Fever-triggered ventricular arrhythmias in Brugada syndrome and type 2 long-QT syndrome

The risk for lethal ventricular arrhythmias is increased in individuals who carry mutations in genes that encode cardiac ion channels. Loss-of-function mutations in SCN5A, the gene encoding the cardiac sodium channel, are linked to Brugada syndrome (BrS). Arrhythmias in BrS are often preceded by coved-type ST-segment elevation in the right-precordial leads V1 and V2. Loss-of-function mutations in KCNH2, the gene encoding the cardiac ion channel that is responsible for the rapidly activating delayed rectifying potassium current, are linked to long-QT syndrome type 2 (LQT-2). LQT-2 is characterised by delayed cardiac repolarisation and rate-corrected QT interval (QTc) prolongation. Here, we report that the risk for ventricular arrhythmias in BrS and LQT-2 is further increased during fever. Moreover, we demonstrate that fever may aggravate coved-type ST-segment elevation in BrS, and cause QTc lengthening in LQT-2. Finally, we describe molecular mechanisms that may underlie the proarrhythmic effects of fever in BrS and LQT-2. (Neth Heart J 2010;18:165-9.)     

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09 ± 0.32 mg/g and 7.25 ± 0.36 mg/g respectively in the leaves and roots annually. The methanolic extracts from 20 g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol–water (1:1, v/v). Totally 42.0 mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, ¹H NMR and ¹³C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.     

Mechanism of CDK5 activation revealed by steered molecular dynamics simulations and energy calculations

In the current work, CDK5/p25 complexes were pulled apart by applying external forces with steered molecular dynamics (SMD) simulations. The crucial interactions between the kinase and the activation protein were investigated and the SMD simulations showed that several activation-relevant motifs of CDK5 leave p25 in sequence during the pulling and lead to an apo-CDK2 like CDK5 structure after separation. Based on systematic examination of hydrogen bond breaking and classical MD/molecular mechanics-generalized Born/surface area) (MM-GBSA) calculations, a CDK5 activation mechanism by p25 is suggested. This is the first step towards the systemic development of CDK inhibitors and the mechanism proposed could lead to a better understanding of the protein–protein recognition characteristics between the kinase and its activator.     

Quantitative proteomic analysis of G‐protein signalling in Stagonospora nodorum using isobaric tags for relative and absolute quantification

The G protein α‐subunit (Gna1) in the wheat pathogen Stagonospora nodorum has previously been shown to be a critical controlling element in disease ontogeny. In this study, iTRAQ and 2‐D LC MALDI‐MS/MS have been used to characterise protein expression changes in the S. nodorum gna1 strain versus the SN15 wild‐type. A total of 1336 proteins were identified. The abundance of 49 proteins was significantly altered in the gna1 strain compared with the wild‐type. Gna1 was identified as having a significant regulatory role on primary metabolic pathways, particularly those concerned with NADPH synthesis or consumption. Mannitol dehydrogenase was up‐regulated in the gna1 strain while mannitol 1‐phosphate dehydrogenase was down‐regulated providing direct evidence of Gna1 regulation over this enigmatic pathway. Enzymatic analysis and growth assays confirmed this regulatory role. Several novel hypothetical proteins previously associated with stress and pathogen responses were identified as positively regulated by Gna1. A short‐chain dehydrogenase (Sch3) was also significantly less abundant in the gna1 strains. Sch3 was further characterised by gene disruption in S. nodorum by homologous recombination. Functional characterisation of the sch3 strains revealed their inability to sporulate in planta providing a further link to Gna1 signalling and asexual reproduction. These data add significantly to the identification of the regulatory targets of Gna1 signalling in S. nodorum and have demonstrated the utility of iTRAQ in dissecting signal transduction pathways.     

Proteomic analysis of human gastric juice: a shotgun approach

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.     

