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.     

Diterpenoids from Caesalpinia pulcherrima

Three new furanoditerpenoids of the caesalpin-type have been isolated from the roots of Caesalpinia pulcherrima. The structures of these compounds, vouacapen-5α-ol, 6β-cinnamoyl-7β-hydroxy-vouacapen-5α-ol and 8,9,11,14-didehydrovouacapen-5α-ol, were elucidated through interpretation of their spectral data. Sitosterol was also obtained.     

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.     

Relationships between inhibitory activity against a cancer cell line panel, profiles of plants collected, and compound classes isolated in an anticancer drug discovery project

In an attempt to determine the relationships between the plant profiles (country of collection, taxonomy, plant part) and the compound classes isolated with cytotoxic activity against a panel of human tumor cell lines, the data compiled from a 15-year anticancer drug-discovery project were subjected to an analysis of variance (ANOVA). The results indicate significant trends in cytotoxic activity relative to collection location, taxonomy, plant part, and compound classes isolated. Plant collections were made in tropical forests in six countries, with collections from Ecuador resulting in higher activity than those from Indonesia and Peru. Interestingly, collections from Florida were not statistically different than those from the countries with higher biodiversity. One hundred and forty-five families were represented in the collections, with the Clusiaceae, Elaeocarpaceae, Meliaceae, and Rubiaceae having low ED50 (half maximal effective dose) values. Especially active genera included Aglaia, Casearia, Exostema, Mallotus, and Trichosanthes. Roots and below-ground plant materials were significantly more active than above-ground materials. Cucurbitacins, flavaglines, anthraquinones, fatty acids, tropane alkaloids, lignans, and sesquiterpenoids were significantly more active than xanthones and oligorhamnosides. The results from this study should serve as a guide for future plant collection endeavors for anticancer drug discovery.     

Koenoline,a further cytotoxic carbazole alkaloid from Murraya koenigii

Koenoline, a carbazole alkaloid, has been isolated from the root bark of Murraya koenigii for the first time as a natural product. Its structure was established as 1-methoxy-3-hydroxymethylcarbazole by analysis of spectroscopic data and was confirmed by partial synthesis from murrayanine isolated from M. siamensis roots. Koenoline exhibited cytotoxic activity against the KB cell-culture test system.     

Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC₅₀ values of 31?nM and 3.5?µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.