Temperature sensitivity of acid-sensitive outwardly rectifying (ASOR) anion channels in cortical neurons is involved in hypothermic neuroprotection against acidotoxic necrosis

The acid-sensitive outwardly rectifying (ASOR) anion channel has been found in non-neuronal cell types and was shown to be involved in acidotoxic death of epithelial cells. We have recently shown that the ASOR channel is sensitive to temperature. Here, we extend those results to show that temperature-sensitive ASOR anion channels are expressed in cortical neurons and involved in acidotoxic neuronal cell death. In cultured mouse cortical neurons, reduction of extracellular pH activated anionic currents exhibiting phenotypic properties of the ASOR anion channel. The neuronal ASOR currents recorded at pH 5.25 were augmented by warm temperature, with a threshold temperature of 26 °C and the Q₁₀ value of 5.6. After 1 h exposure to acidic solution at 37 °C, a large population of neurons suffered from necrotic cell death which was largely protected not only by ASOR channel blockers but also by reduction of temperature to 25 °C. Thus, it is suggested that high temperature sensitivity of the neuronal ASOR anion channel provides, at least in part, a basis for hypothermic neuroprotection under acidotoxic situations associated with a number of pathological brain states.     

Temperature Increase Exacerbates Apoptotic Neuronal Death in Chemically-Induced Ischemia

It is well-established that hyperthermia increases neuronal death and worsens stroke outcome. However, little is known about the mechanisms of how hyperthermia is involved in this neuronal death process. In the present study, we examined how temperature increase exacerbates neuronal death using a model of chemical ischemia. Chemical ischemia was induced by treating SH-SY5Y neuroblastoma cells with sodium azide and deoxyglucose. Temperature increase was treated by placing the cells at 37°C (control) and 41°C (experimental). Cell survival was determined by trypan blue assay and ATP levels were measured with ATP assay kits. Protein expression was detected by western blot. Treatment with sodium azide resulted in cell death in a dose-responsive manner. Increased temperature worsened the ATP depletion and cell volume shrinkage. Temperature increase also enhanced ER stress as demonstrated by the elevated level of phospho-eIF2α and C/EBP homologous protein (CHOP). Inhibition of CHOP expression significantly decreased sodium azide-induced neuronal death. In addition, the increased temperature intensified the activation of caspase-3, an apoptotic effector protease, and inhibition of capspase-3 significantly reduced cell death. These findings support that temperature increase worsened the neuronal death by depleting intracellular ATP, inducing ER stress response and activating apoptotic signal transduction.     

The Effect of Hypothermia Therapy on Cortical Laminar Disruption following Ischemic Injury in Neonatal Mice

Hypothermia has been proposed as a treatment for reducing neuronal damage in the brain induced by hypoxic ischemia. In the developing brain, hypoxic ischemia-induced injury may give rise to cerebral palsy (CP). However, it is unknown whether hypothermia might affect the development of CP. The purpose of this study was to investigate whether hypothermia would have a protective effect on the brains of immature, 3-day old (P3) mice after a challenge of cerebral ischemia. Cerebral ischemia was induced in P3 mice with a right common carotid artery ligation followed by hypoxia (6% O₂, 37°C) for 30 min. Immediately after hypoxic ischemia, mice were exposed to hypothermia (32°C) or normothermia (37°C) for 24 h. At 4 weeks of age, mouse motor development was tested in a behavioral test. Mice were sacrificed at P4, P7, and 5 weeks to examine brain morphology. The laminar structure of the cortex was examined with immunohistochemistry (Cux1/Ctip2); the number of neurons was counted; and the expression of myelin basic protein (MBP) was determined. The hypothermia treatment was associated with improved neurological outcomes in the behavioral test. In the normothermia group, histological analyses indicated reduced numbers of neurons, reduced cortical laminar thickness in the deep, ischemic cortical layers, and significant reduction in MBP expression in the ischemic cortex compared to the contralateral cortex. In the hypothermia group, no reductions were noted in deep cortical layer thickness and in MBP expression in the ischemic cortex compared to the contralateral cortex. At 24 h after the hypothermia treatment prevented the neuronal cell death that had predominantly occurred in the ischemic cortical deep layers with normothermia treatment. Our findings may provide a preclinical basis for testing hypothermal therapies in patients with CP induced by hypoxic ischemia in the preterm period.     