Biochemical studies on malathion resistance,inheritance and association of carboxylesterase activity in brown planthopper,Nilaparvata lugens complex in Peninsular Malaysia

Abstract Two sympatric populations of brown planthopper (BPH), one from rice and the other from Leersia hexandra were collected from each of five locations in Malaysia. All the tested malathion-resistant individuals of the rice BPH population and F₁ generation (cross between malathion-resistant [usually caught on rice] and malathion-susceptible [usually caught on Leersia]) showed high esterase activity, while all malathion-susceptible individuals on L. hexandra showed low esterase activity. In the F₂ generation, all the individuals tested against malathion were approximately 75% resistant and 25% susceptible and the inheritance pattern of esterase activity (high and low esterase activity) segregated in the same manner to a 3: 1 ratio. This confirms that resistance to malathion is mono-factorial and inheritance pattern of esterase activity is also linked to malathion resistance. Carboxylesterase or total esterase activity in BPH is inherited in a simple Mendelian fashion that is encoded by a single dominant gene. For the total esterase assay, average esterase activity levels in the rice-infesting population ranged from 17.64 to 19.37 nmoles 1-napthol/mg protein while that in the Leersia-infesting population ranged from 5.29 to 6.11 nmoles 1-napthol/mg protein. In terms of esterase activity, the two sympatric Nilaparvata lugens populations separated into two distinct groups. Results based on the tube color intensity test showed 96% and 98% resistant and susceptible individuals were present in the rice- and Leersia-infesting populations, respectively. In a filter paper test, the rice-infesting population had 94% with high esterase activity while the Leersia-infesting population had 96% with low esterase activity.     

