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.     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

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.     

The Cf-ECP2 gene is linked to, but not part of, the Cf-4/Cf-9 cluster on the short arm of chromosome 1 in tomato

A gene has been identified in tomato, which confers resistance to Cladosporium fulvum through recognition of the pathogenicity factor ECP2. Segregation analysis of F₂ and F₃ populations showed monogenic dominant inheritance, as for previously reported Cf resistances. The gene has been designated Cf-ECP2. Using several mapping populations, Cf-ECP2 was accurately mapped on chromosome 1, 7.7 cM proximal to TG236 and 6.0 cM distal to TG184. Although Cf-ECP2 is linked to Cf-4, it is not located in the Hcr9 cluster “Milky Way”. Therefore, Cf-ECP2 is the first functional Cf homologue on chromosome 1 that does not belong to this Hcr9 cluster. No recombination events between Cf-ECP2 and CT116 have been observed in three populations tested, representing 282 individuals. The low value for the physical distance per cM around CT116 reported previously and the high probability that Cf-ECP2 is also a member of a Hcr9 cluster will facilitate cloning of the locus. Received: 15 June 1999 / Accepted: 24 August 1999     

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.     

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.     

The patterns of population differentiation in a <Emphasis Type="Italic">Brassica rapa</Emphasis> core collection

With the recent advances in high throughput profiling techniques the amount of genetic and phenotypic data available has increased dramatically. Although many genetic diversity studies combine morphological and genetic data, metabolite profiling has yet to be integrated into these studies. For our study we selected 168 accessions representing the different morphotypes and geographic origins of Brassica rapa. Metabolite profiling was performed on all plants of this collection in the youngest expanded leaves, 5 weeks after transplanting and the same material was used for molecular marker profiling. During the same season a year later, 26 morphological characteristics were measured on plants that had been vernalized in the seedling stage. The number of groups and composition following a hierarchical clustering with molecular markers was highly correlated to the groups based on morphological traits (r = 0.420) and metabolic profiles (r = 0.476). To reveal the admixture levels in B. rapa, comparison with the results of the programme STRUCTURE was needed to obtain information on population substructure. To analyze 5546 metabolite (LC–MS) signals the groups identified with STRUCTURE were used for random forests classification. When comparing the random forests and STRUCTURE membership probabilities 86% of the accessions were allocated into the same subgroup. Our findings indicate that if extensive phenotypic data (metabolites) are available, classification based on this type of data is very comparable to genetic classification. These multivariate types of data and methodological approaches are valuable for the selection of accessions to study the genetics of selected traits and for genetic improvement programs, and additionally provide information on the evolution of the different morphotypes in B. rapa.     

An improved method of partially digesting plant megabase DNA suitable for YAC cloning: application to the construction of a 5.5 genome equivalent YAC library of tomato

An improved method for preparing partially digested tomato DNA has been developed, that is suitable for YAC cloning. It involves (i) isolation of high molecular-weight DNA from agarose-embedded leaf protoplasts, (ii) controlled partial digestion in situ using Eco RI endonuclease in the presence of Eco RI methylase (M. Eco RI), and (iii) fractionation of the partial digest on a Clamped Homogeneous Electric Fields (CHEF) gel. Unlike methods commonly used for generating partial digests, the present method allows one to produce digests in which the bulk of restriction fragments are of the desired size. Use of these partial digests in constructing YAC libraries of the tomato lines Moneymaker- Cf4 and VFNT Cherry resulted in libraries (total 21 060 clones, 5.5 genome equivalents) in which 80% of the YACs have inserts between 200 and 600 kb. Both libraries have been screened with selected RFLP markers linked to the Cladosporium fulvum Cf4 locus on chromosome 1, using a three-dimensional PCR-based screening technique. To this end, the RFLP markers have been sequenced to allow for the synthesis of specific primers. Thus, for each marker tested several YAC clones have been isolated, including a family of clones that carry leucine-rich repeat sequences located around the Cf4/ Cf9 locus.