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91.
J Bramhall 《Biochemistry》1986,25(13):3958-3962
The amphiphilic fluorescent dye N-[(5-dimethylamino)naphth-1-ylsulfonyl]glycine (dansylglycine) can be used to monitor the magnitude and stability of transmembrane proton gradients. Although freely soluble in aqueous media, the dye readily adsorbs to the surfaces of lipid vesicles. Because membrane-bound dye fluoresces at a higher frequency, and with greater efficiency, than dye in aqueous solution, it is easy to isolate the fluorescence emission from those dye molecules adsorbed to the lipid surface. When dansylglycine is mixed with phospholipid vesicles, the dye molecules attain a partition equilibrium between buffer and the outer, proximal surface of the vesicles. This is a rapid, diffusion-limited process that is indicated by a fast phase of fluorescence intensity increase monitored at 510 nm. In a second step, the inner, distal surface of each vesicle becomes populated with dye, a process that involves permeation through the lipid bilayer and that is generally much slower than the original adsorption step. Dansylglycine is a weak acid that permeates as an electrically neutral species; the flux of dye across the bilayer is thus strongly dependent on the degree of protonation of the dye's carboxylate moiety. When the external pH is lower than that of the vesicle lumen, the inward flux of dye is greater than that in the opposite direction, and dye accumulates in the lumen. This leads to a local elevation of dansylglycine concentration in the inner membrane monolayer, which in turn results in an elevated fluorescence intensity proportional to the membrane pH gradient. 相似文献
92.
S S Graves J Bramhall B Bonavida 《Journal of immunology (Baltimore, Md. : 1950)》1986,137(6):1977-1984
Previous studies in our laboratory on the natural killer (NK) lytic mechanism demonstrated that following interaction of target cell with effector cell, the effector cell releases NK cytotoxic factors (NKCF) that can then bind to and lyse the target cell. This study investigates the mechanism by which the target cell signals the effector cell to release NKCF. Studies on other cell systems with secretory functions have indicated that receptor-induced transmembrane signaling leads to the metabolism of phosphatidylinositol and activation of protein kinase C (PKC) by increased cytosolic Ca++ and diacylglycerol (DAG). We tested the hypothesis that a similar sequence of activation events occurs in human NK cells by examining the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), and the calcium ionophores A23187 and ionomycin in their ability to induce release of NKCF. The amount of NKCF released was determined in a 20-hr 51Cr release assay against an NK-sensitive target cell. A23187, ionomycin, or TPA alone did not induce release of NKCF. However, ionophores (200 mM) in conjunction with TPA (20 ng/ml) induced release of NKCF. Several properties of the induced NKCF by TPA and ionophores were concordant with those of the NK cell-mediated cytotoxicity (CMC) reaction. The kinetics of release were faster (less than 1 hr) than when either Con A or target cells were used to stimulate NKCF. Only NK-sensitive target cells were killed by NKCF. Pretreatment of effector cells with interferon enhanced release of NKCF from effector cells. Several lines of evidence suggested that the pathway of activation takes place through phosphatidyl inositol metabolism. Activation of PKC was indicated because TPA and A23187 enhanced protein phosphorylation in the LGL-enriched fraction. Experiments that made use of oleoyl acetyl glycerol, a synthetic DAG, showed release of NKCF in the absence of A23187 but was augmented by the ionophore. The above studies suggest that NKCF is released from NK effector cells within a period of time consistent with NK CMC, and the release of NKCF results either directly or indirectly from protein phosphorylation by PKC. 相似文献
93.
94.
John Bramhall 《生物化学与生物物理学报:生物膜》1984,778(3)
Using fluorescent, anionic dyes such as carboxyfluorescein as model solutes, it is shown that the forces allowing such solutes to be retained within sealed lipid vesicles, against a large concentration gradient, can be primarily electrostatic in nature. At temperatures distant from that of the ordered-fluid lipid phase transition a small number of the anionic dye molecules trapped within lipid vesicles are capable of traversing the lipid bilayer and establishing an electrical diffusion potential across the membrane. Further solute movement can then only occur with the concomitant permeation of ions which restore electrical balance. A significant flux of dye can be triggered by (a) increasing the permeability of the membrane to ions (for example by the addition of ionophores such as gramicidin, or by allowing the lipid to approach a phase transition) or by (b) adding lipophilic counterions such as tetraphenylborate or dinitrophenol to the system. 相似文献
95.
96.
Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA 总被引:2,自引:1,他引:1
Van den Bussche RA; Baker RJ; Wichman HA; Hamilton MJ 《Molecular biology and evolution》1993,10(5):944-959
Within the tribe Stenodermatini the systematics of the complex of species
allied with the genus Artibeus has generated several alternative
phylogenetic hypotheses. The most recent treatment recognized four genera
(Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the
most recent common ancestor of these four genera would include the common
ancestor of all other currently recognized Stenodermatini genera except
Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear
satellite DNA repeat and 402 bp of DNA sequence variation from the
mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern
blot analyses, in situ hybridization, and mitochondrial DNA sequence data
indicate that Enchisthenes is not closely related to Dermanura, Artibeus,
or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common
ancestor after diverging from the remainder of the Stenodermatini. If our
conclusions are correct, then justification for recognizing Dermanura and
Koopmania as generically distinct from Artibeus must be based on the
magnitude of difference that distinguishes each rather than on the
conclusion that to place them as congeneric with Artibeus creates a
paraphyletic taxon.
相似文献
97.
98.
Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli 总被引:11,自引:0,他引:11
Polyclonal antibodies were elicited against seven of the 33 different
proteins of the large subunit of the chloroplast ribosome from
Chlamydomonas reinhardtii. Three of these proteins are synthesized in the
chloroplast and four are made in the cytoplasm and imported. In western
blots, six of the seven antisera are monospecific for their respective
large subunit ribosomal proteins, and none of these antisera cross-reacted
with any chloroplast small subunit proteins from C. reinhardtii. Antisera
to the three chloroplast-synthesized ribosomal proteins cross-reacted with
specific Escherichia coli large subunit proteins of comparable charge and
molecular weight. Only one of the four antisera to the chloroplast
ribosomal proteins synthesized in the cytoplasm cross-reacted with an E.
coli large subunit protein. None of the antisera cross-reacted with any E.
coli small subunit proteins. On the assumption of a procaryotic,
endosymbiotic origin for the chloroplast, those chloroplast ribosomal
proteins still synthesized within the organelle appear to have retained
more antigenic sites in common with E. coli ribosomal proteins than have
those which are now the products of cytoplasmic protein synthesis. Antisera
to this cytoplasmically synthesized group of chloroplast ribosomal proteins
did not recognize any antigenic sites among C. reinhardtii cytoplasmic
ribosomal proteins, suggesting that the genes for the cytoplasmically
synthesized chloroplast ribosomal proteins either are not derived from the
cytoplasmic ribosomal protein genes or have evolved to a point where no
antigenic similarities remain.
相似文献
99.
Correlates of extinction risk in squamate reptiles: the relative importance of biology,geography, threat and range size 下载免费PDF全文
100.