Alternative splicing generates multiple mRNA forms of the acetylcholine receptor gamma-subunit in rat muscle.
(9/28646)
The fetal type acetylcholine receptor, composed of the alphabeta gammadelta subunits, has shown a highly variable channel kinetics during postnatal development. We examine the hypothesis whether such a variability could result from multiple channel forms, differing in the N-terminus of the gamma-subunit. RT-PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4. One of them in addition lacks 19 nucleotides from exon 5, predicting a complete subunit, with a 43 residues shorter N-terminus. A third one lacking the complete exon 5 predicts a subunit without transmembrane segments. These forms, generated by alternative splicing, may account for the kinetic variability of the acetylcholine receptor channel. (+info)
Skeletal muscle type ryanodine receptor is involved in calcium signaling in human B lymphocytes.
(10/28646)
The regulation of intracellular free Ca2+ concentration ([Ca2+]i) in B cells remains poorly understood and is presently explained almost solely by inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ release, followed by activation of a store-operated channel mechanism. In fact, there are reports indicating that IP3 production does not always correlate with the magnitude of Ca2+ release. We demonstrate here that human B cells express a ryanodine receptor (RYR) that functions as a Ca2+ release channel during the B cell antigen receptor (BCR)-stimulated Ca2+ signaling process. Immunoblotting studies showed that both human primary CD19(+) B and DAKIKI cells express a 565-kDa immunoreactive protein that is indistinguishable in molecular size and immunoreactivity from the RYR. Selective reverse transcription-polymerase chain reaction, restriction fragment length polymorphism, and sequencing of cloned cDNA indicated that the major isoform of the RYR expressed in primary CD19(+) B and DAKIKI cells is identical to the skeletal muscle type (RYR1). Saturation analysis of [3H]ryanodine binding yielded Bmax = 150 fmol/mg of protein and Kd = 110 nM in DAKIKI cells. In fluo-3-loaded CD19(+) B and DAKIKI cells, 4-chloro-m-cresol, a potent activator of Ca2+ release mediated by the ryanodine-sensitive Ca2+ release channel, induced Ca2+ release in a dose-dependent and ryanodine-sensitive fashion. Furthermore, BCR-mediated Ca2+ release in CD19(+) B cells was significantly altered by 4-chloro-m-cresol and ryanodine. These results indicate that RYR1 functions as a Ca2+ release channel during BCR-stimulated Ca2+ signaling and suggest that complex Ca2+ signals that control the cellular activities of B cells may be generated by cooperation of the IP3 receptor and RYR1. (+info)
Effects of short term sacral nerve stimulation on anal and rectal function in patients with anal incontinence.
(11/28646)
BACKGROUND: Some patients with faecal incontinence are not amenable to simple surgical sphincter repair, due to sphincter weakness in the absence of a structural defect. AIMS: To evaluate the efficacy and possible mode of action of short term stimulation of sacral nerves in patients with faecal incontinence and a structurally intact external anal sphincter. PATIENTS: Twelve patients with faecal incontinence for solid or liquid stool at least once per week. METHODS: A stimulating electrode was placed (percutaneously in 10 patients, operatively in two) into the S3 or S4 foramen. The electrode was left in situ for a minimum of one week with chronic stimulation. RESULTS: Evaluable results were obtained in nine patients, with early electrode displacement in the other three. Incontinence ceased in seven of nine patients and improved notably in one; one patient with previous imperforate anus and sacral agenesis had no symptomatic response. Stimulation seemed to enhance maximum squeeze pressure but did not alter resting pressure. The rectum became less sensitive to distension with no change in rectal compliance. Ambulatory studies showed a possible reduction in rectal contractile activity and diminished episodes of spontaneous anal relaxation. CONCLUSIONS: Short term sacral nerve stimulation notably decreases episodes of faecal incontinence. The effect may be mediated via facilitation of striated sphincter muscle function, and via neuromodulation of sacral reflexes which regulate rectal sensitivity and contractility, and anal motility. (+info)
Expression of skeletal muscle sarcoplasmic reticulum calcium-ATPase is reduced in rats with postinfarction heart failure.
(12/28646)
OBJECTIVE: To determine whether heart failure in rats is associated with altered expression of the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA). METHODS: SERCA protein and mRNA were examined in the soleus muscles of eight female rats with heart failure induced by coronary artery ligation, six weeks after the procedure (mean (SEM) left ventricular end diastolic pressure 20.4 (2.2) mm Hg) and in six sham operated controls by western and northern analyses, respectively. RESULTS: SERCA-2a isoform protein was reduced by 16% (112 000 (4000) v 134 000 (2000) arbitrary units, p < 0.001), and SERCA-2a messenger RNA was reduced by 59% (0.24 (0. 06) v 0.58 (0.02) arbitrary units, p < 0.001). Although rats with heart failure had smaller muscles (0.54 mg/g v 0.66 mg/g body weight), no difference in locomotor activity was observed. CONCLUSIONS: These results may explain the previously documented abnormalities in calcium handling in skeletal muscle from animals with the same model of congestive heart failure, and could be responsible for the accelerated muscle fatigue characteristic of patients with heart failure. (+info)
Marimastat in recurrent colorectal cancer: exploratory evaluation of biological activity by measurement of carcinoembryonic antigen.
