SUR-dependent modulation of KATP channels by an N-terminal KIR6.2 peptide. Defining intersubunit gating interactions

Ntp and Ctp, synthetic peptides based on the N- and C-terminal sequences of K(IR)6.0, respectively, were used to probe gating of K(IR)6.0/SUR K(ATP) channels. Micromolar Ntp dose-dependently increased the mean open channel probability in ligand-free solution (P(O(max))) and attenuated the ATP inhibition of K(IR)6.2/SUR1, but had no effect on homomeric K(IR)6.2 channels. Ntp (up to approximately 10(-4) m) did not affect significantly the mean open or "fast," K(+) driving force-dependent, intraburst closed times, verifying that Ntp selectively modulates the ratio of mean burst to interburst times. Ctp and Rnp, a randomized Ntp, had no effect, indicating that the effects of Ntp are structure specific. Ntp opened K(IR)6.1/SUR1 channels normally silent in the absence of stimulatory Mg(-) nucleotide(s) and attenuated the coupling of high-affinity sulfonylurea binding with K(ATP) pore closure. These effects resemble those seen with N-terminal deletions (DeltaN) of K(IR)6.0, and application of Ntp to DeltaNK(ATP) channels decreased their P(O(max)) and apparent IC(50) for ATP in the absence of Mg(2+). The results are consistent with a competition between Ntp and the endogenous N terminus for a site of interaction on the cytoplasmic face of the channel or with partial replacement of the deleted N terminus by Ntp, respectively. The K(IR) N terminus and the TMD0-L0 segment of SUR1 are known to control the P(O(max)). The L0 linker has been reported to be required for glibenclamide binding, and DeltaNK(IR)6.2/SUR1 channels exhibit reduced labeling of K(IR) with (125)I-azidoglibenclamide, implying that the K(IR) N terminus and L0 of SUR1 are in proximity. We hypothesize that L0 interacts with the K(IR) N terminus in ligand-inhibited K(ATP) channels and put forward a model, based on the architecture of BtuCD, MsbA, and the KcsA channel, in which TMD0-L0 links the MDR-like core of SUR with the K(IR) pore.     

Phytaspase,a relocalisable cell death promoting plant protease with caspase specificity

Caspases are cysteine‐dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase‐specific proteolytic activity. Nevertheless, plants do display caspase‐like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase‐like proteases. Here, we report the identification and characterisation of a novel PCD‐related subtilisin‐like protease from tobacco and rice named phytaspase (plant aspartate‐specific protease) that possesses caspase specificity distinct from that of other known caspase‐like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD‐related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re‐imported into the cell during PCD providing insights into how phytaspase operates.     

Antizyme frameshifting as a functional probe of eukaryotic translational termination

Translation termination in eukaryotes is mediated by the release factors eRF1 and eRF3, but mechanisms of the interplay between these factors are not fully understood, due partly to the difficulty of measuring termination on eukaryotic mRNAs. Here, we describe an in vitro system for the assay of termination using competition with programmed frameshifting at the recoding signal of mammalian antizyme. The efficiency of antizyme frameshifting in rabbit reticulocyte lysates was reduced by addition of recombinant rabbit eRF1 and eRF3 in a synergistic manner. Addition of suppressor tRNA to this assay system revealed competition with a third event, stop codon readthrough. Using these assays, we demonstrated that an eRF3 mutation at the GTPase domain repressed termination in a dominant negative fashion probably by binding to eRF1. The effect of the release factors and the suppressor tRNA showed that the stop codon at the antizyme frameshift site is relatively inefficient compared to either the natural termination signals at the end of protein coding sequences or the readthrough signal from a plant virus. The system affords a convenient assay for release factor activity and has provided some novel views of the mechanism of antizyme frameshifting.     

Photoperiodic,thermal and trophic responses of a predatory ladybird Cheilomenes propinqua

Large geographic range, wide habitat specificity and broad range of prey (including a number of pests of protected crops) suggest that a predatory ladybird Cheilomenes propinqua can be considered as potential agent for biological control in greenhouses. We investigated the influence of day length (10, 12 and 14 hr), temperature (20 and 24°C) and diet (the green peach aphid Myzus persicae and eggs of the grain moth Sitotroga cerealella) on the rate of maturation, fecundity and induction of reproductive diapause in C. propinqua females of a laboratory population originated from Alexandria, Egypt. The proportion of diapausing females (i.e. those with poorly developed ovaries and well-developed fat body) varied from 5% to 70% being higher at short day, low temperature and feeding on the grain moth eggs. This diapause, however, was not very stable: more than 20% of females kept under diapause-inducing conditions started to lay eggs during 110 days, although their pre-oviposition period was about 5 times longer than that of females which matured at the same temperature but at the long day and on the natural diet. Although not very stable, reproductive diapause significantly increased survival of starving females. Such a short-term reproductive diapause can be considered as an adaptation to mild and short-term subtropical winter. The results of our study suggest that C. propinqua mass rearing will be more intensive at the combination of high temperature, natural food (aphids) and long day (14 hr), whereas individuals intended for long-term storage should be reared under moderate temperature (20°C) and short-day (10 hr) conditions and should be fed on factitious food (the grain moth eggs).     

Crosstalk between AMPK activation and angiotensin II‐induced hypertrophy in cardiomyocytes: the role of mitochondria

AMP‐kinase (AMPK) activation reduces cardiac hypertrophy, although underlying molecular mechanisms remain unclear. In this study, we elucidated the anti‐hypertrophic action of metformin, specifically, the role of the AMPK/eNOS/p53 pathway. H9c2 rat cardiomyocytes were treated with angiotensin II (AngII) for 24 hrs in the presence or absence of metformin (AMPK agonist), losartan [AngII type 1 receptor (AT1R) blocker], Nω‐nitro‐L‐arginine methyl ester (L‐NAME, pan‐NOS inhibitor), splitomicin (SIRT1 inhibitor) or pifithrin‐α (p53 inhibitor). Results showed that treatment with metformin significantly attenuated AngII‐induced cell hypertrophy and death. Metformin attenuated AngII‐induced activation (cleavage) of caspase 3, Bcl‐2 down‐regulation and p53 up‐regulation. It also reduced AngII‐induced AT1R up‐regulation by 30% (P < 0.05) and enhanced AMPK phosphorylation by 99% (P < 0.01) and P‐eNOS levels by 3.3‐fold (P < 0.01). Likewise, losartan reduced AT1R up‐regulation and enhanced AMPK phosphorylation by 54% (P < 0.05). The AMPK inhibitor, compound C, prevented AT1R down‐regulation, indicating that metformin mediated its effects via AMPK activation. Beneficial effects of metformin and losartan converged on mitochondria that demonstrated high membrane potential (Δψ_m) and low permeability transition pore opening. Thus, this study demonstrates that the anti‐hypertrophic effects of metformin are associated with AMPK‐induced AT1R down‐regulation and prevention of mitochondrial dysfunction through the SIRT1/eNOS/p53 pathway.     

Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-gamma. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.