Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium ...homeostasis and reduced coenzyme Q
10
levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q
10
was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q
10
supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q
10
supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q
10
to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q
10
in statin treated patients symptomatic of this condition.
Calcium ions are crucial elements of excitation-contraction coupling in cardiac myocytes. The intracellular Ca(2+ ) concentration changes continously during the cardiac cycle, but the Ca(2+ ) ...entering to the cell serves as an intracellular second messenger, as well. The Ca(2+ ) as a second messenger influences the activity of many intracellular signalling pathways and regulates gene expression. In cardiac myocytes the major pathway for Ca(2+ ) entry into cells is L-type calcium channel (LTCC). The precise control of LTCC function is essential for maintaining the calcium homeostasis of cardiac myocytes. Dysregulation of LTCC may result in different diseases like cardiac hypertrophy, arrhytmias, heart failure. The physiological and pathological structural changes in the heart are induced in part by small G proteins. These proteins are involved in wide spectrum of cell biological functions including protein transport, regulation of cell proliferation, migration, apoptosis, and cytoskeletal rearrangement. Understanding the crosstalk between small G proteins and LTCC may help to understand the pathomechanism of different cardiac diseases and to develop a new generation of genetically-encoded Ca(2+ ) channel inhibitors.
The activity of the transient receptor potential vanilloid type 1 ion channel (TRPV1) that is expressed by the great majority of polymodal nociceptors is pivotal for the development of inflammatory ...heat hyperalgesia. The responsiveness of TRPV1 is regulated by a series of intracellular signalling molecules including the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA); increased or decreased PKA activity results in TRPV1 sensitisation or desensitisation, respectively. Activation of the cannabinoid 1 (CB1) receptor that is expressed by the majority of the TRPV1-expressing primary sensory neurons reduces PKA activity. Therefore, here we studied whether activation of the CB1 receptor resulted in reduced TRPV1-mediated responses in cultured rat primary sensory neurons. We found that TRPV1-mediated whole-cell currents were significantly reduced respectively, by 50% and 25% by 10 nM and 30 nM of the endogenous CB1 receptor agonist, anandamide. The PKA inhibitor, H89 (10 microM) also had a significant inhibitory effect on TRPV1-mediated currents ( approximately 70%). These findings suggest that activation of the CB1 receptor can reduce the activity of TRPV1 in primary sensory neurons, and that this inhibitory effect could be mediated through the reduction of PKA-mediated phosphorylation of TRPV1.
Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium ...homeostasis and reduced coenzyme Q^sub 10^ levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q^sub 10^ was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q^sub 10^ supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q^sub 10^ supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q^sub 10^ to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q^sub 10^ in statin treated patients symptomatic of this condition.
Abstract Clinically relevant concentrations of isoflurane or sevoflurane sensitize transient receptor potential vanilloid type 1 to several of its activators, including capsaicin. It has, moreover, ...been suggested these volatile general anaesthetics may augment nociceptive signalling arising from surgical procedures and thereby contribute to post-operative pain. To investigate this suggestion, we have studied intraplantar capsaicin injection-induced phosphorylation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons (which is a recognized marker of spinal nociceptive processing) in rat during isoflurane or sevoflurane anaesthesia after 60 min under anaesthesia. Control animals were anaesthetized with pentobarbital (which of itself does not activate extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons). Unilateral intraplantar capsaicin injection in control animals evoked extracellular signal-regulated kinase 1/2 phosphorylation in a group of neurons in lamina I and lamina II of the ipsilateral spinal dorsal horn in a somatotopically appropriate area. In contrast, both anaesthetic gases (given for 60 min and without subsequent capsaicin injection) induced extracellular signal-regulated kinase 1/2 activation in a different group of mainly lamina I neurons bilaterally. The total number of spinal dorsal horn neurons labelled on the ipliateral side following capsaicin injection into the isoflurane-, or sevoflurane-, anaesthetized animals was significantly less than that produced by capsaicin alone. Further, capsaicin injection into isoflurane-, or sevoflurane-, anaesthetized animals reduced extracellular signal-regulated kinase 1/2 phosphorylation induced by the gases alone on both sides. These findings do not support the suggestion that isoflurane-, or sevoflurane-, induced sensitization of transient receptor potential vanilloid type 1 by capsaicin, or other agonist, is translated into induction of spinal nociceptive processing and consequential pain sensation.
The first genetic transformation method has been developed for
Cuscuta trifolii, an economically important parasite of alfalfa
(Medicago sativa L.). For transgene delivery,
Agrobacterium tumefaciens ...vector was used according to a standard protocol (Method A) and a modified one, which previously resulted in the successful
Agrobacterium-mediated transformation of monocot cereals (Method B). In Method B the impacts of physical and biochemical stimulation of the bacterial infection were evaluated on the frequency of transformation such as gene gun-mediated microwounding of plant explants, infiltration of
Agrobacterium suspension into target tissues and the influence of in vitro preinduction of
vir genes, respectively. While the physical enhancement of
Agrobacterial infection (microwounding, infiltration) had no positive effect on the transformation frequency, the biochemical one (in vitro preinduction of
vir genes) resulted in 4-fold increase in comparison with the conventional protocol (Method A). Additionally, a PCR-based technique was developed during the transformation work to monitor early transformation events, using primers designed to amplify specific DNA fragments outside of the T-DNA. Our data confirmed that the presence of a given transgene can be evaluated by using this primer pair as a control.
The application of aminoglycoside-3"-adenyltransferase ( aadA) gene-mediated streptomycin resistance for non-lethal selection of transgenic rice resulted in plant regeneration frequencies under ...selection pressure as high as those in non-transformed controls without selection. Since streptomycin does not kill non-transgenic cells, and allows plant regeneration from them, a selection procedure was developed that made the visual identification of transgenic calli and regenerants possible. For callus-level selection, a vital pH indicator-Chlorophenol Red-was applied together with streptomycin, making use of the phenomenon that fast-growing cell lines lower the pH in the culture medium. Transgenic plants were selected according to their main distinctive features; their green colour (photomixotrophic assimilation), and more intense growth. At the same time, non-transgenic regenerants were bleached (heterotrophic assimilation), and growth was retarded in the presence of streptomycin and sucrose. The final efficiency of genetic transformation based on streptomycin resistance was found to be double that of transformations where the selective agent was l-phosphinothricin, and nearly three times more compared to transformations resulting in hygromycin-resistant regenerants. To the best of our knowledge, this is the first report on producing nuclear transformed rice plants by using a non-lethal selection strategy based on the chimaeric aadA gene.
In this paper we discuss the changes in the number of plants developed per embryogenic mass, the percentage of embryogenic masses that developed plants, the percentage of plant regeneration, and the ...index of vigor of regenerated plants for embryogenic masses that were induced from daily samples of a cell suspension culture of rice (Oryza sativa L. cv. Taipei 309). The results indicate that embryogenic masses that matured first and were induced from freshly subcultured cell suspension culture had the highest values for the four parameters. An index of vigor of the regenerated plants was defined.
It has been previously reported that the 5' region of the rice actin 1 gene (Act1) promoted high-level expression of a beta-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper ...we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauliflower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previously been attainable with alternative expression vectors.
