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.     

Folate-liposome-mediated antisense oligodeoxynucleotide targeting to cancer cells: evaluation in vitro and in vivo

The objective of this study was to investigate the use of folate-targeted liposomes for the delivery of encapsulated oligonucleotides to folate receptor (FR)-positive tumor cells in vitro and in vivo. This project involved the synthesis and biological evaluation of many folate-PEG-lipid conjugates, where the chemical form of the folate moiety (pteroate) and the length of the PEG linker chain were varied widely. Folate-targeted oligonucleotide-containing liposomes were prepared using conventional methods, and the extent of cell uptake was evaluated using, among others, the FR positive KB cell line. Oligonucleotide-loaded folate-targeted liposomes were found to rapidly associate with the KB cells, and saturation was typically reached within the first hour of incubation at 37 degrees C. Nearly 100,000 liposomes per cell were bound or internalized at saturation. Importantly, cell association was blocked by a large excess folic acid, thus reflecting the FR-specific nature of the cell interaction. Full targeting potential was achieved with PEG linkers as low as 1000 in molecular weight, and pteroates bearing glycine or gamma-aminobutyryl residues juxtaposed to the pteroic acid moiety were also effective for targeting, provided that a terminal cysteine moiety was present at the distal end of the PEG chain for added hydrophilicity. When tested in vivo, folate-targeted liposomes were found to deliver approximately 1.8-fold more oligonucleotide to the livers of nude mice (relative to the nontargeted PEG-containing formulations); however, no improvement in KB tumor uptake was observed. We conclude from these results that folate liposomes can effectively deliver oligonucleotides into folate receptor-bearing cells in vitro, but additional barriers exist in vivo that prevent or decrease effective tumor uptake and retention.     

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.     

The effect of darbepoetin alfa on growth, oxygenation and radioresponsiveness of a breast adenocarcinoma

Tumor hypoxia is associated with poor clinical outcome in a variety of tumors, including cervical, head/neck and breast cancer. Administration of erythropoietic factors has been suggested as a means of improving tumor oxygenation (pO2). This study randomized rats to treatment with low-dose or high-dose darbepoetin alfa or a placebo to determine the effect of darbepoetin alfa on the pO2, growth and response to radiation therapy of R3230 mammary adenocarcinoma. Rats received 3 microg/kg (high dose) or 0.2 microg/kg (low dose) darbepoetin alfa or placebo for eight doses prior to either (1) pO2 measurement and pimonidazole staining or (2) irradiation or sham irradiation on post-transplant day 20. In the animals randomized to radiation treatment, placebo or darbepoetin alfa administration at a reduced dose was continued for 9 weeks or until the tumor diameter exceeded 15 mm (defined as failure for survival analysis). Treatment with high-dose and low-dose darbepoetin alfa produced hematocrits of 68 and 56% compared to 44 and 45% in their respective control groups (both P < 10(-5)). At 18 days post-transplant, tumor volume was not different for either darbepoetin alfa group compared to the placebo group. Tumor oxygenation, as measured by the fraction of pO2 measurement <10 mmHg and the intensity of pimonidazole staining, was significantly improved in the high-dose group (P = 0.046 and 0.03, respectively, compared with controls) but not in the low-dose group. Growth delay curves after irradiation did not differ significantly for high- or low-dose darbepoetin alfa compared to placebo. In this nonanemic animal model of mammary adenocarcinoma, darbepoetin alfa does not significantly alter tumor growth or radioresponsiveness, even though it improves oxygenation when administered at high doses.     

The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

Background

Inherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa.

Results

Spiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h). Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining.

Conclusion

The Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis.     

Mechanochemical coupling in spin-labeled, active, isometric muscle

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force?. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.