Mutation screening of the RYR1 gene and identification of two novel mutations in Italian malignant hyperthermia families.
(1/1763)
Point mutations in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia, an autosomal dominant disorder triggered in susceptible people (MHS) by volatile anaesthetics and depolarising skeletal muscle relaxants. To date, 17 missense point mutations have been identified in the human RYR1 gene by screening of the cDNA obtained from muscle biopsies. Here we report single strand conformation polymorphism (SSCP) screening for nine of the most frequent RYR1 mutations using genomic DNA isolated from MHS patients. In addition, the Argl63Cys mutation was analysed by restriction enzyme digestion. We analysed 57 unrelated patients and detected seven of the known RYR1 point mutations. Furthermore, we found a new mutation, Arg2454His, segregating with the MHS phenotype in a large pedigree and a novel amino acid substitution at position 2436 in another patient, indicating a 15.8% frequency of these mutations in Italian patients. A new polymorphic site in intron 16 that causes the substitution of a G at position -7 with a C residue was identified. (+info)
A neomorphic syntaxin mutation blocks volatile-anesthetic action in Caenorhabditis elegans.
(2/1763)
The molecular mechanisms underlying general anesthesia are unknown. For volatile general anesthetics (VAs), indirect evidence for both lipid and protein targets has been found. However, no in vivo data have implicated clearly any particular lipid or protein in the control of sensitivity to clinical concentrations of VAs. Genetics provides one approach toward identifying these mechanisms, but genes strongly regulating sensitivity to clinical concentrations of VAs have not been identified. By screening existing mutants of the nematode Caenorhabditis elegans, we found that a mutation in the neuronal syntaxin gene dominantly conferred resistance to the VAs isoflurane and halothane. By contrast, other mutations in syntaxin and in the syntaxin-binding proteins synaptobrevin and SNAP-25 produced VA hypersensitivity. The syntaxin allelic variation was striking, particularly for isoflurane, where a 33-fold range of sensitivities was seen. Both the resistant and hypersensitive mutations decrease synaptic transmission; thus, the indirect effect of reducing neurotransmission does not explain the VA resistance. As assessed by pharmacological criteria, halothane and isoflurane themselves reduced cholinergic transmission, and the presynaptic anesthetic effect was blocked by the resistant syntaxin mutation. A single gene mutation conferring high-level resistance to VAs is inconsistent with nonspecific membrane-perturbation theories of anesthesia. The genetic and pharmacological data suggest that the resistant syntaxin mutant directly blocks VA binding to or efficacy against presynaptic targets that mediate anesthetic behavioral effects. Syntaxin and syntaxin-binding proteins are candidate anesthetic targets. (+info)
Functional brain imaging during anesthesia in humans: effects of halothane on global and regional cerebral glucose metabolism.
(3/1763)
BACKGROUND: Propofol and isoflurane anesthesia were studied previously with functional brain imaging in humans to begin identifying key brain areas involved with mediating anesthetic-induced unconsciousness. The authors describe an additional positron emission tomography study of halothane's in vivo cerebral metabolic effects. METHODS: Five male volunteers each underwent two positron emission tomography scans. One scan assessed awake-baseline metabolism, and the other scan assessed metabolism during halothane anesthesia titrated to the point of unresponsiveness (mean +/- SD, expired = 0.7+/-0.2%). Scans were obtained using a GE2048 scanner and the F-18 fluorodeoxyglucose technique. Regions of interest were analyzed for changes in both absolute and relative glucose metabolism. In addition, relative changes in metabolism were evaluated using statistical parametric mapping. RESULTS: Awake whole-brain metabolism averaged 6.3+/-1.2 mg x 100 g(-1) x min(-1) (mean +/- SD). Halothane reduced metabolism 40+/-9% to 3.7+/-0.6 mg x 100 g(-1) x min(-1) (P< or =0.005). Regional metabolism did not increase in any brain areas for any volunteer. The statistical parametric mapping analysis revealed significantly less relative metabolism in the basal forebrain, thalamus, limbic system, cerebellum, and occiput during halothane anesthesia. CONCLUSIONS: Halothane caused a global whole-brain metabolic reduction with significant shifts in regional metabolism. Comparisons with previous studies reveal similar absolute and relative metabolic effects for halothane and isoflurane. Propofol, however, was associated with larger absolute metabolic reductions, suppression of relative cortical metabolism more than either inhalational agent, and significantly less suppression of relative basal ganglia and midbrain metabolism. (+info)
Primary and secondary hyperalgesia in a rat model for human postoperative pain.
(4/1763)
BACKGROUND: Previously, the authors developed and characterized a rat model for postoperative pain to learn more about pain produced by incisions. In this study, the responses to heat and mechanical stimuli were evaluated directly on or adjacent to the incision and at varying distances from the incision. METHODS: Rats were anesthetized with halothane and incisions were made at different locations in the plantar aspect of the foot. The response frequency to a blunt mechanical stimulus, the withdrawal threshold to von Frey filaments (15-522 mN), and the withdrawal latency to radiant heat were measured. Rats were tested before surgery, 2 h later, and then daily through postoperative day 9. RESULTS: After plantar incision, persistent hyperalgesia was observed immediately adjacent to or directly on the incision to punctate and blunt mechanical stimuli, respectively. The withdrawal threshold to punctate stimuli applied 1 cm from the incision was decreased through postoperative day 1. In a transitional area, between the distant and adjacent sites, the withdrawal threshold was intermediate and the duration of hyperalgesia was transient. Heat hyperalgesia was persistent but present when the stimulus was applied to the site of injury but not to a distant site. CONCLUSION: Robust primary hyperalgesia to punctate and blunt mechanical stimuli was present. Hyperalgesia distant to the wound, or secondary hyperalgesia, occurred in response to punctate mechanical stimuli, was short-lived, and required greater forces. These results suggest that the most persistent pain behaviors in this model are largely primary hyperalgesia. (+info)
Halothane, an inhalational anesthetic agent, increases folding stability of serum albumin.
