Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment

Plant helicases are known to be involved in salinity and low-temperature tolerance. However, a functional involvement of helicases in the antioxidative response of plants has not been described. We have isolated a DEAD-box-containing cDNA sequence from Medicago sativa (alfalfa) that is a homolog of the pea DNA helicase 45 (PDH45) and named it M. sativa helicase 1 (MH1). Transient transfection of 35S::MH1-GFP to onion epidermis revealed that MH1 was localized in the nucleus. Expression of MH1 was detected in roots, stems and leaves of alfalfa. Furthermore, real-time PCR analysis revealed that mannitol, NaCl, methyl viologen and abscisic acid induced the expression of MH1. The ectopic expression of MH1 in Arabidopsis improved seed germination and plant growth under drought, salt and oxidative stress. The capacity for osmotic adjustment, superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and proline content were also elevated in the transgenic Arabidopsis plants. Our results suggest that MH1 responds to reactive oxygen species (ROS) and functions in drought and salt stress tolerance by enhancing the capacities for ROS scavenging and osmotic adjustment.     

Role of transcription factor Sp1 in the 4-O-methylhonokiol-mediated apoptotic effect on oral squamous cancer cells and xenograft

Recently, biphenolic components derived from the Magnolia family have been studied for anti-cancer, anti-stress, and anti-inflammatory pharmacological effects. However, the pharmacological mechanism of action of 4-O-methylhonokiol (MH) is not clear in oral cancer. The aim of this study was to investigate the role of MH in apoptosis and its molecular mechanism in oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, as well as tumor xenografts. Here, we demonstrated that MH decreased cell growth and induced apoptosis in HN22 and HSC4 cells through the regulation of specificity protein 1 (Sp1). We employed several experimental techniques such as MTS assay, DAPI staining, PI staining, Annexin-V/7-ADD staining, RT-PCR, western blot analysis, immunocytochemistry, immunohistochemistry, TUNEL assay and in vivo xenograft model analysis. MH inhibited Sp1 protein expression and reduced Sp1 protein levels via both proteasome-dependent protein degradation and inhibition of protein synthesis in HN22 and HSC4 cells; MH did not alter Sp1 mRNA levels. We found that MH directly binds Sp1 by Sepharose 4B pull-down assay and molecular modeling. In addition, treatment with MH or knocking down Sp1 expression suppressed oral cancer cell colony formation. Moreover, MH treatment effectively inhibited tumor growth and Sp1 levels in BALB/c nude mice bearing HN22 cell xenografts. These results indicated that MH inhibited cell growth, colony formation and also induced apoptosis via Sp1 suppression in OSCC cells and xenograft tumors. Thus, MH is a potent anti-cancer drug candidate for oral cancer.     

中国北方主产地苹果冷热积累变化及其对始花期的影响

开展气候变化背景下苹果冷热积累变化及其对始花期的影响研究,对指导苹果种植及生产具有重要意义。本研究选取山东福山、山西万荣、甘肃西峰和新疆阿克苏代表中国北方苹果主产地,利用1996—2018年红富士苹果的始花期观测资料和逐时气温数据,采用动态模型、生长度小时模型分别计算逐日冷积累量(CP)和热积累量(GDH),并利用偏最小二乘回归法,对逐日冷、热积累量和各地苹果始花期进行相关分析,以明确各地苹果冷、热积累起止日期和积累量,以及冷、热积累期内温度变化对始花期的影响规律。结果表明: 我国北方主产地苹果冷积累时段集中于10月1日前后至2月中下旬或3月中旬,积累量为74.1～89.3 CP;热积累时段集中于1月下旬前后至始花期,积累量为4010～5770 GDH。西峰和阿克苏冷积累期内平均气温每升高1 ℃,冷积累量将分别增加3.8和5.0 CP;各地热积累期内平均气温每升高1 ℃,热积累量将增加725～967 GDH。与冷积累期内温度变化的影响效应相比,热积累期内温度变化主控我国北方主产地苹果始花期,且气候变暖总体有利于冷积累期内平均气温较低地区的苹果开花和生产。     

Methyl helicterate inhibits hepatic stellate cell activation through downregulating the ERK1/2 signaling pathway

The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.     

Reciprocal coactivation patterns of the medial and lateral quadriceps and hamstrings during slow, medium and high speed isokinetic movements.

