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.     

Genistein prevents the glucose autoxidation mediated atherogenic modification of low density lipoprotein

Hyperglycemia has been assumed to be responsible for oxidative stress in diabetes. In this respect, glucose autoxidation and advanced glycation end products (AGE) may play a causal role in the etiology of diabetic complications as e.g. atherosclerosis. There is now growing evidence that the oxidative modification of LDL plays a potential role in atherogenesis. Glucose derived oxidants have been shown to peroxidise LDL. In the present study, genistein, a compound derived from soy with a flavonoid chemical structure (4', 5, 7-trihydroxyisoflavone) has been evaluated for its ability to act as an antioxidant against the atherogenic modification of LDL by glucose autoxidation radical products. Daidzein, (4', 7-dihydroxyisoflavone) an other phytoestrogen of soy, was tested in parallel. Genistein — in contrast to daidzein — effectively prevented the glucose mediated LDL oxidation as measured by thiobarbituric acid-reactive substance formation (TBARS), alteration in electrophoretic mobility, lipid hydroperoxides and fluorescence quenching of tryptophan residues of the lipoprotein. In addition the potential of glucose-oxidized LDL to increase tissue factor (TF) synthesis in human endothelial cells (HUVEC) was completely inhibited when genistein was present during LDL oxidative modification by glucose. Both phytoestrogens did not influence the nonenzymatic protein glycation reaction as measured by the in vitro formation of glycated LDL. As the protective effect of genistein on LDL atherogenic modification was found at glucose/genistein molar ratios which may occur in vivo, our findings support the suggested beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Novoste Beta-Cath integrity test with the active source train

Intravascular brachytherapy (IVB) to prevent restenosis is currently being performed using several different commercial delivery devices. The Novoste Beta-Cath system uses a source train of 90Sr/90Y pure beta emitters and two gold radiopaque markers. A nonactive transfer device with dummy sources is also supplied to test the delivery catheter. We have developed an alternate procedure using an acrylic shield to test both the active transfer device and delivery catheter prior to patient treatment.     

The Legionella pneumophila CpxRA two‐component regulatory system: new insights into CpxR's function as a dual regulator and its connection to the effectors regulatory network

Legionella pneumophila utilizes the Icm/Dot type‐IV secretion system to translocate approximately 300 effector proteins into host cells, and the CpxRA two‐component system (TCS) was previously shown to regulate the expression of several of these effectors. In this study, we expanded the pool of L. pneumophila CpxR‐regulated genes to 38, including 27 effector‐encoding genes. Our study demonstrates for the first time that the CpxR dual regulator has different requirements for activation and repression of target genes. These differences include the positioning of the CpxR regulatory element relative to the promoter element, and the effect of CpxR phosphate donors on the expression of CpxR target genes. In addition, unlike most response regulators, a mutant form of the L. pneumophila CpxR which cannot be phosphorylated was found to self‐interact, and to repress gene expression similarly to wild‐type CpxR, even though its ability to activate gene expression was reduced. Moreover, the CpxRA TCS was found to activate the expression of LetE which was found to function as a connector protein between the CpxRA TCS and the LetAS‐RsmYZ‐CsrA regulatory cascade. Our results show that CpxR plays a major role in L. pneumophila pathogenesis gene expression and functions as part of a regulatory network.     

A chiton uses aragonite lenses to form images

Hundreds of ocelli are embedded in the dorsal shell plates of certain chitons. These ocelli each contain a pigment layer, retina, and lens, but it is unknown whether they provide chitons with spatial vision. It is also unclear whether chiton lenses are made from proteins, like nearly all biological lenses, or from some other material. Electron probe X-ray microanalysis and X-ray diffraction revealed that the chiton Acanthopleura granulata has the first aragonite lenses ever discovered. We found that these lenses allow A. granulata's ocelli to function as small camera eyes with an angular resolution of about 9°-12°. Animals responded to the sudden appearance of black, overhead circles with an angular size of 9°, but not to equivalent, uniform decreases in the downwelling irradiance. Our behavioral estimates of angular resolution were consistent with estimates derived from focal length and receptor spacing within the A. granulata eye. Behavioral trials further indicated that A. granulata's eyes provide the same angular resolution in both air and water. We propose that one of the two refractive indices of the birefringent chiton lens places a focused image on the retina in air, whereas the other does so in water.