Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by x-ray diffraction.
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The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state. (+info)
Primary structure of a visual pigment in bullfrog green rods.
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In frog retina there are special rod photoreceptor cells ('green rods') with physiological properties similar to those of typical vertebrate rods ('red rods'). A cDNA fragment encoding the putative green rod visual pigment was isolated from a retinal cDNA library of the bullfrog, Rana catesbeiana. Its deduced amino acid sequence has more than 65% identity with those of blue-sensitive cone pigments such as chicken blue and goldfish blue. Antisera raised against its C-terminal amino acid sequence recognized green rods. It is concluded that bullfrog green rods contain a visual pigment which is closely related to the blue-sensitive cone pigments of other non-mammalian vertebrates. (+info)
Regulation of photoreceptor phosphodiesterase catalysis by its non-catalytic cGMP-binding sites.
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The photoreceptor 3':5'-cyclic nucleotide phosphodiesterase (PDE) is the central enzyme of visual excitation in rod photoreceptors. The hydrolytic activity of PDE is precisely regulated by its inhibitory gamma subunit (Pgamma), which binds directly to the catalytic site. We examined the inhibition of frog rod outer segment PDE by endogenous Pgamma, as well as by synthetic peptides corresponding to its central and C-terminal domains, to determine whether the non-catalytic cGMP-binding sites on the catalytic alphabeta dimer of PDE allosterically regulate PDE activity. We found that the apparent binding affinity of Pgamma for PDE was 28 pM when cGMP occupied the non-catalytic sites, whereas Pgamma had an apparent affinity only 1/16 of this when the sites were empty. The elevated basal activity of PDE with empty non-catalytic sites can be decreased by the addition of nanomolar levels of cGMP, demonstrating that the high-affinity non-catalytic sites on the PDE catalytic dimer mediate this effect. No evidence for a direct allosteric effect of the non-catalytic sites on catalysis could be detected for the activated enzyme lacking bound Pgamma. The intrinsic affinity of a synthetic C-terminal (residues 63-87) Pgamma peptide to bind and to inhibit the hydrolytic activity of activated PDE was enhanced 300-fold in the presence of cGMP compared with cAMP. We conclude that the binding of cGMP to the non-catalytic sites of PDE induces an allosteric change in the structure of the catalytic domain that greatly enhances the interaction of the C-terminus of Pgamma with the catalytic domain. (+info)
Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.
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Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca(2+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location. (+info)
Behavioral reduction of infection risk.
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Evolutionary biologists have long postulated that there should be fitness advantages to animals that are able to recognize and avoid conspecifics infected with contact-transmitted disease. This avoidance hypothesis is in direct conflict with much of epidemiological theory, which is founded on the assumptions that the likelihood of infection is equal among members of a population and constant over space. The inconsistency between epidemiological theory and the avoidance hypothesis has received relatively little attention because, to date, there has been no evidence that animals can recognize and reduce infection risk from conspecifics. We investigated the effects of Candida humicola, a pathogen that reduces growth rates and can cause death of tadpoles, on associations between infected and uninfected individuals. Here we demonstrate that bullfrog (Rana catesbeiana) tadpoles avoid infected conspecifics because proximity influences infection. This avoidance behavior is stimulated by chemical cues from infected individuals and thus does not require direct contact between individuals. Such facultative modulations of disease infection risk may have critical consequences for the population dynamics of disease organisms and their impact on host populations. (+info)
Activation by bitter substances of a cationic channel in membrane patches excised from the bullfrog taste receptor cell.
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1. The response to bitter-tasting substances was recorded in outside-out membrane patches excised from the taste receptor cell of the bullfrog fungiform papilla. 2. Application of a bitter-tasting substance, quinine or denatonium, induced channel openings under conditions in which none of the second messenger candidates or their precursors (e.g. cyclic nucleotide, inositol 1,4,5-trisphosphate, Ca2+, ATP and GTP) were present on either side of the membrane. The response could be recorded > 10 min after excision of the patch membrane. These data suggest that the channel was directly gated by the bitter-tasting substances. 3. No change in response was detected upon addition to the cytoplasmic side of either GDPbetaS (1 mM) or GTPgammaS (1 mM), suggesting that the G protein cascade has no direct relation to response generation. 4. The quinine-induced current was dose dependent. The lowest effective concentration was approximately 0.1 mM, and the saturating concentration was near 1 mM. The dose-response curve was fitted by the Hill equation with a K of 0.52 mM and a Hill coefficient of 3.8. 5. The single channel conductance measured in 120 mM NaCl solution was 10 pS. The channel was cation selective, and the ratio of the permeabilities for Na+, K+ and Cs+ (PNa : PK : PCs) was 1 : 0.48 : 0.39. The unitary conductance was dependent on the extracellular Ca2+ concentration ([Ca2+]o); 9.2 pS in a nominally Ca2+-free solution, and 4.5 pS in 1. 8 mM [Ca2+]o. 6. The dose dependence, the ion selectivity and the dependence of the unitary conductance on [Ca2+]o were almost identical to those of the quinine-induced whole-cell current reported previously, indicating that the channel activity observed in the excised membrane is the basis of the whole-cell current. 7. The present observations suggest the new possibility that the cationic channel directly gated by bitter substances is involved in the bitter taste transduction mechanism. (+info)
Acute effects of inhalable particles on the frog palate mucociliary epithelium.
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This work was designed to evaluate the toxicity of inhalable particles [less than/equal to] 10 microm in aerodynamic diameter (PM(10)) collected from the urban air in Sao Paulo, Brazil, to the mucociliary apparatus using the frog palate preparation. Seven groups of frog palates were immersed in different concentrations of PM(10) diluted in Ringer's solution during 120 min: 0 (control, n = 31); 50 (n = 10); 100 (n = 9); 500 (n = 28); 1,000 (n = 10); 5,000 (n = 11); and 10,000 microg/m(3) (n = 10). Mucociliary transport and transepithelial potential difference were determined at 0, 30, 60, and 120 min exposure. Additional groups (control and 500 microg/m(3)) were studied by means of morphometric analyses (quantification of the amount of intraepithelial and surface mucins), measurement of cilia beat frequency, and quantification of total glutathione. Mucociliary transport and transepithelial potential difference were significantly decreased at higher concentrations of PM(10) (p = 0.03 and p = 0.02, respectively). Exposure to PM(10) also elicited a significant decrease of total glutathione (p = 0. 003) and depletion of neutral intraepithelial mucins (p = 0.0461). These results show that PM(10) can promote significant alterations in ciliated epithelium in vitro. (+info)
Water diffusion, T(2), and compartmentation in frog sciatic nerve.
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A potential relationship between structural compartments in neural tissue and NMR parameters may increase the specificity of MRI in diagnosing diseases. Nevertheless, our understanding of MR of nerves and white matter is limited, particularly the influence of various water compartments on the MR signal is not known. In this study, components of the (1)H transverse relaxation decay curve in frog peripheral nerve were correlated with the diffusion characteristics of the water in the nerve. Three T(2) values were identified with nerve. Water mobility was found to be unrestricted on the timescale of 100 msec in the component of the signal with the intermediate T(2) time, suggesting some contribution from the interstitial space to this T(2) component. Restricted diffusion was observed in the component with the longest T(2) time, supporting the assignment of at least part of the spins contributing to this component to an intracellular compartment. The observed nonexponential behavior of the diffusion attenuation curves was investigated and shown to be potentially caused by the wide range of axon sizes in the nerve. Magn Reson Med 42:911-918, 1999. (+info)