieties. Shoot Cd and leaf chlorosis increased with a concomitant reduction in shoot dry weight, leaf area, relative growth rate (RGR), net assimilation rate (NAR) and relative leaf expansion rate. Reduction in transpiration rate (E) and stomatal conductance (gs) and increase in substomatal CO2 level (Ci), indicated that Cd reduced net photosynthesis (Pn) by reducing CO2 fixation by Rubisco, albeit these changes were less pronounced in NM‐98. A positive correlation of chlorosis with shoot Cd, and negative relationships of chlorosis and shoot Cd with Pn revealed that Cd damages the photosynthetic apparatus in mungbean. Time course decrease in in vivo nitrate reductase activity (NRA) and an increase in soluble nitrate in NM‐28 revealed that Cd markedly hampers nitrogen assimilation. Positive correlations of RGR and NAR with Pn and NRA and negative ones with chlorosis, shoot dry weight, shoot Cd and Ci in NM‐98 suggested that mungbean sensitivity to Cd is due to perturbed C and N assimilation.
Influence of Growth Stage on Fusarium Head Blight and Deoxynivalenol Production in Wheat
Authors: Del Ponte E. M. , Fernandes J. M. C. , Bergstrom G. C. 
Published: July 2007

AbstractFusarium head blight is a major concern for wheat production worldwide. The fungi that cause the disease may infect head tissues from flowering to late stages of kernel development, but a better understanding of the influence of the time of infection on grain weight reduction and mycotoxin accumulation resulting from the infection process is needed. We investigated the influence of wheat reproductive stage at the time of inoculation on disease and grain quality parameters, especially production of deoxynivalenol (DON) in mature grains. Heads of Norm wheat were spray inoculated with a macroconidial suspension of a DON‐producing isolate of Fusarium graminearum at each of six reproductive stages from flowering to hard dough. Plants were incubated in a mist chamber for 48 h and then moved to the greenhouse until maturity. Norm wheat was susceptible at all stages inoculated but the highest grain weight reduction and DON accumulation occurred in plants inoculated past flowering to late milk stages. However, high incidences of kernel infection and significant levels of DON accumulation resulted from inoculations as late as the hard dough stage, even though there was no corresponding reduction in grain weight compared to non‐inoculated plants. The occurrence of commercially significant levels of DON in plump, high‐yielding wheat may result from infections that occur during favourable environments well after the flowering stages. Late infection and DON production should therefore be a future research focus for wheat breeding and integrated management of FHB and an important consideration for grading systems that employ the presence of visibly damaged kernels as a means of estimating DON content of wheat.
In vitro and ex vitro Selection of Potato Plantlets for Resistance to Early Blight
Authors: Rodríguez N. V. , Kowalski B. , Rodríguez L. G. , Caraballoso I. B. , Suárez M. A. , Pérez P. O. , Quintana C. R. , González N. , Ramos R. Q. 
Published: July 2007

AbstractReaction to the culture filtrate of Alternaria solani (Sorauer) was used as an indicator in an in vitro screening test to select lines with decreased field susceptibility to early blight from a population of 1000 putative mutants. Plantlets of cv. ‘Desirée derived from irradiated callus of potato were inoculated in vitro with a culture filtrate of A. solani (Sorauer). Of the 45 lines selected and subsequently evaluated under conditions of natural infection in the greenhouse six showed lesser degrees of early blight infection than the cv. Desirée control. The six lines selected in the greenhouse retained lower degrees of infection during 2 years of field trials.
J‐integral determination in riveted beams of a structural old steel by estimation methods*
Authors: MORENO J. , VALIENTE A. 
Published: July 2007