(5/1763)
Inhalational anesthetic agents are known to alter protein function, but the nature of the interactions underlying these effects remains poorly understood. We have used differential scanning calorimetry to study the effects of the anesthetic agent halothane on the thermally induced unfolding transition of bovine serum albumin. We find that halothane (0.6-10 mM) stabilizes the folded state of this protein, increasing its transition midpoint temperature from 62 to 71 degrees C. Binding of halothane to the native state of serum albumin thus outweighs any non-specific interactions between the thermally unfolded state of serum albumin and halothane in this concentration range. Based on the average enthalpy change DeltaH for unfolding of 170 kcal/mol, the increase from 62 to 71 degrees C corresponds to an additional Gibbs energy of stabilization (DeltaDeltaG) due to halothane of more than 4 kcal/mol. Analysis of the dependence of DeltaDeltaG on halothane concentration shows that thermal unfolding of a bovine serum albumin molecule is linked to the dissociation of about one halothane molecule at lower halothane concentrations and about six at higher halothane concentrations. Serum albumin is the first protein that has been shown to be stabilized by an inhalational anesthetic. (+info)
A mutation in the transmembrane/luminal domain of the ryanodine receptor is associated with abnormal Ca2+ release channel function and severe central core disease.
(6/1763)
Central core disease is a rare, nonprogressive myopathy that is characterized by hypotonia and proximal muscle weakness. In a large Mexican kindred with an unusually severe and highly penetrant form of the disorder, DNA sequencing identified an I4898T mutation in the C-terminal transmembrane/luminal region of the RyR1 protein that constitutes the skeletal muscle ryanodine receptor. All previously reported RYR1 mutations are located either in the cytoplasmic N terminus or in a central cytoplasmic region of the 5,038-aa protein. The I4898T mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. The response of the mutant RyR1 Ca2+ channel to the agonists halothane and caffeine in a Ca2+ photometry assay was completely abolished. Coexpression of normal and mutant RYR1 cDNAs in a 1:1 ratio, however, produced RyR1 channels with normal halothane and caffeine sensitivities, but maximal levels of Ca2+ release were reduced by 67%. [3H]Ryanodine binding indicated that the heterozygous channel is activated by Ca2+ concentrations 4-fold lower than normal. Single-cell analysis of cotransfected cells showed a significantly increased resting cytoplasmic Ca2+ level and a significantly reduced luminal Ca2+ level. These data are indicative of a leaky channel, possibly caused by a reduction in the Ca2+ concentration required for channel activation. Comparison with two other coexpressed mutant/normal channels suggests that the I4898T mutation produces one of the most abnormal RyR1 channels yet investigated, and this level of abnormality is reflected in the severe and penetrant phenotype of affected central core disease individuals. (+info)
Effects of the halothane genotype and slaughter weight on texture of pork.
(7/1763)
The objective of this study was to investigate the effects of the halothane (HAL) genotype, slaughter weight (SW), and the HAL x SW interaction on compositional and textural traits of raw and cooked pork. Pigs were bred to exhibit one of the three HAL genotypes (NN, Nn, and nn) with otherwise equivalent genomes. The nn halothane reactors are known to typically produce PSE pork, whereas NN pigs do not typically produce PSE pork. Pietrain x Large White gilts and boars, all with verified Nn genotype (by DNA test), were mated to obtain F2 littermates of the three HAL genotypes. These pigs were slaughtered at either 101 +/- 3 ("light") or 127 +/- 3 ("heavy") kg BW and were evaluated for longissimus muscle traits. The pH at .5 h after death (pH1) was 6.35, 6.13, and 5.68 in NN, Nn, and nn pigs, respectively. Sarcomere length was greater in nn than in NN and Nn pigs (1.94 vs 1.83 and 1.85 microm, respectively). Mechanical resistance was higher in nn than in NN pigs for both raw and cooked meat. Meat from nn pigs was judged by a trained panel to be less rough, more cohesive, harder, more fibrous, less granular, more elastic, and less easy to swallow than meat from NN pigs. For most traits under study, the heterozygotes were intermediate between the homozygotes but closer to NN than to nn pigs. Muscle from heavy pigs had longer sarcomeres and less moisture than muscle from light pigs. The n allele of the HAL gene unfavorably affects pork texture, and this effect is maintained throughout the range of 101 to 127 kg BW. (+info)
Halothane effect on formalin-induced paw edema and flinching in rat.
(8/1763)
The formalin test is a model of injury-produced inflammatory pain. Anesthetics, in clinically relevant concentrations, affect neutrophils and immune suppression. This study was to determine whether halothane reliably inhibits inflammatory reaction and formalin induced pain behavior or does not. Rats were exposed to 100% oxygen (control) or halothane, respectively for 30 min and then 24 hr later five percent formalin test was assessed. The base values of the paw's diameter were obtained earlier, and then formalin induced edema was assessed by measuring diameters of the injected paws at 5 min, 1 hr, 4 hr and 24 hr after the injection. Nociceptive behavior was quantified by counting the number of times with the paw flinched at 5 min intervals for 60 min. The diameters of edema in the halothane group lessened more than those in the oxygen group at 1 and 24 hr in each following of the injection (p<0.05). The rats pre-administered with oxygen or halothane were similar appearances in nociceptive behaviors. It suggests that halothane anesthesia might inhibit slightly the inflammatory reaction with the formalin-induced edema but might not inhibit the formalin-induced pain behavior in the event of pre-administration halothane 24 hr earlier before the formalin test of rat. (+info)