The effect of movement velocity and fatigue on the reciprocal coactivation of the quadriceps and hamstrings was investigated through analysis of the root mean square (RMS) and the median frequency (MDF) of surface electromyography for the vastus medialis (VM), vastus lateralis (VL), medial hamstrings (MH) and biceps femoris (BF). Fourteen subjects performed six continuous isokinetic knee extension and flexion movements at 60 degrees, 180 degrees and 300 degrees s(-1), and 30 continuous movements at 300 degrees s(-1) to examine muscular fatigue patterns. Statistical analyses revealed that the RMS activity of the VM displayed greater coactivation than the VL (P<0.01) and the BF displayed greater coactivation than the MH (P<0.0001). There was no effect of velocity on the coactivation levels of the VM, the VL, or the MH; however, there was an effect of velocity on the coactivation levels of the BF (P<0.0001). Relative to MDF activity, the MH shifted upward as velocity increased (P<0. 01) while the BF decreased between 180 and 300 degrees s(-1) (P<0. 01). Results of the muscular fatigue test indicated that the RMS activity of the VM showed a higher degree of coactivation than the VL (P<0.01) and the BF showed approximately three times the coactivation level of the MH (P<0.001). The MDF of the VL and MH shifted downward as the repetitions progressed (P<0.01) with no changes for the VM or for the BF. Results of this study suggest that during isokinetic testing, both the VM and BF have significantly greater reciprocal coactivation levels when compared to the VL and MH, respectively. In addition, these results suggest that motor unit recruitment patterns of the VM and VL and the MH and BF differ with regard to the effects of velocity and fatigue.     

Changes of polyamines and ethylene in cucumber seedlings in response to chilling stress

Cucumber ( Cucumis sativus L. cv. Victory) seedlings were exposed to chilling at 5°C and endogenous levels of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) were measured after chilling and after warming at 20°C. The level of spermidine was higher in the chilled seedlings than in the non-chilled seedlings. Treatment with a plant bioregulator, (2RS,3RS)-1-(4-cholorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol (paclobutrazol), reduced the chilling injury and the levels of spermidine in the chilled seedlings. The levels of ACC and production of ethylene showed sharp increases after warming following exposure to chilling. These increases were suppressed by the application of aminooxyacetic acid (AOA). However, AOA treatment did not reduce chilling injury or affect the levels of polyamines in the tissue. These data indicate that the increase in ACC and ethylene is a response of the tissue to the chilling exposure and is not a cause of the injury. The data also suggest that the syntheses of polyamines and ethylene are not competitive with each other even under chilling stress conditions.     

Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport

The free thiols 3-mercapto-hexanol (3MH) and its acetate, practically absent from musts, are liberated by yeast during fermentation from a cysteinylated precursor [S-3-(hexan-1-ol)-l-cysteine (Cys-3MH)] present in the grape must and contribute favorably to the flavor of Sauvignon white wines. Production of 3MH is increased when urea is substituted for diammonium phosphate (DAP) as the sole nitrogen source on a synthetic medium. On grape must, complementation with DAP induces a decrease of 3MH production. This observation is reminiscent of nitrogen catabolite repression (NCR). The production of 3MH is significantly lower for a gap1Delta mutant compared with the wild type, during fermentation of a synthetic medium containing Cys-3MH as the precursor and urea as the sole nitrogen source. Mutants isolated from an enological strain with a relief of NCR on GAP1 produce significantly higher amounts of 3MH on synthetic medium than the parental strain. These phenotypes were not confirmed on grape must. It is concluded that on synthetic medium, Cys-3MH enters the cell through at least one identified transporter, GAP1p, whose activity is limiting the release of volatile thiols. On grape must, the uptake of the precursor through GAP1p is not confirmed, but the effect of addition of DAP, eventually prolonging NCR, is shown to decrease thiol production.     

Relationship between alcohol dehydrogenase activity and low-temperature in two maize genotypes, Silverado F1 and Adh1-Adh2- doubly null.

We have examined the role of alcohol dehydrogenase (ADH, E.C.1.1.1.1) in chilling tolerance using maize (Zea mays L.) Adh1(-)Adh2(-) doubly null mutant. Adh1(-)Adh2(-) doubly null seedlings were found to have lowered survival rates compared to non-doubly null maize seedlings (Silverado F(1)) when held at 2 degrees C for varying periods. Exposure to ethanol did not increase the chilling tolerance in either Silverado F(1) or Adh1(-)Adh2(-) doubly null. ADH activity in Silverado F(1) remained steady when held at 2 degrees C for up to 3 d. ADH1 protein accumulation in chilled Silverado F(1) seedlings remained unchanged throughout the period of cold exposure. Chilling led to a significant inhibition of the P-H(+)-ATPase (E.C. 3.6.3.6) activity in Adh1(-)Adh2(-)doubly null, but minimal inhibition was seen in Silverado F(1). Though P-H(+)-ATPase activity in Adh1(-)Adh2(-) decreased, P-H(+)-ATPase protein levels remained constant during the chilling period. Levels of ATP slightly fluctuated in both types of seedlings during the duration of chilling. Lipid peroxidation levels in Adh1(-)Adh2(-) doubly null increased with chilling exposure, but not in the Silverado F(1). We suggest that ADH activity may play a role in chilling tolerance that is not related to maintenance of glycolysis and ATP production as has been observed during oxygen depravation.     

