A specific binding site for K+ channel openers in rat aorta.

The K+ channel openers, including cromakalim, pinacidil, minoxidil sulfate, diazoxide, and nicorandil, form a chemically heterogeneous group of compounds, which relax smooth muscle by opening plasmalemmal K+ channels. At present it is not known whether these drugs elicit their effects by binding to the same target, presumably the K+ channel. In order to address this question, a binding assay for K+ channel openers has been developed in vascular smooth muscle. The novel tritiated K+ channel opener, [3H]P1075, an analogue of pinacidil, binds with high affinity (KD = 6 +/- 1 nM) to endothelium-denuded rings of rat aorta. Inhibition studies indicate that the different families of K+ channel openers bind to a common target. Evidence is presented to suggest that the binding site for the sulfonylurea, glibenclamide, the major blocker of the K+ channel openers, is coupled in a negative allosteric manner to the binding site(s) for the openers. The binding assay described here may open the way to the biochemical characterization of the drug receptor for the K+ channel openers.     

ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis

The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies.     

Hydroxyl Radical Formation in Hyperglycemic Rats During Middle Cerebral Artery Occlusion/Reperfusion

Preexisting hyperglycemia is associated with enhanced reperfusion injury in the postischemic rat brain. The goal of this study was to evaluate whether the hyperglycemic exacerbation of brain injury is associated with enhanced generation of hydroxyl radicals in rats subjected to middle cerebral artery occlusion (2 h), followed by reperfusion (2 h). Magnetic resonance images revealed the exacerbation of focal brain injury in hyperglycemic rats. The salicylate trapping method was used in conjunction with microdialysis to continuously estimate hydroxyl radical production by measurement of the stable adducts 2,3- and 2,5-dihydroxybenzoic acid (DHBA) during ischemia/reperfusion. In normoglycemic rats, from a mean baseline level of 130 nmol/l, 2,3-DHBA levels surged to peak levels of 194 nmol/l 45 min into ischemia and to 197 nmol/l 15–30 min into the reperfusion period, returning to baseline by 2 h into reperfusion. A similar temporal profile was observed in hyperglycemic rats, except that absolute 2,3-DHBA levels were higher (165 nmol/l at baseline, 317 nmol/l peak during ischemia, 333 nmol/l peak during reperfusion), and levels remained significantly high (p < .05) throughout the reperfusion period. These results suggest that hydroxyl radical is an important contributor to the exacerbation of neuronal and cerebrovascular injury after focal ischemia/reperfusion in hyperglycemic rats.     

Comparison of complications and shocks in paediatric and young transvenous and subcutaneous implantable cardioverter-defibrillator patients

Background

Young implantable cardioverter-defibrillator (ICD) patients are prone to complications and inappropriate shocks (IAS). The subcutaneous ICD (S-ICD) may avoid lead-related complications. This study aims to describe the incidence and nature of device-related complications in young transvenous ICD (TV-ICD) and S?ICD patients.

Methods

Single-chamber TV-ICD and S?ICD patients up to and including the age of 25 years implanted between 2002 and 2015 were retrospectively analysed. Complications were defined as device-related complications requiring surgical intervention. IAS were defined as shocks for anything other than ventricular tachycardia or ventricular fibrillation. Follow-up data were collected 5 years post-implantation. Kaplan-Meier estimates for complications at 5?year follow-up were calculated with a corresponding 95% confidence interval.

Results

Eighty-one patients (46 TV-ICD, 35 S-ICD) were included (median age 19.0 (IQR 16.0–23.0) and 16.5 (IQR 13.0–20.2) years respectively). Median follow-up was 60 and 40 months respectively. All-cause complication rate was 34% in the TV-ICD group and 25% in the S?ICD group (p?=?0.64). TV-ICD patients had more lead complications: 23% (10–36%) versus 0% (p?=?0.02). The rate of infections did not differ between TV-ICD and S?ICD: 2% (0–6%) versus 10% (0–21%) (p?=?0.15). No systemic infections occurred in the S?ICD patients. The rates of IAS were similar, TV-ICD 22% (9–35%) versus S?ICD 14% (0–30%) (p?=?0.40), as were those for appropriate shocks: 25% (11–39%) versus 27% (6–48%) (p?=?0.92).

Conclusion

The rates of all-cause complications in this cohort were equal, though the nature of the complications differed. S?ICD patients did not suffer lead failures or systemic infections. An era effect is present between the two groups.

     

Interactions between functionally diverse fungal mutualists inconsistently affect plant performance and competition

Plants form mutualistic relationship with a variety of belowground fungal species. Such a mutualistic relationship can enhance plant growth and resistance to pathogens. Yet, we know little about how interactions between functionally diverse groups of fungal mutualists affect plant performance and competition. We experimentally determined the effects of interaction between two functional groups of belowground fungi that form mutualistic relationship with plants, arbuscular mycorrhizal (AM) fungi and Trichoderma, on interspecific competition between pairs of closely related plant species from four different genera. We hypothesized that the combination of two functionally diverse belowground fungal species would allow plants and fungi to partition their symbiotic relationships and relax plant–plant competition. Our results show that: 1) the AM fungal species consistently outcompeted the Trichoderma species independent of plant combinations; 2) the fungal species generally had limited effects on competitive interactions between plants; 3) however, the combination of fungal species relaxed interspecific competition in one of the four instances of plant–plant competition, despite the general competitive superiority of AM fungi over Trichoderma. We highlight that the competitive outcome between functionally diverse fungal species may show high consistency across a broad range of host plants and their combinations. However, despite this consistent competitive hierarchy, the consequences of their interaction for plant performance and competition can strongly vary among plant communities.     

