Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Losing the desire: selection can promote obligate asexuality

Whilst parthenogenesis has evolved multiple times from sexual invertebrate and vertebrate lineages, the drivers and consequences of the sex-asex transition remain mostly uncertain. A model by Stouthamer et al. recently published in BMC Evolutionary Biology shows a pathway by which obligate asexuality could be selected for following endosymbiont infection.     

Interspecific competition between entomopathogenic nematodes (Steinernema) is modified by their bacterial symbionts (Xenorhabdus)

Background  

Symbioses between invertebrates and prokaryotes are biological systems of particular interest in order to study the evolution of mutualism. The symbioses between the entomopathogenic nematodes Steinernema and their bacterial symbiont Xenorhabdus are very tractable model systems. Previous studies demonstrated (i) a highly specialized relationship between each strain of nematodes and its naturally associated bacterial strain and (ii) that mutualism plays a role in several important life history traits of each partner such as access to insect host resources, dispersal and protection against various biotic and abiotic factors. The goal of the present study was to address the question of the impact of Xenorhabdus symbionts on the progression and outcome of interspecific competition between individuals belonging to different Steinernema species. For this, we monitored experimental interspecific competition between (i) two nematode species: S. carpocapsae and S. scapterisci and (ii) their respective symbionts: X. nematophila and X. innexi within an experimental insect-host (Galleria mellonella). Three conditions of competition between nematodes were tested: (i) infection of insects with aposymbiotic IJs (i.e. without symbiont) of both species (ii) infection of insects with aposymbiotic IJs of both species in presence of variable proportion of their two Xenorhabdus symbionts and (iii) infection of insects with symbiotic IJs (i.e. naturally associated with their symbionts) of both species.     

Root-induced modifications of the exchange of phosphate ion between soil solution and soil solid phase

The uptake of phosphorus (P) by roots results in a depletion of phosphate ions (PO₄) in the rhizosphere. The corresponding decrease in PO₄ concentration in the soil solution (C_P) gives rise to a replenishment of P from the solid phase which is time- and C_P-dependent. This PO₄ exchange which reflects the buffer power of the soil for PO₄ also varies with the composition and the physico-chemical conditions of the soil. As root activity can modify these physico-chemical conditions in the rhizosphere, the question arises whether these modifications affect the ability of PO₄ bound to the soil solid phase to exchange with PO₄ in soil solution. The aim of the present work was to measure and compare the parameters which describe the amount of PO₄ bound to soil solid phase that is capable to replenish solution P for both rhizosphere and bulk soils. The soil sample was a P-enriched, calcareous topsoil collected from a long-term fertiliser trial. Rhizosphere soil samples were obtained by growing dense mats of roots at the surface of 3 mm thick soil layer for one week. Three plant species were compared: oilseed rape (Brassica napus L., cv Goeland) pea (Pisum sativum L., cv. Solara) and maize ( Zea mays L., cv. Volga). The time- and C_P-dependence of the PO₄ exchange from soil to solution were described using an isotopic dilution method. The measured C_P values were 0.165 mg P L⁻¹ for bulk soil and 0.111, 0.101 and 0.081 mg P L⁻¹ for rhizosphere soils of maize, pea and rape, respectively. The kinetics of the PO₄ exchange between liquid and solid phases of soil were significantly different between rhizosphere and bulk soils. However, when changes in C_P were accounted for, the parameters describing the PO₄ exchange with time and C_P between soil solution and soil solid phase were found to be very close for bulk and rhizosphere soils. For this calcareous and P-enriched soil, plant species differed in their ability to deplete PO₄ in solution. The resulting changes in the ability of the soil solid phase to replenish solution PO₄ were almost fully explained by the depletion of soil solution P. This revised version was published online in June 2006 with corrections to the Cover Date.     