Gas Exchange and Co-regulation of Photochemical and Nonphotochemical Quenching in Bean during Chilling at Ambient and Elevated Carbon Dioxide

The effects of elevated (700 micromol mol(-1)) and ambient (350 micromol mol(-1)) CO(2) on gas exchange parameters and chlorophyll fluorescence were measured on bean (Phaseolus vulgaris) during 24 h chilling treatments at 6.5 degrees C. Consistent with previous research on this cultivar, photosynthetic decline during chilling was not significantly affected by CO(2) while post-chilling recovery was more rapid at elevated compared to ambient CO(2). Our primary focus was whether there were also CO(2)-mediated differences in demand on nonphotochemical quenching (NPQ) processes during the chilling treatments. We found that photosystem II quantum yield and total NPQ were similar between the CO(2) treatments during chilling. In both CO(2) treatments, chilling caused a shift from total NPQ largely composed of q(E), the protective, rapidly responding component of NPQ, to total NPQ dominated by the more slowly relaxing q(I), related to both protective and damage processes. The switch from q(E) to q(I) during chilling was more pronounced in the elevated CO(2) plants. Using complementary plots of the quantum yields of photochemistry and NPQ we demonstrate that, despite CO(2) effects on the partitioning of NPQ into q(E) and q(I) during chilling, total NPQ was regulated at both CO(2) levels to maximize photochemical utilization of absorbed light energy and dissipate only that fraction of light energy that was in excess of the capacity of photosynthesis. Photodamage did occur during chilling but was repaired within 3 h recovery from chilling in both CO(2) treatments.     

Synergistic action of rapid chilling and nisin on the inactivation of Escherichia coli

The effect of rapid and slow chilling on survival and nisin sensitivity was investigated in Escherichia coli. Membrane permeabilization induced by cold shock was assessed by uptake of the fluorescent dye 1-N-phenylnapthylamine. Slow chilling (2°C min⁻¹) did not induce transient susceptibility to nisin. Combining rapid chilling (2,000°C min⁻¹) and nisin causes a dose-dependent reduction in the population of cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU ml⁻¹ nisin. Cells were more sensitive if nisin was present during stress. Nevertheless, addition of nisin to cell suspension after the rapid chilling produced up to 5 log of cell inactivation for exponentially growing cells and 1 log for stationary growing cells. This suggests that the rapid chilling strongly damaged the cell membrane by disrupting the outer membrane barrier, allowing the sensitization of E. coli to nisin post-rapid chilling. Measurements of membrane permeabilization showed a good correlation between the membrane alteration and nisin sensitivity. Application involving the simultaneous treatment with nisin and rapid cold shock could thus be of value in controlling Gram negatives, enhancing microbiological safety and stability.     

Co-segregation of the malignant hyperthermia and the Arg615-Cys615 mutation in the skeletal muscle calcium release channel protein in five European Landrace and Pietrain pig breeds

A total of 392 pigs of European Landrace and Pietrain origin segregating for malignant hyperthermia (MH) were genotyped using a polymerase chain reaction (PCR)/restriction endonuclease test for the C—T mutation at nucleotide (nt) 1843 in the skeletal muscle ryanodine receptor (RYR1) gene, earlier identified as the causal mutation for MH. All pigs had been halothane tested and genotyped at linked polymorphic marker loci. There was complete correlation between MH status of the 392 animals, as diagnosed by a combination of the halothane challenge test with S, GPI, H, A1BG, PGD haplotyping, and the DNA-based test. DNA-based detection of the MH status in 238 MH-susceptible heterozygous (N/n) and homozygous (n/n) pigs was shown to be accurate, eliminating the 2% diagnostic error that is associated with the halothane challenge test. The mutation was also associated with an allele of a polymorphic microsatellite (ETH5 001) at the RYR1 locus.     