PKC–NF-κB are involved in CCL2-induced Nav1.8 expression and channel function in dorsal root ganglion neurons

CCL2 [chemokine (C–C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)–NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG (dorsal root ganglion) neurons were prepared from adult male Sprague–Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C–C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Na_v1.8 elevated from the baseline 46.53±4.53 pA/pF to 64.28±3.12 pA/pF following 10 nM CCL2 (P<0.05). Compared with the control, significant change in Na_v1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Na_v1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC–NF-κB are involved in CCL2-induced elevation of Na_v1.8 current density by promoting the phosphorylation of Na_v1.8 and its expression.     

Modeling the Recovery of Heat-Treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 Spores at Suboptimal Temperature and pH Using Growth Limits

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.     

Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke

Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD).Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively.Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline.Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.     

Thermophilic Sulfate Reduction in Hydrothermal Sediment of Lake Tanganyika, East Africa

In environments with temperatures above 60°C, thermophilic prokaryotes are the only metabolically active life-forms. By using the ³⁵SO₄^2- tracer technique, we studied the activity of sulfate-reducing microorganisms (SRM) in hot sediment from a hydrothermal vent site in the northern part of freshwater Lake Tanganyika (East Africa). Incubation of slurry samples at 8 to 90°C demonstrated meso- and thermophilic sulfate reduction with optimum temperatures of 34 to 45°C and 56 to 65°C, respectively, and with an upper temperature limit of 80°C. Sulfate reduction was stimulated at all temperatures by the addition of short-chain fatty acids and benzoate or complex substrates (yeast extract and peptone). A time course experiment showed that linear thermophilic sulfate consumption occurred after a lag phase (12 h) and indicated the presence of a large population of SRM in the hydrothermal sediment. Thermophilic sulfate reduction had a pH optimum of about 7 and was completely inhibited at pH 8.8 to 9.2. SRM could be enriched from hydrothermal chimney and sediment samples at 60 and 75°C. In lactate-grown enrichments, sulfide production occurred at up to 70 and 75°C, with optima at 63 and 71°C, respectively. Several sporulating thermophilic enrichments were morphologically similar to Desulfotomaculum spp. Dissimilatory sulfate reduction in the studied hydrothermal area of Lake Tanganyika apparently has an upper temperature limit of 80°C.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Halide Permeation in Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels

Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl⁻ pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22°C or 8.7 pS at 37°C. The current–voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22°C to 10.9 pS at 37°C. The conductance at 22°C was ∼1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl⁻ activity was hyperbolic and well fitted by a Michaelis-Menten–type function having a K _m of ∼38 mM and maximum conductance of 10 pS at 22°C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P_Na/P_Cl = 0.003–0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P_I (1.8) > P_Br (1.3) > P_Cl (1.0) > P_F (0.17), consistent with a “weak field strength” selectivity site. The same sequence was obtained for external halides, although inward F⁻ flow was not observed. Iodide currents were protocol dependent and became blocked after 1–2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I⁻/Cl⁻ permeability ratio (P_I/P_Cl < 0.4). The switch to low I⁻ permeability was enhanced at potentials that favored Cl⁻ entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl⁻ and I⁻ ions may influence I⁻ permeation and be responsible for the wide range of P_I/P_Cl ratios that have been reported for the CFTR channel. The low P_I/P_Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a “weak field strength” anion selectivity sequence.     

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na⁺ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na⁺ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.     

Neuronal necrosis and spreading death in a Drosophila genetic model

Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death.     