Root uptake and distribution of radiocaesium from contaminated soils and the enhancement of Cs adsorption in the rhizosphere

Uptake by roots from contaminated soil is one of the key steps in the entry of radiocaesium into the food chain. We have measured the uptake by roots of radiocaesium and its transfer to shoots of a heathland grass, sheep fescue (Festuca ovina L.) from two contrasting agricultural soils, a sandy podzol and a clayey calcareous soil. A culture device which keeps the roots separate from the soil was used thus allowing rhizosphere soil to be obtained easily and enhancing the effect of root action. Biomass production and ¹³⁷Cs in shoots and roots were recorded. Cs adsorption was studied on both the initial, nonrhizosphere soil and on rhizosphere soil in dilute soil suspension. Cs desorption was measured by resuspending subsamples of contaminated soil in solutions containing various concentrations of stable Cs. The proportion of Cs fixed, i.e. not readily desorbable, was calculated by comparison of the adsorption and desorption isotherms. Uptake by roots varied considerably between soils and did not appear to be diffusion limited. Root-to-shoot transfer did not differ for the two soils studied. Root action considerably enhanced Cs adsorption on the soils, particularly the in sandy podzol with a low Cs affinity. The value of K_d was increased by up to an order of magnitude. A large proportion of adsorbed Cs was found to be fixed, the K_d was up to seven times greater on desorption than adsorption, indicating that up to 80% of adsorbed Cs was not readily exchangeable. Root action had little effect on the fixed fraction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rainfall reduction impacts rhizosphere biogeochemistry in eucalypts grown in a deep Ferralsol in Brazil

Plant and Soil - Comparing root functioning under contrasting rainfall regimes can help assessing the capacity of plant species to cope with more intense and frequent drought predicted under...     

Dynamics of phosphorus fractions in the rhizosphere of common bean (Phaseolus vulgaris L.) and durum wheat (Triticum turgidum durum L.) grown in monocropping and intercropping systems

The main objective of the present study was to investigate phosphorus (P) dynamics in the rhizosphere of durum wheat (Triticum turgidum durum L.) and common bean (Phaseolus vulgaris L.) grown in monocropping and intercropping systems with nitrate supply. Wheat and common bean were grown either alone or in association in a cropping device with a thin (1 mm) soil layer sandwiched between large root mats. Wheat intercropped with common bean exhibited a 33% increase in shoot biomass and a 22% increased root biomass, without significantly affecting common bean growth. After 12 days of culture, rhizosphere pH decreased by 1.66 and 1.13 units in monocropping system of common bean and intercropping system, respectively. Wheat increased intercropped common bean proton release by 36% compared with monocropped beans. Common bean and wheat exhibited different behaviors in rhizosphere P dynamics. Monocropped wheat decreased Resin-P, NaHCO₃-P and NaOH-P in its rhizosphere by 24, 96 and 10%, respectively. However, NaHCO₃-P and NaOH-P were increased by 61 and 10% in the rhizosphere of intercropping. Almost all values about P fraction in intercropping system were between those in monocropped common bean and monocropped wheat. Through taping different P fraction, different plants species possibly can alleviate competition for phosphorus in intercropping system.     

A Phylogenetic Re-Analysis of Groupers with Applications for Ciguatera Fish Poisoning

Background

Ciguatera fish poisoning (CFP) is a significant public health problem due to dinoflagellates. It is responsible for one of the highest reported incidence of seafood-borne illness and Groupers are commonly reported as a source of CFP due to their position in the food chain. With the role of recent climate change on harmful algal blooms, CFP cases might become more frequent and more geographically widespread. Since there is no appropriate treatment for CFP, the most efficient solution is to regulate fish consumption. Such a strategy can only work if the fish sold are correctly identified, and it has been repeatedly shown that misidentifications and species substitutions occur in fish markets.

Methods

We provide here both a DNA-barcoding reference for groupers, and a new phylogenetic reconstruction based on five genes and a comprehensive taxonomical sampling. We analyse the correlation between geographic range of species and their susceptibility to ciguatera accumulation, and the co-occurrence of ciguatoxins in closely related species, using both character mapping and statistical methods.

Results

Misidentifications were encountered in public databases, precluding accurate species identifications. Epinephelinae now includes only twelve genera (vs. 15 previously). Comparisons with the ciguatera incidences show that in some genera most species are ciguateric, but statistical tests display only a moderate correlation with the phylogeny. Atlantic species were rarely contaminated, with ciguatera occurrences being restricted to the South Pacific.

Conclusions

The recent changes in classification based on the reanalyses of the relationships within Epinephelidae have an impact on the interpretation of the ciguatera distribution in the genera. In this context and to improve the monitoring of fish trade and safety, we need to obtain extensive data on contamination at the species level. Accurate species identifications through DNA barcoding are thus an essential tool in controlling CFP since meal remnants in CFP cases can be easily identified with molecular tools.