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.     

La protein is associated with terminal oligopyrimidine mRNAs in actively translating polysomes

La is an abundant, mostly nuclear, RNA-binding protein that interacts with regions rich in pyrimidines. In the nucleus it has a role in the metabolism of several small RNAs. A number of studies, however, indicate that La protein is also implicated in cytoplasmic functions such as translation. The association of La in vivo with endogenous mRNAs engaged with polysomes would support this role, but this point has never been addressed yet. Terminal oligopyrimidine (TOP) mRNAs, which code for ribosomal proteins and other components of the translational apparatus, bear a TOP stretch at the 5' end, which is necessary for the regulation of their translation. La protein can bind the TOP sequence in vitro and activates TOP mRNA translation in vivo. Here we have quantified La protein in the cytoplasm of Xenopus oocytes and embryo cells and have shown in embryo cells that it is associated with actively translating polysomes. Disruption of polysomes by EDTA treatment displaces La in messenger ribonucleoprotein complexes sedimenting at 40-60 S. The results of polysome treatment with either low concentrations of micrococcal nuclease or with high concentrations of salt indicate, respectively, that La association with polysomes is mediated by mRNA and that it is not an integral component of ribosomes. Moreover, the analysis of messenger ribonucleoprotein complexes dissociated from translating polysomes shows that La protein associates with TOP mRNAs in vivo when they are translated, in line with a positive role of La in the translation of this class of mRNAs previously observed in cultured cells.     

Treatment of Solitary Painful Osseous Metastases with Radiotherapy,Cryoablation or Combined Therapy: Propensity Matching Analysis in 175 Patients

Purpose

aim of this study was to identify outcomes in pain relief and quality of life in patients with a solitary painful osseous metastasis treated by radiotherapy, cryoablation or the combination using a propensity score matching study design.

Materials and Methods

175 patients with painful bone metastases were included in the study. Twenty-five of them underwent a radiation course (20 Gy in five daily fractions) 15 days after the cryoablation. These subjects were retrospectively matched by propensity analysis with a group of subjects treated by radiotherapy (125 subjects) and with a group treated byCryoablation (25 subjects). The pain relief in terms of complete response, rate of subjects requiring analgesics after treatments and the changes in self-rated quality of life were measured. Informed consent was obtained from the subject and the study was approved by the local Ethical Committee.

Results

An higher proportion of subjects treated by cryoablation (32%) or cryoablation followed by RT (72%;) experienced a complete response compared with patients treated by radiotherapy alone (11.2%). After Bonferroni correction strategy, the addition of radiotherapy to cryoablation significantly improved the rate of complete response compared with cryoablation alone (p = 0.011) and this paralleled with an improved self-rated quality of life. Seventeen subjects (13.6%) of patients in the radiotherapy group, 9 (36%) in the cryoablation group, and 19 (76)% in the cryoablation- radiotherapy group did not require narcotic medications.

Conclusions

The addition of radiotherapy to cryoablation favorably impacts on perceived pain, with a favorable toxicity profile. However, our data should be interpreted with caution and could serve as a framework around which to design future trials.     

Analyzing the hydrodynamic and crowding evolution of aqueous hydroxyapatite‐gelatin networks: Digging deeper into bone scaffold design variables

The hydration of the polypeptide network is a determinant factor to be controlled on behalf of the design of precise functional tissue scaffolding. Here we present an exhaustive study of the hydrodynamic and crowding evolution of aqueous gelatin‐hydroxyapatite systems with the aim of increasing the knowledge about the biomimesis of collagen mineralization; and how it can be manipulated for the preparation of collagenous derived frameworks with specific morphological characteristics. The solution's density and viscosity evaluation measurements in combination with spectroscopic techniques revealed that there is a progressive association of protein chain that can be influenced by the amount of hydroxyapatite nanorods. Gelatin and additives’ concentration effect on the morphology of the gelatin scaffolds was investigated. Transverse and longitudinal sections of the obtained scaffolds were taken and analyzed using optical microscopy. It can be seen that the porous size and shape of gelatin assemblies can be easily adjusted by controlling the gelatin/HAp ratio in the solution used as template in agreement with our statement. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 393–405, 2015.     

Hormonal and Nutritional Changes in the Flavedo Regulating Rind Color Development in Sweet Orange [Citrus sinensis (L.) Osb.]

The objective of this research was to determine the changes in the levels of endogenous gibberellins GA₁ and GA₄, abscisic acid (ABA), and ethylene during fruit coloring of on-tree fruits of sweet orange. The time course of carbohydrates and nitrogen content in the flavedo prior to fruit color break and during peel ripening were also studied. To identify nutritional and hormonal changes in the fruit, 45?days before fruit color break the peduncles of 15?C30 fruits per tree of ??Washington?? navel, ??Navelate,?? and ??Valencia Delta Seedless?? sweet orange, located in single-fruited shoots, were girdled to intercept phloem transport. A set of 15?C30 fruits per tree remained intact on the peduncle for control. Girdling significantly delayed fruit coloration for more than 2?months; the delay paralleled higher GA₁ and GA₄ concentrations in the flavedo and retarded the rise of ABA concentration prior to color break. Girdling also reduced carbohydrate concentrations and increased N concentrations in the flavedo compared to control fruits; no ethylene production was detected. Therefore, in sweet orange, fruit changes color by reducing active gibberellin concentrations in the flavedo, which are involved in regulating sugars and ABA accumulation and in reducing N fraction concentration as rind color develops. This was demonstrated in vivo without removing the fruit from the tree. Comparable results were obtained with experiments carried out over four consecutive years in two countries (Spain and Uruguay).     

Neutrophil paralysis in Plasmodium vivax malaria

Background

The activation of innate immune responses by Plasmodium vivax results in activation of effector cells and an excessive production of pro-inflammatory cytokines that may culminate in deleterious effects. Here, we examined the activation and function of neutrophils during acute episodes of malaria.

Materials and Methods

Blood samples were collected from P. vivax-infected patients at admission (day 0) and 30–45 days after treatment with chloroquine and primaquine. Expression of activation markers and cytokine levels produced by highly purified monocytes and neutrophils were measured by the Cytometric Bead Assay. Phagocytic activity, superoxide production, chemotaxis and the presence of G protein-coupled receptor (GRK2) were also evaluated in neutrophils from malaria patients.

Principal Findings

Both monocytes and neutrophils from P. vivax-infected patients were highly activated. While monocytes were found to be the main source of cytokines in response to TLR ligands, neutrophils showed enhanced phagocytic activity and superoxide production. Interestingly, neutrophils from the malaria patients expressed high levels of GRK2, low levels of CXCR2, and displayed impaired chemotaxis towards IL-8 (CXCL8).

Conclusion

Activated neutrophils from malaria patients are a poor source of pro-inflammatory cytokines and display reduced chemotactic activity, suggesting a possible mechanism for an enhanced susceptibility to secondary bacterial infection during malaria.