Effect of Nitric Oxide on Anammox Bacteria

The effects of nitrogen oxides on anammox bacteria are not well known. Therefore, anammox bacteria were exposed to 3,500 ppm nitric oxide (NO) in the gas phase. The anammox bacteria were not inhibited by the high NO concentration but rather used it to oxidize additional ammonium to dinitrogen gas under conditions relevant to wastewater treatment.Nitric oxide (NO) has several different roles in bacteria, fungi, and mammals (24). In nitrogen cycle bacteria, it acts as an intermediate and cell communication/signal transduction molecule. On the other hand, NO is a highly reactive and toxic compound that contributes to ozone depletion and air pollution (5). Due to its reactive nature, many bacteria employ an arsenal of proteins (those encoded by norVW, as well as bacterial globins, heme proteins, etc.) that are used to detoxify NO to the less-reactive and more-stable nitrous oxide (N₂O) (24). Still, N₂O is a very effective greenhouse gas and an unfavorable constituent in the off-gases from nitrification/denitrification nitrogen removal systems (4). The presence of gene(s) encoding cytochrome cd₁ nitrite reductase (EMBL accession no. {"type":"entrez-protein","attrs":{"text":"CAJ74898","term_id":"91201838","term_text":"CAJ74898"}}CAJ74898), flavorubredoxin NorVW (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ73918","term_id":"91200863","term_text":"CAJ73918"}}CAJ73918 and {"type":"entrez-protein","attrs":{"text":"CAJ73688","term_id":"91200638","term_text":"CAJ73688"}}CAJ73688), and bacterial hemoglobin (accession no. {"type":"entrez-protein","attrs":{"text":"CAJ72702","term_id":"91203063","term_text":"CAJ72702"}}CAJ72702) in the genome of Kuenenia stuttgartiensis led to the proposal that NO also plays this dual role (metabolic versus toxic) in anammox bacteria (Fig. ​(Fig.1)1) (10, 20). This has ramifications for both application and metabolism of anammox bacteria. The source of NO in an anammox reactor could be the activity of other community members (ammonium-oxidizing or denitrifying bacteria) or high concentrations of nitrite in the influent wastewater stream. Full-scale anammox reactors typically contain a significant population of ammonium-oxidizing bacteria (AOB). In the single nitritation-anammox reactors, these carry out the conversion of 50% of the ammonium in the wastewater to nitrite (6). It has been shown that AOB may produce significant amounts of NO (2, 7), and recently it was reported that NO and N₂O could be emitted from these reactors up to 0.005 and 1.2% of the total nitrogen load to the reactor, respectively (6, 23). NO may inhibit the anammox bacteria and could also be further reduced to N₂O in these reactors (6, 23). It is presently unknown whether anammox bacteria contribute to the NO or N₂O emissions, although it has been suggested previously that anammox bacteria do not produce N₂O under physiologically relevant conditions (10). Nevertheless, if conversion of NO could be coupled to anaerobic ammonium oxidation, the toxic air pollutant NO would facilitate further removal of ammonium in full-scale anammox bioreactors. In the present study, we investigated the effect of very high NO fluxes on anammox bacteria.Open in a separate windowFIG. 1.The hypothetical anammox pathway with possible routes of NO removal. Solid black arrows: anammox pathway, including nitrite oxidation to nitrate; gray arrow, possible detoxification pathway to N₂O (not observed in the bioreactor); dashed gray arrow, NO oxidation to nitrite/nitrate (not possible under anoxic conditions).NO has been described many times as a potent inhibitor of nitrogen cycle bacteria; aerobic ammonium oxidizers, nitrite oxidizers, and denitrifiers were all inhibited by concentrations as low as a few micromolar units (1, 18, 24). In a previous study, it was suggested that “Candidatus Brocadia anammoxidans” could tolerate up to 600 ppm NO (approximately 1 mg NO·day⁻¹ NO load) (16). In the reported experiments, without direct measurement of nitrous oxide (N₂O) in the effluent gas stream, it was postulated that NO was reduced to N₂O (16). In the present study, we used a carefully monitored sequencing batch reactor (SBR) to further our understanding of the effect and fate of NO in a laboratory-scale anammox reactor under conditions which are relevant in wastewater treatment plants.An SBR (working volume, 3.5 liters) consisting of approximately 80% of the anammox bacterium “Candidatus Brocadia fulgida” and no detectable aerobic ammonium oxidizers (determined by fluorescence in situ hybridization (FISH) as described previously [15]) was used in the present study. Before the first introduction of NO into the reactor, the influent (synthetic wastewater) (21) was supplied to the reactor at a flow rate of 1.4 ml·min⁻¹ with nitrite and ammonium concentrations (assayed as previously described [9]) at 45 and 39 mM, respectively (corresponding to a total of 2,370 mg N·day⁻¹). All nitrite was consumed in the reactor, while 2 mM ammonium was still present in the effluent. For every 1 mol of ammonium, 1.22 mol of nitrite was consumed, similar to the previously determined anammox stoichiometry (19). NO was first introduced at a concentration of 400 to 600 ppm in the gas phase at a flow rate of 10 ml/min (CLD 700EL chemiluminescence NOx analyzer, detection limit of 0.1 ppm NO, with 15 ml/min Ar/CO₂ as the dilution gas [a load of 25 to 28 mg NO·day⁻¹]; EcoPhysics, Michigan). During this period, 45% (±6%) of the supplied NO was removed from the system. Initially, there was no detectable change in the ammonium and nitrite removal efficiencies and no detectable nitrous oxide (N₂O) in the flue gas (analyzed with an Agilent 6890 gas chromatograph). It is most likely that NO was converted to N₂, but the increase in the N₂ concentrations in the off-gas was below the detection limit (1,000 ppm).At day 49, the influent NO concentration was increased to 3,500 ppm (640 mg NO·day⁻¹ load). Simultaneously, the stirring speed of the reactor was increased from 200 to 600 rpm to enable better mass transfer to the flocculent anammox biomass. The increase in the stirring speed did not result in any disturbance in the floc size and settling ability of the biomass but did lead to a much higher level of NO removal (128 mg NO·day⁻¹) by the anammox bacteria. The converted NO could theoretically be converted to N₂O via detoxification enzymes or coupled to ammonium oxidation (Fig. ​(Fig.1).1). Surprisingly, there was no change in the nitrite removal capacity of the bioreactor, suggesting that NO was not a substrate preferred over nitrite. Nitrate concentrations (assayed according to the method in reference 9) were stable around 7.2 mM (±0.7 mM). Theoretically, as anammox bacteria reduce NO, they could oxidize a larger proportion of nitrite to nitrate (Fig. ​(Fig.1)1) to increase their capacity for CO₂ fixation; however, such an increase in nitrate production was not observed (or could not be discriminated by the method used [sensitivity, 100 μM]). During this phase of the experiment, the effluent ammonium concentration gradually decreased to below the detection limit (Fig. ​(Fig.2).2). There was only a minimal N₂O (0.6 ppm) emission from the system, and the total N₂ production increased from 3,060 to 3,680 mg N₂·day⁻¹. This indicated that NO reduction was coupled to the catabolism of the anammox bacteria rather than being detoxified by anammox or other community members. To the best of our knowledge, this was the first time that such a high load of NO was not found to be toxic to the nitrogen cycle bacteria. In a previous study, an NO load of 1 mg NO·day⁻¹ was reported to be toxic to anammox bacteria, most probably due to the fact that the experiments were conducted with biomass that had a 100-fold lower cell density and 10-fold lower activity compared to the current enrichment cultures. Furthermore, the NO conversion in the current experiments was stoichiometrically coupled to ammonium oxidation and not converted to N₂O, indicating that the previously reported N₂O emissions from full-scale anammox bioreactors originated not with the anammox bacteria but rather with other community members as hypothesized previously (8).Open in a separate windowFIG. 2.Ammonium concentration in the effluent of the anammox bioreactor. Dashed lines indicate the trend of effluent ammonium concentration during different phases of the reactor operation. Black arrows indicate the manipulations to influent NO stream, and the gray arrow points to an increase in the influent ammonium concentration. d, day.To determine if there could be more NO-dependent ammonium removal, the influent ammonium concentration was first increased to 41 mM (day 80) and then to 43 mM (day 81). This resulted in a slow but gradual increase in the effluent ammonium concentration, and additional ammonium did not appear to be completely converted, most probably due to NO mass transfer limitations. As a result of the higher level of ammonium removal, the observed anammox stoichiometry in the reactor decreased from 1.22 to 0.91 (nitrite/ammonium). Between days 95 and 131, the NO supply to the reactor was turned off, which resulted in an average ammonium concentration of 3.3 mM (±0.9 mM) in the effluent. Following this period, on day 132, the NO load on the reactor was increased back to 640 mg NO·day⁻¹ (Fig. ​(Fig.2).2). As a result, the effluent ammonium concentration gradually decreased again to an average of 1.5 mM (±0.36 mM). The highest level of NO removal achieved in this period was 371 mg NO·day⁻¹. When the NO supply was turned off on day 165, ammonium concentrations increased back to 3.5 mM (±0.71 mM).During the course of the experiment, the biodiversity of the reactor was monitored using FISH and 16S rRNA gene sequence analysis as described previously (15) with probes specific to eubacteria (3), Planctomycetes (13), anammox bacteria (15), “Ca. Brocadia fulgida” (11), and a variety of aerobic ammonium-oxidizing bacteria (12, 22). Before the experiments started and throughout the cultivation of the anammox bacteria with NO, the only detectable anammox species (with FISH and 16S rRNA gene sequence analysis) was “Candidatus Brocadia fulgida.”In the present study, we showed that 2 mM ammonium (4.5% of the influent concentration) could be removed by anammox bacteria via direct coupling to NO reduction. These observations support the proposal of NO as an intermediate of the anammox reaction and have two consequences for application of the anammox process for nitrogen removal. First, we obtained strong indications that previously reported N₂O emissions (6, 8) from full-scale anammox reactors were not generated by anammox bacteria. In our experiments, even under a very high load of NO, there was hardly any detectable N₂O in the effluent gas stream. The competition for nitrogen oxides by denitrifying and anammox bacteria needs further study but may ultimately be used to design operational conditions that would reduce or even prevent NO and N₂O emissions from full-scale nitritation-anammox reactors. Second, by implementing the results of this study, in the future the anammox process could be designed to remove NO from flue gases. Since NO is mostly emitted together with O₂, this could be achieved by the combination of anammox and aerobic ammonium-oxidizing bacteria, for example, with CANON (completely autotrophic nitrogen removal over nitrite)- or OLAND (oxygen-limited autotrophic nitrification-denitrification)-type reactor systems (14, 17).     

