| 1. |
- Arner, Anders, et al.
(author)
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Calcium transients and the effect of a photolytically released calcium chelator during electrically induced contractions in rabbit rectococcygeus smooth muscle
- 1998
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In: Biophysical Journal. - 1542-0086 .- 0006-3495. ; 75:4, s. 1895-1903
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Journal article (peer-reviewed)abstract
- Intracellular Ca2+ was determined with the fura-2 technique during electrically induced contractions in the rabbit rectococcygeus smooth muscle at 22 degreesC. The muscles were electrically activated to give short, reproducible contractions. Intracellular [Ca2+] increased during activation; the increase in [Ca2+] preceded force development by approximately 2 s. After cessation of stimulation Ca2+ fell, preceding the fall in force by approximately 4 s. The fluorescence properties of fura-2 were determined with time-resolved spectroscopy using synchrotron light at the MAX-storage ring, Lund, Sweden. The fluorescence decay of free fura-2 was best described by two exponential decays (time constants approximately 0.5 and 1.5 ns) at low Ca2+ (pCa 9). At high Ca2+ (pCa 4.5), fluorescence decay became slower and could be fitted by one exponential decay (1.9 ns). Time-resolved anisotropy of free fura-2 was characteristic of free rotational motion (correlation time 0.3 ns). Motion of fura-2 could be markedly inhibited by high concentrations of creatine kinase. Time-resolved spectroscopy measurements of muscle fibers loaded with fura-2 showed that the fluorescence lifetime of the probe was longer, suggesting an influence of the chemical environment. Anisotropy measurements revealed, however, that the probe was mobile in the cells. The Ca2+-dependence of contraction and relaxation was studied using a photolabile calcium chelator, diazo-2, which could be loaded into the muscle cells in a similar manner as fura-2. Photolysis of diazo-2 leads to an increase in its Ca2+-affinity and a fall in free Ca2+. When muscles that had been loaded with diazo-2 were illuminated with UV light flashes during the rising phase of contraction, the rate of contraction became slower, suggesting a close relation between intracellular Ca2+ and the cross-bridge interaction. In contrast, photolysis during relaxation did not influence the rate of force decay, suggesting that relaxation of these contractions is not determined by the rate of Ca2+ removal or due to an increased Ca2+ sensitivity, but instead is limited by other processes such as deactivation by dephosphorylation or detachment of tension-bearing cross-bridges, possibly regulated by thin filament systems.
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| 2. |
- Basanez, G, et al.
(author)
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Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles : a cryo-transmission electron microscopy study of liposome fusion.
- 1997
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 72:6, s. 2630-2637
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Journal article (peer-reviewed)abstract
- Cryo-transmission electron microscopy has been applied to the study of the changes induced by phospholipase C on large unilamellar vesicles containing phosphatidylcholine, as well as to the action of sphingomyelinase on vesicles containing sphingomyelin. In both cases vesicle aggregation occurs as the earliest detectable phenomenon; later, each system behaves differently. Phospholipase C induces vesicle fusion through an intermediate consisting of aggregated and closely packed vesicles (the ''honeycomb structure'') that finally transforms into large spherical vesicles. The same honeycomb structure is also observed in the absence of enzyme when diacylglycerols are mixed with the other lipids in organic solution, before hydration. In this case the sample then evolves toward a cubic phase. The fact that the same honeycomb intermediate can lead to vesicle fusion (with enzyme-generated diacylglycerol) or to a cubic phase (when diacylglycerol is premixed with the lipids) is taken in support of the hypothesis according to which a highly curved lipid structure (''stalk'') would act as a structural intermediate in membrane fusion, Sphingomyelinase produces complete leakage of vesicle aqueous contents and an increase in size of about one-third of the vesicles. A mechanism of vesicle opening and reassembling is proposed in this case.
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| 3. |
- Beugin, S, et al.
(author)
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New sterically stabilized vesicles based on nonionic surfactant, cholesterol, and poly(ethylene glycol)-cholesterol conjugates.
- 1998
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 74:6, s. 3198-3210
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Journal article (other academic/artistic)abstract
- Monomethoxypoly(ethylene glycol) cholesteryl carbonates (M-PEG-Chol) with polymer chain molecular weights of 1000 (M-PEG1000-Chol) and 2000 (M-PEG2000-Chol) have been newly synthesized and characterized. Their aggregation behavior in mixture with diglycerol hexadecyl ether (C(16)G(2)) and cholesterol has been examined by cryotransmission electron microscopy, high-performance gel exclusion chromatography, and quasielastic light scattering. Nonaggregated, stable, unilamellar vesicles were obtained at low polymer levels with optimal shape and size homogeneity at cholesteryl conjugate/ lipids ratios of 10 mol% M-PEG1000-Chol or 5 mol% M-PEG2000-Chol, corresponding to the theoretically predicted brush conformational state of the PEG chains. At 20 mol% M-PEG1000-Chol or 10 mol% M-PEG2000-Chol, the saturation threshold of the C(16)G(2)/cholesterol membrane in polymer is exceeded, and open disk-shaped aggregates are seen in coexistence with closed vesicles. Higher levels up to 30 mol% lead to the complete solubilization of the vesicles into disk-like structures of decreasing size with increasing PEG content. This study underlines the bivalent role of M-PEG-Chol derivatives: while behaving as solubilizing surfactants, they provide an efficient steric barrier, preventing the vesicles from aggregation and fusion over a period of at least 2 weeks.
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| 4. |
- Copello, J A, et al.
