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.     

Duplex formation of a nonionic oligo(deoxythymidylate) analogue (heptadeoxythymidylyl-(3'-5')-deoxythymidine heptaethyl ester (d-(Tp(Et))7T)) with poly(deoxyadenylate). Evaluation of the electrostatic interaction.

The heptaethyl ester of heptadeoxythymidylyl-(3'-5')-deoxythymidine (d-[Tp(Et)]7T or d-T8-Et) has been prepared by chemical methods. The material, consisting of a mixture of diastereoisomers, forms a 1:1 complex with (dA)n in neutral aqueous buffer; this interaction is virtually independent of ionic strength. The octamer triester does not bind to (dA)n-(dT)n, and it interacts with (rA)n only at low temperatures. By cochromatography with (dA)n on Sephadex G-50, d-T8-Et fractions with different binding affinities for the polyadenylates were obtained. This heterogeneity in binding affinity is ascribed to the diastereoisomerism of d-T8-Et. Enthalpies of dupoex formation were determined by the concentration variation method. At 0.1 M sodium ion concentration, the enthalpy of binding of the various d-T8-Et fractions to (dA)n is essentially invariant (-8.1 kcal/mol of base pairs at 0 degrees C to -8.6 kcal at 25 degrees C) and 1.6 kcal/mol of base pairs more negative than the enthalpy of binding of the phosphodiester analogue, d-(Tp)7T, to (dA)n (-6.8 kcal/mol of base pairs at 11 degrees C). This difference is the electrostatic contribution to the enthalpy of duplex formation, arising from the interstrand electrostatic repulsion and the intrastrand repulsion in d-(Tp)7T. The entropy of binding to (dA)n is more negative for the octamer triesters than for the diester analogue, and is different for the various d-T8-Et fractions. This is interpreted in terms of varying degrees of restriction of rotational freedom for the ethyl substituents upon double helix formation.     

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.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis. Characterization of [Man-14C]glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-[14C]Man.

Endogenous proteins of cell-free preparations of hen oviduct labeled from GDP-[14C]Man or from [Man-14C]oligosaccharide-lipid have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under the conditions tested, a polypeptide chain of molecular weight about 25,000 was the principle acceptor for the oligosaccharide moiety of exogenous [Man-14C]oligosaccharide-lipid. The product labeled by [Man-14C]oligosaccharide-lipid appeared identical with one of three glycoproteins formed when GDP-[14C]Man was incubated with a crude membrane fraction. These three proteins (apparent molecular weight of 75,000, 55,000, and 25,000) accounted for nearly two-thirds of the [14C]mannose-labeled glycoprotein products using GDP-[14C]Man and either the crude membrane fraction or a total oviduct homogenate. Thus, all of the mannose acceptor proteins present in the oviduct homogenate appear to be membrane-bound. Analyses of the [Man-14C]glycoproteins labeled from GDP-[14C]Man in membrane fractions from hen kidney, liver, brain, and oviduct indicated that a labeled polypeptide of apparent molecular weight 25,000 was the only major protein product common to the four preparations.     

Sunshine versus gold: The effect of population age on genetic structure of an invasive mosquito

The genetic diversity and structure of invasive species are affected by the time since invasion, but it is not well understood how. We compare likely the oldest populations of Aedes aegypti in continental North America with some of the newest to illuminate the range of genetic diversity and structure that can be found within the invasive range of this important disease vector. Aedes aegypti populations in Florida have probably persisted since the 1600‐1700s, while populations in southern California derive from new invasions that occurred in the last 10 years. For this comparison, we genotyped 1,193 individuals from 28 sites at 12 highly variable microsatellites and a subset of these individuals at 23,961 single nucleotide polymorphisms (SNPs). This is the largest sample analyzed for genetic structure for either region, and it doubles the number of southern California populations previously analyzed. As predicted, the older populations (Florida) showed fewer indicators of recent founder effect and bottlenecks; in particular, these populations have dramatically higher genetic diversity and lower genetic structure. Geographic distance and driving distance were not good predictors of genetic distance in either region, especially southern California. Additionally, southern California had higher levels of genetic differentiation than any comparably sized documented region throughout the worldwide distribution of the species. Although population age and demographic history are likely driving these differences, differences in climate and transportation practices could also play a role.