Mutations in the RYR1 gene in Italian patients at risk for malignant hyperthermia: evidence for a cluster of novel mutations in the C-terminal region

Mutations in the ryanodine receptor type 1 (RYR1) gene are associated with Malignant Hyperthermia (MH) and Central Core Disease (CCD). We report here on the molecular analysis of the RYR1 gene in Italian families referred as potential cases of MH or in patients with CCD or multicore/minicore myopathy. Of a total of 20 individuals with mutations in the RYR1 gene, 14 were part of a group of 47 MH susceptible (MHS) patients, 4 of 34 individuals diagnosed as MH equivocal (MHE), and 2 were patients diagnosed with minicore myopathy and CCD, respectively. Mutations were found to segregate with the MHS or MHE phenotype within the families of the probands. A discordance between phenotype and genotype was observed in a family where a mutation detected in an MHS proband was also found in the father who had been diagnosed MH normal (MHN) at the IVCT. In addition to known mutations, seven novel mutations were found, five of which occurred in exons encoding the C-terminal region of RYR1. These results indicate that the C-terminal region of RYR1 represents an additional hot spot for mutations in patients with MH, similar to what has been reported for patients with CCD.     

Estimation of Chilling Sensitivity and Injury in Gloxinia Leaves by the Thermal Hysteresis of NMR Relaxation Times of Water Protons

Thermal hysteresis of longitudinal relaxation times (T₁) ofwater protons in leaf tissues of gloxinia was investigated bypulse nuclear magnetic resonance (NMR) spectroscopy. The profilesof T₁ hysteresis during a slow cool-warm cycle (20 to 0?C) variedwith the degree of chilling injury. General trends of T₁ changesupon warming were as follows: (1) a small increase of T₁ wasobserved when no chilling injury occurred or the degree of injurywas minor, (2) a large increase of T₁ occurred when injury increased,(3) a large decrease of T₁ occurred from the earlier stage ofwarming when serious injury occured. The sum of T₁ ratio (theratio obtained from the difference between the T₁ value in thecooling process and that of the wanning process for eight stepsof cool-warm cycle, divided by the former) reflecting T₁ increaseor decrease during the warming process was related to varietaldifference and seasonal changes of chilling sensitivity. Therefore,T₁ hysteresis can be used as a diagnostic tool in detectingthe chilling sensitivity and the degree of chilling injury. (Received August 27, 1986; Accepted February 24, 1987)     

Chilling-Induced Ethylene Production in the Peel and Pulp of Banana

Ethylene production rates and 1-aminocyclopropane-1-carboxylic acid (ACC) synthetase activities were 0. 78,0.91 nl· g-l ·h-land 0.02,0.05 nmol·g-1·h-1 respectively in the peel and pulp of newly harvested banana fruits(Musa acuminata Colla “warf cavendish”),their ethylene-forming enzyme(EFE)activities were yet as high as 10.5 and 5.1 nl·g-1·h-1. When the fruits were chilled at 1.5℃ ,the ethylene production and EFE activities of the peel and pulp kept decreasing with the time course of chilling treatment. However, after these chilled fruits were transferred to 20℃ for 24 h,their ACC synthetase activities increased markedly,and ethylene production had separate peaks(1.75 and 2.45 nl·g-1 ·h-1) in the peel and pulp. In this case,the endogenous low content of S-adenosylmethionine (SAM)in vivo was insufficient for its ACC synthesis, The inhibitory effect of cycloheximide on ACC synthesis showed that chilling-induced ethylene production was mainly the result of activity of the resynthesized ACC synthetase induced by chilling treatment. The production of chilling-induced ethylene could be good indicator of chilling injury, but it is unlikely an indicator of chilling damage during ripening process in banana. In the severly chilling-injured fruits, both the peel and pulp still had the capability of converting ACC to ethylene.     

Relationship between Proline and Abscisic Acid in the Induction of Chilling Tolerance in Maize Suspension-Cultured Cells

Both proline and abscisic acid (ABA) induce chilling tolerance in chilling-sensitive plants. However, the relationship between proline and ABA in the induction of chilling tolerance is unclear. We compared the time course of the increase in chilling tolerance induced by proline and ABA, and the time course of the uptake of both into the cultured cells of maize (Zea mays L. cv Black Mexican Sweet) at 28[deg]C. The plateau of proline-induced chilling tolerance preceded by 12 h the plateau of ABA-induced chilling tolerance. The uptake of exogenous ABA into the cells reached a plateau in 1 h, whereas the uptake of exogenous proline gradually increased throughout the 24-h culture period. Although the proline content in ABA-treated cells was 2-fold higher than in untreated cells at the end of the 24-h ABA treatment at 28[deg]C, the correlation between the endogenous free proline content and the chilling tolerance in the ABA-treated cells was insignificant. Isobutyric acid treatment, which resulted in a larger accumulation of proline in the cells than ABA treatment, did not increase chilling tolerance. The induction of chilling tolerance by proline and ABA appeared to be additive. Cycloheximide inhibited ABA-induced chilling tolerance, but it did not inhibit proline-induced chilling tolerance. Newly synthesized proteins accumulate in ABA-treated cells at 28[deg]C while the chilling tolerance is developing (Z. Xin and P.H. Li [1993] Plant Physiol 101: 277-284), but none of these proteins were observed in the proline-treated cells. Results suggest that proline and ABA induce chilling tolerance in maize cultured cells by different mechanisms.     