(author)
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Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors.
- 1997
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:1, s. 141-56
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Journal article (peer-reviewed)abstract
- The single-channel activity of rabbit skeletal muscle ryanodine receptor (skeletal RyR) and dog cardiac RyR was studied as a function of cytosolic [Ca2+]. The studies reveal that for both skeletal and cardiac RyRs, heterogeneous populations of channels exist, rather than a uniform behavior. Skeletal muscle RyRs displayed two extremes of behavior: 1) low-activity RyRs (LA skeletal RyRs, approximately 35% of the channels) had very low open probability (Po < 0.1) at all [Ca2+] and remained closed in the presence of Mg2+ (2 mM) and ATP (1 mM); 2) high-activity RyRs (HA skeletal RyRs) had much higher activity and displayed further heterogeneity in their Po values at low [Ca2+] (< 50 nM), and in their patterns of activation by [Ca2+]. Hill coefficients for activation (nHa) varied from 0.8 to 5.2. Cardiac RyRs, in comparison, behaved more homogeneously. Most cardiac RyRs were closed at 100 nM [Ca2+] and activated in a cooperative manner (nHa ranged from 1.6 to 5.0), reaching a high Po (> 0.6) in the presence and absence of Mg2+ and ATP. Heart RyRs were much less sensitive (10x) to inhibition by [Ca2+] than skeletal RyRs. The differential heterogeneity of heart versus skeletal muscle RyRs may reflect the modulation required for calcium-induced calcium release versus depolarization-induced Ca2+ release.
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| 5. |
- Edwards, Katarina, et al.
(author)
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Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes
- 1997
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:1, s. 258-266
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Journal article (other academic/artistic)abstract
- Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present.
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| 6. |
- Elinder, Fredrik, et al.
(author)
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Role of individual surface charges of voltage-gated K channels
- 1999
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In: Biophysical Journal. - : Elsevier Science B.V., Amsterdam. - 0006-3495 .- 1542-0086. ; 77:3, s. 1358-1362
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Journal article (peer-reviewed)abstract
- Fixed charges on the extracellular surface of voltage-gated ion channels influence the gating. In previous studies of cloned voltage-gated K channels, we found evidence that the functional surface charges are located on the peptide loop between the fifth transmembrane segment and the pore region (the S5-P loop). In the present study, we determine the role of individual charges of the S5-P loop by correlating primary structure with experimentally calculated surface potentials of the previously investigated channels. The results suggest that contributions to the surface potential at the voltage sensor of the different residues varies in an oscillating pattern, with the first residue of the N-terminal end of the S5-P loop, an absolutely conserved glutamate, contributing most. An analysis yields estimates of the distance between the residues and the voltage sensor, the first N-terminal residue being located at a distance of 5-6 Angstrom. To explain the results, a structural hypothesis, comprising an a-helical N-terminal end of the S5-P loop, is presented.
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| 7. |
- Karolin, J, et al.
(author)
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Donor-donor energy migration for determining intramolecular distances in proteins : I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).
- 1998
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 74:1, s. 11-21
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Journal article (peer-reviewed)abstract
- A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.
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| 8. |
- Song, Z., et al.
(author)
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Conformational transitions of the phosphodiester backbone in native DNA: two-dimensional magic-angle-spinning 31P-NMR of DNA fibers
- 1997
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 73:3, s. 1539-1552
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Journal article (peer-reviewed)abstract
- Solid-state 31P-NMR is used to investigate the orientation of the phosphodiester backbone in NaDNA-, LiDNA-, MgDNA-, and NaDNA-netropsin fibers. The results for A- and B-DNA agree with previous interpretations. We verify that the binding of netropsin to NaDNA stabilizes the B form, and find that in NaDNA, most of the phosphate groups adopt a conformation typical of the A form, although there are minor components with phosphate orientations close to the B form. For LiDNA and MgDNA samples, on the other hand, we find phosphate conformations that are in variance with previous models. These samples display x-ray diffraction patterns that correspond to C-DNA. However, we find two distinct phosphate orientations in these samples, one resembling that in B-DNA, and one displaying a twist of the PO4 groups about the O3-P-O4 bisectors. The latter conformation is not in accordance with previous models of C-DNA structure.
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| 9. |
- Thomson, Neil H., et al.
(author)
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Oriented, active Escherichia coli RNA polymerase : an atomic force microscope study
- 1999
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In: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 76:2, s. 1024-1033
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Journal article (peer-reviewed)abstract
- RNA polymerase (RNAP) molecules to be imaged under aqueous buffer using tapping-mode atomic force microscopy (AFM). Recombinant RNAP molecules containing histidine tags (hisRNAP) on the C-terminus were specifically immobilized on ultraflat gold via a mixed monolayer of two different Ω-functionalized alkanethiols. One alkanethiol was terminated in an ethylene-glycol (EG) group, which resists protein adsorption, and the other was terminated in an N-nitrilotriacetic acid (NTA) group, which binds the histidine tag through two coordination sites with a nickel ion. AFM images showed that these two alkanethiols phase-segregate. Specific binding of the hisRNAP molecules was followed in situ by injecting proteins directly into the AFM fluid cell. The activity of the hisRNAP bound to the NTA groups was confirmed with a 42-base circular single-stranded DNA template (rolling circle), which the RNAP uses to produce huge RNA transcripts. These transcripts were imaged in air after the samples were rinsed and dried, since RNA also has low affinity for the EG-thiol and cannot be imaged under the buffers we used.
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