Forced flowering of pineapple (<Emphasis Type="Italic">Ananas comosus</Emphasis> cv. Tainon 17) in response to cold stress,ethephon and calcium carbide with or without activated charcoal

Ethylene, a gaseous plant hormone, is responsible for the initiation of reproductive development in pineapple. Reproductive development can be forced in pineapple (Ananas comosus var. comosus) throughout the year with ethylene. Inhibition of natural flowering initiation with aviglycine [(S)-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride], an inhibitor of ethylene biosynthesis, provides evidence that reproductive development in response to cold stress and short daylength is also in response to ethylene production. We studied the effect of cold treatment of pineapple on ethylene production and flower induction by applying a short-term cold stress to stem apices. Shoot apices of pineapple treated with ice crystals also produced twice as much ethylene as did those of control plants and significantly more than was produced by “D” leaf basal tissue. Moreover, pineapple plants treated four times with ice crystals or ice water were induced to flower under field conditions and the forcing efficiency, as evaluated by the percentages of inflorescence emergence and fruit harvest, was comparable to forcing with calcium carbide (CaC₂) and ethephon. In another field experiment two applications of a 1.0% solution of CaC₂ or 0.15% ethephon applied at 48 h intervals was sufficient to force reproductive development of ‘Tainon 17’. Furthermore, 0.5 or 1.0% solutions of CaC₂ supplemented with 0.5% activated charcoal (AC) significantly improved the forcing effectiveness of CaC₂. This could/would make it possible to reduce the number or concentration, or both, of CaC₂ required to effect forcing in pineapple.