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141.
Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry. 相似文献
142.
Ying Wang Brian D. Carter Susan M. Gapstur Marjorie L. McCullough Mia M. Gaudet Victoria L. Stevens 《Metabolomics : Official journal of the Metabolomic Society》2018,14(10):129
Introduction
Processing delays after blood collection is a common pre-analytical condition in large epidemiologic studies. It is critical to evaluate the suitability of blood samples with processing delays for metabolomics analysis as it is a potential source of variation that could attenuate associations between metabolites and disease outcomes.Objectives
We aimed to evaluate the reproducibility of metabolites over extended processing delays up to 48 h. We also aimed to test the reproducibility of the metabolomics platform.Methods
Blood samples were collected from 18 healthy volunteers. Blood was stored in the refrigerator and processed for plasma at 0, 15, 30, and 48 h after collection. Plasma samples were metabolically profiled using an untargeted, ultrahigh performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) platform. Reproducibility of 1012 metabolites over processing delays and reproducibility of the platform were determined by intraclass correlation coefficients (ICCs) with variance components estimated from mixed-effects models.Results
The majority of metabolites (approximately 70% of 1012) were highly reproducible (ICCs?≥?0.75) over 15-, 30- or 48-h processing delays. Nucleotides, energy-related metabolites, peptides, and carbohydrates were most affected by processing delays. The platform was highly reproducible with a median technical ICC of 0.84 (interquartile range 0.68–0.93).Conclusion
Most metabolites measured by the UPLC–MS/MS platform show acceptable reproducibility up to 48-h processing delays. Metabolites of certain pathways need to be interpreted cautiously in relation to outcomes in epidemiologic studies with prolonged processing delays.143.
We examined alternative hypotheses for the benefits of footdrummingin the presence of snakes by the banner-tailed kangaroo rat,Dipodomys spectabitis, by testing whether the target of thesignal includes conspecifics, the predator or both. Footdrummingrecorded in the field revealed that rats altered their footdrummingsignatures when drumming at snakes. In playback tests, however,neighbors failed to show any measurable change in behavior tobroadcasts of the snake drumming pattern, but mothers footdrummedsignificantly more than nonmothen in the presence of a tetheredsnake. Gopher snakes, Pituophis melanolsucus affinis, respondedto footdrumming vibrations created by a mechanical thumper.Nonhungry snakes avoided footdrumming, while hungry snakes approachedthe seismic footdrumming. Snakes decreased stalking rates asfootdrumming increased, but they spent more time stalking drummingthan nondnimming rats. We conclude that D. spectabilis footdrumsin individual defense and in parental care, rather than to warnadult conspecfics. Footdrumming deters pursuit by informingthe snake that the rat is alert and the chances of predationare low. We find little evidence that footdrumming startles,confuses, or harasses the snake. Hungry gopher snakes, however,may locate prey by eavesdropping on territorial footdrumming 相似文献
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Recently two articles on the use of thionin as a cell stain for neurological materials have appeared. One utilizes a solution buffered in the acid range3; the other uses a “steaming” staining solution4. For some time we have been using thionin as a routine stain after either formalin or alcohol fixation and our method is so simple and has given such satisfactory results with a variety of brands of thionin that it seemed to be worthy of more general use. Briefly the method consists of placing the celloidin sections in a 0.05% solution of Li2CO3 (the percentage of Li2CO3 is non-critical) for about 5 minutes and then grossly overstaining in a 0.25% solution of thionin in a 0.05% solution of Li2CO3 in distilled water. The overstaining is necessary if all the stain is to be removed from the background. The sections are then passed through distilled water, 70 or 80% alcohol, two changes of butyl alcohol, two changes of xylene and mounted with Clarite. For most material, split mica cover-slips are quite satisfactory. The time of differentiation may be considerably lessened by the use of the differentiator recommended by Neumann (1942) except that we find the chloroform superfluous and transfer the sections to the aniline solution from 95% alcohol. Less fading seems to occur if the aniline differentiator is followed by a saturated solution of Li2CO3 in 95% alcohol. 相似文献
149.
Intraspecies and Interspecies Transformation Reactions in Pneumococcus and Streptococcus 总被引:1,自引:0,他引:1
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The efficiency of transformation of pneumococcus and a strain of viridans streptococcus (strain D) to streptomycin resistance is influenced by the species in which the mutation to resistance occurred, as well as by the species in which the mutated gene has been replicated. Pneumococcus and streptococcus strain D transform in higher frequency with DNA that has been replicated in bacteria of the same species than with DNA from the heterologous species. However, the difference between the frequencies of interspecific and intraspecific transformation is much greater with pneumococcus as receptor than with streptococcus. In addition pneumococcus transforms in higher frequency with wholly homologous (pneumococcal) DNA than with DNA from pneumococci that have replicated the streptococcal Smr gene. Pneumococcus is transformed in lower frequency by wholly heterologous (streptococcal) DNA than by DNA from streptococci that have replicated the pneumococcal Smr gene. Streptococcus behaves similarly in that wholly homologous (streptococcal) DNA transforms it more efficiently than when the transforming fragment contains a pneumococcal moiety. Streptococcus is transformed in the same or lower frequency by wholly heterologous (pneumococcal) DNA than by DNA from pneumococci that have replicated the streptococcal Smr gene. When erythromycin resistance was used as genetic marker instead of streptomycin resistance, similar results were found. 相似文献