ABSTRACTThis work deals with the application of the J‐integral estimation method to cracked riveted beams of old puddle iron. The ductility of some of these materials makes it necessary to use elasto‐plastic fracture criteria to assess damage tolerance. This requires a high computational effort because it involves the determination of the J‐integral in beams with plates and angles that interact by means of redundant forces dependent on crack size. The J‐integral estimation method proposed by the Electric Power Research Institute could reduce this effort provided it were shown to be valid for this type of beam. This work contributes to this end by comparing the J‐integral values resultant from the estimation method and a complete finite element modelling of a riveted beam consisting of two angles and a cracked plate made of actual puddle iron from a road bridge more than a century old but still in service. A number of samples removed from the bridge showed that this material failed in a ductile manner when it was fracture tested with fatigue precracked compact specimens, even at temperatures as low as −20 °C. The two types of J‐integral values found are in good agreement over the ranges of load and crack size explored.
Hypothermia treatment potentiates ERK1/2 activation after traumatic brain injury
Authors: Atkins Coleen M. , Oliva Anthony A. , Alonso Ofelia F. , Chen Shaoyi , Bramlett Helen M. , Hu Bing‐Ren , Dietrich W. Dalton 
Published: July 2007

AbstractTraumatic brain injury (TBI) results in significant hippocampal pathology and hippocampal‐dependent memory loss, both of which are alleviated by hypothermia treatment. To elucidate the molecular mechanisms regulated by hypothermia after TBI, rats underwent moderate parasagittal fluid‐percussion brain injury. Brain temperature was maintained at normothermic or hypothermic temperatures for 30 min prior and up to 4 h after TBI. The ipsilateral hippocampus was assayed with Western blotting. We found that hypothermia potentiated extracellular signal‐regulated kinase 1/2 (ERK1/2) activation and its downstream effectors, p90 ribosomal S6 kinase (p90RSK) and the transcription factor cAMP response element‐binding protein. Phosphorylation of another p90RSK substrate, Bad, also increased with hypothermia after TBI. ERK1/2 regulates mRNA translation through phosphorylation of mitogen‐activated protein kinase‐interacting kinase 1 (Mnk1) and the translation factor eukaryotic initiation factor 4E (eIF4E). Hypothermia also potentiated the phosphorylation of both Mnk1 and eIF4E. Augmentation of ERK1/2 activation and its downstream signalling components may be one molecular mechanism that hypothermia treatment elicits to improve functional outcome after TBI.
Presynaptic protein distribution and odour mapping in glomeruli of the olfactory bulb of Xenopus laevis tadpoles
Authors: Manzini Ivan , Heermann Stephan , Czesnik Dirk , Brase Christoph , Schild Detlev , Rössler Wolfgang 
Published: July 2007

AbstractThe sensory input layer in the olfactory bulb (OB) is typically organized into spheroidal aggregates of dense neuropil called glomeruli. This characteristic compartmentalization of the synaptic neuropil is a typical feature of primary olfactory centres in vertebrates and most advanced invertebrates. In the present work we mapped the location of presynaptic sites in glomeruli across the OB using antibodies to presynaptic vesicle proteins and presynaptic membrane proteins in combination with confocal microscopy. In addition the responses of glomeruli upon mucosal application of amino acid‐odorants and forskolin were monitored using functional calcium imaging. We first describe the spatial distribution of glomeruli across the main olfactory bulb (MOB) in premetamorphic Xenopus laevis. Second, we show that the heterogeneous organization of glomeruli along the dorsoventral and mediolateral axes of the MOB is associated with a differential distribution of synaptic vesicle proteins. While antibodies to synaptophysin, syntaxin and SNAP‐25 uniformly labelled glomeruli in the whole MOB, intense synaptotagmin staining was present only in glomeruli in the lateral, and to a lesser extent in the intermediate, part of the OB. Interestingly, amino acid‐responsive glomeruli were always located in the lateral part of the OB, and glomeruli activated by mucosal forskolin application were exclusively located in the medial part of the OB. This correlation between odour mapping and presynaptic protein distribution is an additional hint on the existence of different subsystems within the main olfactory system in larval Xenopus laevis.