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Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work. 相似文献
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The F-spondin genes are a family of extracellular matrix molecules united
by two conserved domains, FS1 and FS2, at the amino terminus plus a
variable number of thrombospondin repeats at the carboxy terminus.
Currently, characterized members include a single gene in Drosophila and
multiple genes in vertebrates. The vertebrate genes are expressed in the
midline of the developing embryo, primarily in the floor plate of the
neural tube. To investigate the evolution of chordate F-spondin genes, I
have used the basal position in chordate phylogeny of the acraniate
amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was
isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin
is closely related to a particular subgroup of vertebrate F-spondin genes
that encode six thrombospondin repeats. However, unlike these genes,
expression of AmphiF-spondin is not confined to the midline but is found
through most of the central nervous system. Additionally, AmphiF-spondin
has lost three thrombospondin repeats and gained two fibronectin type III
repeats, one of which has strong identity to a fibronectin type III repeat
from Deleted in Colorectal Cancer (DCC). Taken together, these results
suggest a complex evolutionary history for chordate F-spondin genes that
includes (1) domain loss, (2) domain gain by tandem duplication and
divergence of existing domains, and (3) gain of heterologous domains by
exon shuffling.
相似文献
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Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins. 相似文献
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BLA Verçosa CM Lemos IL Mendonça SMMS Silva SM de Carvalho H Goto FAL Costa 《BMC veterinary research》2008,4(1):45