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.     

Ligand-induced conformation changes in Torpedo californica membrane-bound acetylcholine receptor

A time-dependent increase in ligand affinity has been studied in cholinergic ligand binding to Torpedocalifornica acetylcholine receptor by inhibition of the kinetics of of [125I]-alpha-bungarotoxin-receptor complex formation. The conversion of the acetylcholine receptor from low to high affinity form was induced by both agonists and antagonists of acetylcholine and was reversible upon removal of the ligand. The slow ligand induced affinity change in vitro resembled electrophysiological desensitization observed at the neuromuscular junction and described by a two-state model (Katz, B., & Thesleff, S. (1957) J. Physiol. 138, 63). A quantitative treatment of the rate and equilibrium constants determined for binding of the agonist carbamoylcholine to membrane bound acetylcholine receptor indicated that the two-state model is not compatible with the in vitro results.     

The molecular structures of pyridine-chromiumpentacarbonyl and bis(pyridine)-chromiumtetracarbonyl

The structures of pyridinechromiumpentacarbonyl, (1), and bis(pyridine)chromiumtetracarbonyl,(2) have been determined. (1) crystallizes in the space group Pbam with a = 15.289(3) Å, b = 19.276(5) Å and c = 7.677(6) Å. (2) crystallizes in the space group P$\text{1}$ with a = 7.365(2) Å, b = 8.136(2) Å, c = 13.491(4) Å, α = 89.49(2)°, β = 88.89(2)°, and γ = 63.09(2)°. The structures refined to R_w values of 0.020 and 0.034 for (1) and (2), respectively. In both cases the pyridine rings are planar and stagger the cis CrCO bonds. A comparison of the structural results from these two compounds to piperidinechromiumpentacarbonyl and Cr(CO)₆ seems to indicate that the pyridine ligand is a weaker σ-donor and stronger π-acceptor than the saturated analog, piperidine.     

The Corynebacterium glutamicum insertion sequence ISCg2 prefers conserved target sequences located adjacent to genes involved in aspartate and glutamate metabolism

An IS element, termed ISCg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of ISCg2 including their flanking regions were sequenced and analyzed. ISCg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS elements, ISCg2 was classified as a member of the IS30 family of insertion sequences. The six copies of ISCg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in which the six copies of ISCg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be the target region for ISCg2. Interestingly, five of the six copies of ISCg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation of the distribution of ISCg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of ISCg2 in C. glutamicum. Received: 7 April 1999 / Accepted: 17 June 1999     

Two neonatal diabetes mutations on transmembrane helix 15 of SUR1 increase affinity for ATP and ADP at nucleotide binding domain 2

K(ATP) channels, (SUR1/Kir6.2)(4) (sulfonylurea receptor type 1/potassium inward rectifier type 6.2) respond to the metabolic state of pancreatic β-cells, modulating membrane potential and insulin exocytosis. Mutations in both subunits cause neonatal diabetes by overactivating the pore. Hyperactive channels fail to close appropriately with increased glucose metabolism; thus, β-cell hyperpolarization limits insulin release. K(ATP) channels are inhibited by ATP binding to the Kir6.2 pore and stimulated, via an uncertain mechanism, by magnesium nucleotides at SUR1. Glibenclamide (GBC), a sulfonylurea, was used as a conformational probe to compare nucleotide action on wild type versus Q1178R and R1182Q SUR1 mutants. GBC binds with high affinity to aporeceptors, presumably in the inward facing ATP-binding cassette configuration; MgATP reduces binding affinity via a shift to the outward facing conformation. To determine nucleotide affinities under equilibrium, non-hydrolytic conditions, Mg(2+) was eliminated. A four-state equilibrium model describes the allosteric linkage. The K(D) for ATP(4-) is ~1 versus 12 mM, Q1178R versus wild type, respectively. The linkage constant is ~10, implying that outward facing conformations bind GBC with a lower affinity, 9-10 nM for Q1178R. Thus, nucleotides cannot completely inhibit GBC binding. Binding of channel openers is reported to require ATP hydrolysis, but diazoxide, a SUR1-selective agonist, concentration-dependently augments ATP(4-) action. An eight-state model describes linkage between diazoxide and ATP(4-) binding; diazoxide markedly increases the affinity of Q1178R for ATP(4-) and ATP(4-) augments diazoxide binding. NBD2, but not NBD1, has a higher affinity for ATP (and ADP) in mutant versus wild type (with or without Mg(2+)). Thus, the mutants spend more time in nucleotide-bound conformations, with reduced affinity for GBC, that activate the pore.