Heat stress reveals a specialized variant of the pachytene checkpoint in meiosis of Arabidopsis thaliana

Plant growth and fertility strongly depend on environmental conditions such as temperature. Remarkably, temperature also influences meiotic recombination and thus, the current climate change will affect the genetic make-up of plants. To better understand the effects of temperature on meiosis, we followed male meiocytes in Arabidopsis thaliana by live cell imaging under three temperature regimes: at 21°C; at heat shock conditions of 30°C and 34°C; after an acclimatization phase of 1 week at 30°C. This work led to a cytological framework of meiotic progression at elevated temperature. We determined that an increase from 21°C to 30°C speeds up meiosis with specific phases being more amenable to heat than others. An acclimatization phase often moderated this effect. A sudden increase to 34°C promoted a faster progression of early prophase compared to 21°C. However, the phase in which cross-overs mature was prolonged at 34°C. Since mutants involved in the recombination pathway largely did not show the extension of this phase at 34°C, we conclude that the delay is recombination-dependent. Further analysis also revealed the involvement of the ATAXIA TELANGIECTASIA MUTATED kinase in this prolongation, indicating the existence of a pachytene checkpoint in plants, yet in a specialized form.

Live cell imaging of plants exposed to different heat stresses provides a temporal framework of meiosis at high temperatures in wild-type and mutants for several meiotic recombination factors.     

Lignocellulolytic Enzymes Produced by Volvariella volvacea, the Edible Straw Mushroom

Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25°C; optimal temperature, 37°C; maximal temperature, 40°C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50°C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and β-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin.     

Combinations of Intervention Treatments Resulting in 5-Log10-Unit Reductions in Numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 Organisms in Apple Cider

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log₁₀ units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log₁₀ units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35°C and/or freeze-thawing (48 h at −20°C; 4 h at 4°C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log₁₀-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35°C treatments. In pH 3.7 cider the 5-log₁₀-unit reduction followed freeze-thawing combined with either 6 h at 4°C, 2 h at 25°C, or 1 h at 35°C or 6 h at 35°C alone. A 5-log₁₀-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35°C plus freeze-thawing, SA plus 12 h at 25°C plus freeze-thawing, SA plus 6 h at 35°C, and SA plus 4 h at 35°C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35°C. Cider with no added organic acids treated with either 6 h at 35°C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.     

Adaptation in the Visual Cortex: Influence of Membrane Trajectory and Neuronal Firing Pattern on Slow Afterpotentials

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p<0.05). Spike frequency adaptation had a faster time course and was larger during plateau (square pulse) than during intermittent (sinusoidal) depolarizations. Similar results were obtained in 17 neurons intracellularly recorded from the visual cortex in vivo. The differences in the afterpotentials evoked with both protocols were abolished by removing calcium from the extracellular medium or by application of the L-type calcium channel blocker nifedipine, suggesting that the activation of a calcium-dependent current is at the base of this afterpotential difference. These findings suggest that not only the spikes, but the membrane potential values and firing patterns evoked by a particular stimulation protocol determine the responses to any subsequent incoming input in a time window that spans for tens of seconds to even minutes.     

Synthesis and evaluation of a novel analgesic conotoxin Lt7b that inhibits calcium currents and increases sodium currents

