Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating

1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl^–, Na⁺, K⁺, Li⁺, Rb⁺, Ca²⁺, Mg²⁺, NH₄ ⁺ , TMA⁺, TEA⁺. However, asymmetric charge movements similar to the gating currents of the Na⁺-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX tetrodotoxin; Na⁺, sodium; K⁺, potassium; - NFR normal frog Ringer - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid - TEA tetraethylammonium - TMA tetramethylammonium;I _g , gating current; , single-channel conductance     

Effect of cleaning, milling, and baking on deoxynivalenol in wheat.

Samples of wheat naturally infected by Fusarium graminearum Schwabe were obtained from mills in Oklahoma, Missouri, Kansas, and Minnesota and fields in Nebraska and Kansas in 1982; they were analyzed for deoxynivalenol (DON). The wheat was milled, and DON was found throughout all the milling fractions (bran, shorts, reduction flour, and break flour). The DON recoveries for each mill run ranged from 90 to 98%. These samples, regardless of DON concentration, also gave similar fractional distributions of DON. The greatest (21 ppm [21 micrograms/g]) concentration of DON was found in the bran, and the smallest (1 ppm) was found in the break flour. Cleaning and milling were not effective in removing DON; DON was not destroyed in the bread baked from the naturally contaminated whole wheat flour, but the effect on its concentration in the samples analyzed varied, the reduction ranging from 19 to 69%. The percent reduction found in the cleaned wheat ranged from 6 to 19%. DON concentrations in the following commercially made breads, caraway rye, seedless rye, and pumpernickel, were 45 ppb (ng/g), 39 ppb, and 0 ppb, respectively. The limits of detection by gas chromatography-mass spectrometry and high-pressure liquid chromatography for DON were 0.5 and 10 ng, respectively.     

Effect of cleaning, milling, and baking on deoxynivalenol in wheat

Samples of wheat naturally infected by Fusarium graminearum Schwabe were obtained from mills in Oklahoma, Missouri, Kansas, and Minnesota and fields in Nebraska and Kansas in 1982; they were analyzed for deoxynivalenol (DON). The wheat was milled, and DON was found throughout all the milling fractions (bran, shorts, reduction flour, and break flour). The DON recoveries for each mill run ranged from 90 to 98%. These samples, regardless of DON concentration, also gave similar fractional distributions of DON. The greatest (21 ppm [21 micrograms/g]) concentration of DON was found in the bran, and the smallest (1 ppm) was found in the break flour. Cleaning and milling were not effective in removing DON; DON was not destroyed in the bread baked from the naturally contaminated whole wheat flour, but the effect on its concentration in the samples analyzed varied, the reduction ranging from 19 to 69%. The percent reduction found in the cleaned wheat ranged from 6 to 19%. DON concentrations in the following commercially made breads, caraway rye, seedless rye, and pumpernickel, were 45 ppb (ng/g), 39 ppb, and 0 ppb, respectively. The limits of detection by gas chromatography-mass spectrometry and high-pressure liquid chromatography for DON were 0.5 and 10 ng, respectively.     

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.     

The Tick-Tock of Language: Is Language Processing Sensitive to Circadian Rhythmicity and Elevated Sleep Pressure?

The master circadian pacemaker emits signals that trigger organ-specific oscillators and, therefore, constitutes a basic biological process that enables organisms to anticipate daily environmental changes by adjusting behavior, physiology, and gene regulation. Although circadian rhythms are well characterized on a physiological level, little is known about circadian modulations of higher cognitive functions. Thus, we investigated circadian repercussions on language performance at the level of minimal syntactic processing by means of German noun phrases in ten young healthy men under the unmasking conditions of a 40 h constant-routine protocol. Language performance for both congruent and incongruent noun phrases displayed a clear diurnal rhythm with a peak performance decrement during the biological night. The nadirs, however, differed such that worst syntactic processing of incongruent noun phrases occurred 3 h earlier (07:00 h) than that of congruent noun phrases (10:00 h). Our results indicate that language performance displays an internally generated circadian rhythmicity with optimal time for parsing language between 3 to 6 h after the habitual wake time, which usually corresponds to 10:00–13:00 h. These results may have important ramifications for establishing optimal times for shiftwork changes or testing linguistically impaired people.     

Quantitation of heteroplasmy in mitochondrial DNA mutations by primer extension using Vent(R)(exo-) DNA polymerase and RFLP analysis

In this report we describe a simple and rapid protocol for reliable quantitation of mitochondrial DNA (mtDNA) mutations, which is basically a modification of the traditional polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis technique. Up to now, the PCR/RFLP method has been of limited use for the accurate determination of ratios of mutant and wild type molecules, largely owing to the formation of heteroduplex molecules by PCR and incompleteness of restriction digestion. In order to overcome this problem, we have introduced a single-step primer extension reaction using Vent(R)(exo-) DNA polymerase and a fluorescence-labeled primer to the standard assay. The labeled homoduplex molecules are then digested with a restriction endonuclease, and the nucleic acids fractionated on an automated DNA sequencer equipped with GENESCAN analysis software. The amount of mutant mtDNA is readily estimated from fluorescence intensities of the wild-type and mutant mtDNA fragments corrected for incomplete digestion as monitored by a homologous control fragment. The accuracy of the improved protocol was determined by constructing standard curves obtained from defined mixtures of genomic DNA containing homoplasmic wild-type and mutant mtDNA. The expected values were obtained, with an observed correlation coefficient of 0.997 and a typical variability of +/-5% between repeated measurements. Further validation of the protocol is provided by the screening of five patients and unaffected subjects carrying the guanine to adenine transition at the nucleotide 3460 of the mitochondrial genome responsible for the mitochondrial disorder of Leber's hereditary optic neuropathy.     

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.     

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity

C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.