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91.
Stretching or aligning DNA molecules onto a surface by means of molecular combing techniques is one of the critical steps in single DNA molecule analysis. However, many of the current studies have focused on λ-DNA, or other large DNA molecules. There are very few studies on stretching methodologies for DNA molecules generated via PCR (typically smaller than 20 kb). Here we describe a simple method of stretching DNA molecules up to 18 kb in size on a modified glass surface. The very low background fluorescence allows efficient detection of single fluorescent dye labels incorporated into the stretched DNA molecules. 相似文献
92.
93.
B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC). To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(-) IgM(-) CD19(+) B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1)-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+) plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25) or secondary (day 10) infection. CD138(+) plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood. 相似文献
94.
Rangarajan ES Li Y Ajamian E Iannuzzi P Kernaghan SD Fraser ME Cygler M Matte A 《The Journal of biological chemistry》2005,280(52):42919-42928
Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases. 相似文献
95.
Ángel Eliezer Bravo-Monzón Eunice Ríos-Vásquez Guillermo Delgado-Lamas 《Biocontrol Science and Technology》2016,26(3):419-425
We assessed the preference of two populations of the specialist beetle Stolas punicea, on plants from three phytochemically differentiated geographical mosaics of the American weed Mikania micrantha. Our results show a significant geographic variation in host preference in S. punicea that could affect its success as a biocontrol agent. 相似文献
96.
Grishin AM Ajamian E Tao L Zhang L Menard R Cygler M 《The Journal of biological chemistry》2011,286(12):10735-10743
The utilization of phenylacetic acid (PA) in Escherichia coli occurs through a hybrid pathway that shows features of both aerobic and anaerobic metabolism. Oxygenation of the aromatic ring is performed by a multisubunit phenylacetyl-coenzyme A oxygenase complex that shares remote homology of two subunits to well studied bacterial multicomponent monooxygenases and was postulated to form a new bacterial multicomponent monooxygenase subfamily. We expressed the subunits PaaA, B, C, D, and E of the PA-CoA oxygenase and showed that PaaABC, PaaAC, and PaaBC form stable subcomplexes that can be purified. In vitro reconstitution of the oxygenase subunits showed that each of the PaaA, B, C, and E subunits are necessary for catalysis, whereas PaaD is not essential. We have determined the crystal structure of the PaaAC complex in a ligand-free form and with several CoA derivatives. We conclude that PaaAC forms a catalytic core with a monooxygenase fold with PaaA being the catalytic α subunit and PaaC, the structural β subunit. PaaAC forms heterotetramers that are organized very differently from other known multisubunit monooxygenases and lacks their conservative network of hydrogen bonds between the di-iron center and protein surface, suggesting different association with the reductase and different mechanisms of electron transport. The PaaA structure shows adaptation of the common access route to the active site for binding a CoA-bound substrate. The enzyme-substrate complex shows the orientation of the aromatic ring, which is poised for oxygenation at the ortho-position, in accordance with the expected chemistry. The PA-CoA oxygenase complex serves as a paradigm for the new subfamily multicomponent monooxygenases comprising several hundred homologs. 相似文献
97.
98.
James CD Reuel N Lee ES Davalos RV Mani SS Carroll-Portillo A Rebeil R Martino A Apblett CA 《Biosensors & bioelectronics》2008,23(6):845-851
We report here a non-invasive, reversible method for interrogating single cells in a microfluidic flow-through system. Impedance spectroscopy of cells held at a micron-sized pore under negative pressure is demonstrated and used to determine the presence and viability of the captured cell. The cell capture pore is optimized for electrical response and mechanical interfacing to a cell using a deposited layer of parylene. Changes in the mechanical interface between the cell and the chip due to chemical exposure or environmental changes can also be assessed. Here, we monitored the change in adhesion/spreading of RAW264.7 macrophages in response to the immune stimulant lipopolysaccharide (LPS). This method enables selective, reversible, and quantitative long-term impedance measurements on single cells. The fully sealed electrofluidic assembly is compatible with long-term cell culturing, and could be modified to incorporate single cell lysis and subsequent intracellular separation and analysis. 相似文献
99.
Gruneberg U Neef R Li X Chan EH Chalamalasetty RB Nigg EA Barr FA 《The Journal of cell biology》2006,172(3):363-372
Multiple mitotic kinesins and microtubule-associated proteins (MAPs) act in concert to direct cytokinesis (Glotzer, M. 2005. Science. 307:1735-1739). In anaphase cells, many of these proteins associate with an antiparallel array of microtubules termed the central spindle. The MAP and microtubule-bundling protein PRC1 (protein-regulating cytokinesis 1) is one of the key molecules required for the integrity of this structure (Jiang, W., G. Jimenez, N.J. Wells, T.J. Hope, G.M. Wahl, T. Hunter, and R. Fukunaga. 1998. Mol. Cell. 2:877-885; Mollinari, C., J.P. Kleman, W. Jiang, G. Schoehn, T. Hunter, and R.L. Margolis. 2002. J. Cell Biol. 157:1175-1186). In this study, we identify an interaction between endogenous PRC1 and the previously uncharacterized kinesin KIF14 as well as other mitotic kinesins (MKlp1/CHO1, MKlp2, and KIF4) with known functions in cytokinesis (Hill, E., M. Clarke, and F.A. Barr. 2000. EMBO J. 19:5711-5719; Matuliene, J., and R. Kuriyama. 2002. Mol. Biol. Cell. 13:1832-1845; Kurasawa, Y., W.C. Earnshaw, Y. Mochizuki, N. Dohmae, and K. Todokoro. 2004. EMBO J. 23:3237-3248). We find that KIF14 targets to the central spindle via its interaction with PRC1 and has an essential function in cytokinesis. In KIF14-depleted cells, citron kinase but not other components of the central spindle and cleavage furrow fail to localize. Furthermore, the localization of KIF14 and citron kinase to the central spindle and midbody is codependent, and they form a complex depending on the activation state of citron kinase. Contrary to a previous study (Di Cunto, F., S. Imarisio, E. Hirsch, V. Broccoli, A. Bulfone, A. Migheli, C. Atzori, E. Turco, R. Triolo, G.P. Dotto, et al. 2000. Neuron. 28:115-127), we find a general requirement for citron kinase in human cell division. Together, these findings identify a novel pathway required for efficient cytokinesis. 相似文献
100.
Quality along the Continuum: A Health Facility Assessment of Intrapartum and Postnatal Care in Ghana
Robin C. Nesbitt Terhi J. Lohela Alexander Manu Linda Vesel Eunice Okyere Karen Edmond Seth Owusu-Agyei Betty R. Kirkwood Sabine Gabrysch 《PloS one》2013,8(11)