Conotoxins are promising neuropharmacological tools and drug candidates due to their high efficiency and specificity in targeting ion channels or neurotransmitter receptors. In this study, a novel O₂‐superfamily conotoxin, Lt7b, was synthesized and its pharmacological functions were evaluated. Lt7b with three modified amino acids and three disulfide bonds was successfully synthesized. CD spectra showed that Lt7b had a typical α‐helix in the secondary structure. Patch clamp experiments on rat DRG neurons showed that Lt7b could significantly inhibit calcium currents with an IC₅₀ value of 856 ± 95 nM. Meanwhile, 10 μM Lt7b could significantly increase the sodium currents by 77 ± 8%, but it had no obvious effects on the potassium currents in DRG neurons. In addition, patch clamp experiments on ion channel subtypes showed that 10 μM Lt7b could inhibit 7.0 ± 1.2%, 8.0 ± 1.5%, 4.6 ± 3.4%, and 9.5 ± 0.1% of the hCa_v1.2, hCa_v2.1, hCa_v2.2, and hCa_v3.2 currents, respectively, while it did not increase the rNa_v1.7, rNa_v1.8, hNa_v1.5, hNa_v1.7, and hNa_v1.8 currents. Lt7b had no obvious toxicity to HaCaT and ND7/23 cells up to 1 mM and significantly increased the pain threshold at the testing time of 0.5–4 h in a dose‐dependent manner in the mouse hotplate assay. This novel conotoxin Lt7b may be a useful tool for ion channel studies and analgesic drug development.     

Febrile temperatures unmask biophysical defects in Nav1.1 epilepsy mutations supportive of seizure initiation

Generalized epilepsy with febrile seizures plus (GEFS+) is an early onset febrile epileptic syndrome with therapeutic responsive (a)febrile seizures continuing later in life. Dravet syndrome (DS) or severe myoclonic epilepsy of infancy has a complex phenotype including febrile generalized or hemiclonic convulsions before the age of 1, followed by intractable myoclonic, complex partial, or absence seizures. Both diseases can result from mutations in the Na_v1.1 sodium channel, and initially, seizures are typically triggered by fever. We previously characterized two Na_v1.1 mutants—R859H (GEFS+) and R865G (DS)—at room temperature and reported a mixture of biophysical gating defects that could not easily predict the phenotype presentation as either GEFS+ or DS. In this study, we extend the characterization of Na_v1.1 wild-type, R859H, and R865G channels to physiological (37°C) and febrile (40°C) temperatures. At physiological temperature, a variety of biophysical defects were detected in both mutants, including a hyperpolarized shift in the voltage dependence of activation and a delayed recovery from fast and slow inactivation. Interestingly, at 40°C we also detected additional gating defects for both R859H and R865G mutants. The GEFS+ mutant R859H showed a loss of function in the voltage dependence of inactivation and an increased channel use-dependency at 40°C with no reduction in peak current density. The DS mutant R865G exhibited reduced peak sodium currents, enhanced entry into slow inactivation, and increased use-dependency at 40°C. Our results suggest that fever-induced temperatures exacerbate the gating defects of R859H or R865G mutants and may predispose mutation carriers to febrile seizures.     

Isolation of Bacteria Which Can Grow at Both High pH and Low Temperature

Fifty bacterial strains able to grow at pH 10 and 0°C were isolated from soils, and growth characteristics of three selected strains were investigated. Strain 207, which showed the best growth rate of all the isolates at the conditions described above, could grow at a temperature of −5 to 39°C at pH 8.5. The optimum pH for this strain changed from 9.5 at 10°C to 9.0 at 20°C.     

Growth of Candida ingens on Supernatant from Anaerobically Fermented Pig Waste: Effects of Temperature and pH

Candida ingens, a pellicle-forming yeast utilizing volatile fatty acids, grew over a pH range of 4.1 to 6.0 on nonsterile supernatants from anaerobically fermented pig wastes; growth was inconsistent between pH 4.1 and 4.6. When ambient temperature above the pellicle was 21°C and the temperature of the medium was 29 to 32°C, a pH range of 4.8 to 5.0 gave yields of 1.90 to 3.31 g of dry matter per liter, and 0.059 to 0.065 mol of volatile fatty acids was utilized per liter. There was no advantage in utilization of volatile fatty acids and yield of dry matter in keeping the pH constant during a 24-h growth period. C. ingens grew at pH 4.8 and 5.0 when both ambient and medium temperatures were 30°C. When ambient temperature was 10°C, maximum yield and utilization of volatile fatty acids occurred at a medium temperature of 28 to 30°C.