Role of the Aquaporin PIP1 Subfamily in the Chilling Tolerance of Rice

Although an association between chilling tolerance and aquaporinshas been reported, the exact mechanisms involved in this relationshipremain unclear. We compared the expression profiles of aquaporingenes between a chilling-tolerant and a low temperature-sensitiverice variety using real-time PCR and identified seven genesthat closely correlated with chilling tolerance. Chemical treatmentexperiments, by which rice plants were induced to lose theirchilling tolerance, implicated the PIP1 (plasma membrane intrinsicprotein 1) subfamily member genes in chilling tolerance. Ofthese members, changes in expression of the OsPIP1;3 gene suggestedthis to be the most closely related to chilling tolerance. AlthoughOsPIP1;3 showed a much lower water permeability than membersof the OsPIP2 family, OsPIP1;3 enhanced the water permeabilityof OsPIP2;2 and OsPIP2;4 when co-expressed with either of theseproteins in oocytes. Transgenic rice plants (OE1) overexpressingOsPIP1;3 showed an enhanced level of chilling tolerance andthe ability to maintain high OsPIP1;3 expression levels underlow temperature treatment, similar to that of chilling-tolerantrice plants. We assume that OsPIP1;3, constitutively overexpressedin the leaf and root of transgenic OE1 plants, interacts withmembers of the OsPIP2 subfamily, thereby improving the plants’water balance under low temperatures and resulting in the observedchilling tolerance of the plants.     

Comparison of effects of a single and thrice-repeated moderate hypobaric hypoxia upon expression of thioredoxine-1 in the rat hypothalamus

We have previously shown that severe acute hypobaric hypoxia (SH) increases the expression of several endogenous antioxidants including thioredoxin-1 (Trx-1) in hippocampal neurons of rats. Preconditioning by three sessions of mild hypobaric hypoxia (MH) significantly augments this increase at the early period after subsequent SH, but MH itself without subsequent SH, in contrast, decreases expression of Trx-1. The dynamics of Trx-1 expression between the first and the last (third) sessions of preconditioning remains, however, unclear. In the present work, the previously studied Trx-1 expression in different areas of the hippocampus at hours 3 and 24 after thrice-repeated MH is compared to its expression at hours 3 and 24 after a single MH. It is shown that both a single and a thrice-repeated MH have similar effects on the Trx-1 expression. Since their neuroprotective effects in subsequent SH significantly differ, it is possible to conclude that hypoxic tolerance of neurons is determined not by the "background" level of antioxidants expression itself but has rather more complex regulatory mechanisms. These mechanisms may be associated with the wave-like oscillations of Trx-1 expression during preconditioning which was described in the present study.     

Searching the Cytochrome P450 Enzymes for the Metabolism of Meranzin Hydrate: A Prospective Antidepressant Originating from Chaihu-Shugan-San

Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH in vitro by human liver microsomes (HLMs) or human recombinant CYPs. MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The K_m and V_max values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.     

Calcium release from sarcoplasmic reticulum is facilitated in human myotubes derived from carriers of the ryanodine receptor type 1 mutations Ile2182Phe and Gly2375Ala

Malignant hyperthermia (MH) is caused by increased calcium release from sarcoplasmic reticulum, triggered by volatile anesthetics or depolarizing muscle relaxants. Numerous mutations associated with MH have been detected in the skeletal muscle type ryanodine receptor gene (RyR1), but so far facilitated calcium release has only been demonstrated for a few of them. This is a prerequisite for confirming the causative role of an RyR1 mutation for MH. Calcium release from sarcoplasmic reticulum induced by 4-chloro-m-cresol (4CmC), caffeine, and halothane was determined in human myotubes by calcium imaging. The RyR1 Ile2182Phe mutation and the RyR1 Gly2375Ala mutation have been identified in individuals susceptible to MH. In myotubes of individuals carrying the RyR1 Ile2182Phe or the RyR1 Gly2375Ala mutation, the EC(50) for caffeine and halothane was reduced; in the Ile2182Phe myotubes, the EC(50) for 4CmC was also reduced, all consistent with facilitated calcium release from the sarcoplasmic reticulum. From these data we conclude that both mutations are pathogenic for MH.