(13/28646)
Marimastat is a specific inhibitor of matrix metalloproteinases that has been shown to be effective in cancer models. A pilot, escalating-dose study of oral marimastat was performed in patients with recurrent colorectal cancer, in whom evaluation of serological response was made by measurement of carcinoembryonic antigen (CEA) levels. The study assessed the safety and tolerability of 4 weeks administration of marimastat, and determined a dose range producing detectable serological effects. Patients were recruited with a serum CEA level greater than 5 ng ml(-1), and rising by more than 25% over a 4-week screening period. Patients were treated for 28 days and entered into a continuation protocol if a serological response or clinical benefit was observed. Pharmacokinetic and safety data determined that groups of patients were recruited sequentially at 25 mg and 50 mg twice daily, and, thereafter, 10 mg twice daily, 10 mg once daily, 5 mg once daily and 20 mg once daily. A biological effect (BE) was defined as a CEA value on day 28 no greater than on day 0; a partial biological effect (PBE) was defined as a rise in CEA over the 28-day treatment period of less than 25%. Of 70 patients recruited, 63 completed the 28-day treatment period, and 55 were eligible for cancer antigen analysis. Examination of the dose-effect relationships provides evidence for a causal relationship between marimastat and biological effects: the proportion of patients with BE or PBE was higher with twice daily dosing (16 out of 25, 64%) than with once daily dosing (11 out of 30, 37%) (P = 0.043, chi2 test). Furthermore, the median rates of rise of CEA fell markedly during treatment compared with the screening period for patients receiving twice daily marimastat (P<0.0001), but not for patients receiving marimastat once daily (P = 0.25). Musculoskeletal adverse events emerged as the principal drug-related toxicity of marimastat, occurring in a dose- and time-dependent fashion. It was concluded that marimastat was associated with dose-dependent biological effects in cancer patients. The occurrence of musculoskeletal side-effects define 25 mg twice daily as the upper limit of the dose range for continuous use in further studies. Therefore, a dose range of 20 mg once daily to 25 mg twice daily seems appropriate for further studies, which should aim to demonstrate the efficacy of the drug in terms of conventional clinical end points and describe the long-term tolerability of this novel agent. (+info)
Rescue of diabetes-related impairment of angiogenesis by intramuscular gene therapy with adeno-VEGF.
(14/28646)
Diabetes is a major risk factor for coronary and peripheral artery diseases. Although diabetic patients often present with advanced forms of these diseases, it is not known whether the compensatory mechanisms to vascular ischemia are affected in this condition. Accordingly, we sought to determine whether diabetes could: 1) impair the development of new collateral vessel formation in response to tissue ischemia and 2) inhibit cytokine-induced therapeutic neovascularization. Hindlimb ischemia was created by femoral artery ligation in nonobese diabetic mice (NOD mice, n = 20) and in control C57 mice (n = 20). Hindlimb perfusion was evaluated by serial laser Doppler studies after the surgery. In NOD mice, measurement of the Doppler flow ratio between the ischemic and the normal limb indicated that restoration of perfusion in the ischemic hindlimb was significantly impaired. At day 14 after surgery, Doppler flow ratio in the NOD mice was 0.49+/-0.04 versus 0.73+/-0.06 for the C57 mice (P< or =0.005). This impairment in blood flow recovery persisted throughout the duration of the study with Doppler flow ratio values at day 35 of 0.50+/-0.05 versus 0.90+/-0.07 in the NOD and C57 mice, respectively (P< or =0.001). CD31 immunostaining confirmed the laser Doppler data by showing a significant reduction in capillary density in the NOD mice at 35 days after surgery (302+/-4 capillaries/mm2 versus 782+/-78 in C57 mice (P< or =0.005). The reduction in neovascularization in the NOD mice was the result of a lower level of vascular endothelial growth factor (VEGF) in the ischemic tissues, as assessed by Northern blot, Western blot and immunohistochemistry. The central role of VEGF was confirmed by showing that normal levels of neovascularization (compared with C57) could be achieved in NOD mice that had been supplemented for this growth factor via intramuscular injection of an adenoviral vector encoding for VEGF. We conclude that 1) diabetes impairs endogenous neovascularization of ischemic tissues; 2) the impairment in new blood vessel formation results from reduced expression of VEGF; and 3) cytokine supplementation achieved by intramuscular adeno-VEGF gene transfer restores neovascularization in a mouse model of diabetes. (+info)
Nerve terminal damage by beta-bungarotoxin: its clinical significance.
(15/28646)
We report here original data on the biological basis of prolonged neuromuscular paralysis caused by the toxic phospholipase A2 beta-bungarotoxin. Electron microscopy and immunocytochemical labeling with anti-synaptophysin and anti-neurofilament have been used to show that the early onset of paralysis is associated with the depletion of synaptic vesicles from the motor nerve terminals of skeletal muscle and that this is followed by the destruction of the motor nerve terminal and the degeneration of the cytoskeleton of the intramuscular axons. The postjunctional architecture of the junctions were unaffected and the binding of fluorescein-isothiocyanate-conjugated alpha-bungarotoxin to acetylcholine receptor was not apparently affected by exposure to beta-bungarotoxin. The re-innervation of the muscle fiber was associated by extensive pre- and post-terminal sprouting at 3 to 5 days but was stable by 7 days. Extensive collateral innervation of adjacent muscle fibers was a significant feature of the re-innervated neuromuscular junctions. These findings suggest that the prolonged and severe paralysis seen in victims of envenoming bites by kraits (elapid snakes of the genus Bungarus) and other related snakes of the family Elapidae is caused by the depletion of synaptic vesicles from motor nerve terminals and the degeneration of the motor nerve terminal and intramuscular axons. (+info)
Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle.
(16/28646)
The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers. (+info)