hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

Cooperativity of peptidoglycan synthases active in bacterial cell elongation

Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.     

Stigmastane derivatives from the roots of Vernonia guineensis and their antimicrobial activity

Chemical investigation of the roots of Vernonia guineensis (Asteraceae) afforded a new stigmastane derivative, vernoguinoside A (1) and the known vernoguinoside (2), stigmasterol 3-O-β-d-glucoside (3) and sitosterol 3-O-β-d-glucoside (4). Their structures were elucidated by spectroscopic analysis. Antimicrobial activities of 1–3 and CH₂Cl₂–MeOH (1:1) extract were evaluated against three bacteria species (Salmonella typhi, Staphylococcus aureus and Shigella flexneri) and three yeasts species (Candida albicans, Candida parapsilosis and Cryptococcus neoformans). Compounds 1 and 2 exhibited both antibacterial and antifungal activities that varied between the microbial species (MIC = 7.81–125 μg/mL) while S. flexneri and C. albicans were sensitive to all the tested compounds.     

Dysregulation of Cytokine Response in Canadian First Nations Communities: Is There an Association with Persistent Organic Pollutant Levels?

In vitro and animal studies report that some persistent organic pollutants (POPs) trigger the secretion of proinflammatory cytokines. Whether POP exposure is associated with a dysregulation of cytokine response remains to be investigated in humans. We studied the strength of association between plasma POP levels and circulating cytokines as immune activation markers. Plasma levels of fourteen POPs and thirteen cytokines were measured in 39 Caucasians from a comparator sample in Québec City (Canada) and 72 First Nations individuals from two northern communities of Ontario (Canada). Caucasians showed significantly higher levels of organochlorine insecticides (β-HCH, p,p'-DDE and HCB) compared to First Nations. Conversely, First Nations showed higher levels of Mirex, Aroclor 1260, PCB 153, PCB 170, PCB 180 and PCB 187 compared to Caucasians. While there was no difference in cytokine levels of IL-4, IL-6, IL-10 and IL-22 between groups, First Nations had significantly greater average levels of IFNγ, IL-1β, IL-2, IL-5, IL-8, IL-12p70, IL-17A, TNFα and TNFβ levels compared to Caucasians. Among candidate predictor variables (age, body mass index, insulin resistance and POP levels), high levels of PCBs were the only predictor accounting for a small but significant effect of observed variance (～7%) in cytokine levels. Overall, a weak but significant association is detected between persistent organochlorine pollutant exposure and elevated cytokine levels. This finding augments the already existing information that environmental pollution is related to inflammation, a common feature of several metabolic disorders that are known to be especially prevalent in Canada's remote First Nations communities.     

Repolarisation and vulnerability to re-entry in the human heart with short QT syndrome arising from KCNQ1 mutation--a simulation study

Idiopathic short QT syndrome (SQTS) is a recently identified, genetically heterogeneous condition characterised by abbreviated QT intervals and an increased susceptibility to arrhythmia and sudden death. This simulation study identifies mechanisms by which cellular electrophysiological changes in the SQT2 (slow delayed rectifier, I_Ks, -linked) SQTS variant increases arrhythmia risk. The channel kinetics of the V307L mutation of the KCNQ1 subunit of the I_Ks channel were incorporated into human ventricular action potential (AP) models and into 1D and 2D transmural tissue simulations. Incorporating the V307L mutation into simulations reproduced defining features of the SQTS: abbreviation of the QT interval, and increases in T wave amplitude and T_peak–T_end duration. In the single-cell model, the V307L mutation abbreviated ventricular cell AP duration at 90% repolarisation (APD₉₀) and increased the maximal transmural voltage heterogeneity (δV) during APs; this resulted in augmented transmural heterogeneity of APD₉₀ and of the effective refractory period (ERP). In the intact tissue model, the vulnerable window for unidirectional conduction block was also increased. In 2D tissue the V307L mutation facilitated and maintained reentrant excitation. Thus, in SQT2 increases in transmural heterogeneity of APD, δV, ERP and an increased vulnerable window for unidirectional conduction block generate an electrical substrate favourable to reentrant arrhythmia.     

The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation

Eukaryotic peptidoglycan recognition proteins (PGRPs) are related to bacterial amidases. In Drosophila, PGRPs bind peptidoglycan and function as central sensors and regulators of the innate immune response. PGRP-LC/PGRP-LE constitute the receptor complex in the immune deficiency (IMD) pathway, which is an innate immune cascade triggered upon Gram-negative bacterial infection. Here, we present the functional analysis of the nonamidase, membrane-associated PGRP-LF. We show that PGRP-LF acts as a specific negative regulator of the IMD pathway. Reduction of PGRP-LF levels, in the absence of infection, is sufficient to trigger IMD pathway activation. Furthermore, normal development is impaired in the absence of functional PGRP-LF, a phenotype mediated by the JNK pathway. Thus, PGRP-LF prevents constitutive activation of both the JNK and the IMD pathways. We propose a model in which PGRP-LF keeps the Drosophila IMD pathway silent by sequestering circulating peptidoglycan.     

The effects of fire, local environment and time on ant assemblages in fens and forests

We investigated the effects of the abiotic environment, plant community composition and disturbance by fire on ant assemblages in two distinct habitat types in the Siskiyou Mountains in northern California and southern Oregon, USA. Sampling over 2 years in burned and unburned Darlingtonia fens and their adjacent upland forests, we found that the effects of disturbance by fire depended on habitat type. In forests, fire intensity predicted richness in ant assemblages in both years after the fire, and plant community composition predicted richness 2 years after the fire. No factors were associated with richness in the species‐poor fen ant assemblages. Species‐specific responses to both habitat type and disturbance by fire were idiosyncratic. Assemblage composition depended on habitat type, but not disturbance by fire, and the composition of each assemblage between years was more dissimilar in burned than unburned sites.