Termination of endothelin signaling: Role of nitric oxide

Cellular mechanisms responsible for the termination of ET-1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET- 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ET_A receptor cDNA (CHO-ET) were studied. CHO-ET responded to ET-1 with robust [Ca²⁺], transients and developed a long-lasting homologous desensitization. Donors of nitric oxide (NO), 3-morpholino-sydnonimine HCl(SIN-1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca²⁺], recovery, and counteracted the development of homologous desensitization by a cyclic GMP-independent mechanism, suggesting an alternative mode for NO modulation of ET-1 responses. Stimulation of CHO-ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO-induced modulation of [Ca²⁺] mobilization. Using a lys⁹-biotinylated ET-1 (ET-1 [BtK⁹]), binding sites were “mapped” in CHO-ET cells. Receptor-ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN-1 caused a rapid, concentration-dependent, and reversible dissociation of biotinylated ET- 1 from ET_A receptor (EC₅₀ = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8-bromo-cyclic GMP. “Sandwich” co-culture of endothelial cells with CHO-ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET-1 [BtK⁹] from ET_A receptors. We hypothesize that NO plays a role in physiological termination of ET-1 signalling by dual mechanisms: (1) displacement of bound ET-1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca²⁺] mobilization, hence inhibiting end-responses to ET-1. © 1994 Wiley-Liss, Inc.     

Use of simultaneous pressure and velocity measurements to estimate arterial wave speed at a single site in humans

It has not been possible to measure wave speed in the human coronary artery, because the vessel is too short for the conventional two-point measurement technique used in the aorta. We present a new method derived from wave intensity analysis, which allows derivation of wave speed at a single point. We apply this method in the aorta and then use it to derive wave speed in the human coronary artery for the first time. We measured simultaneous pressure and Doppler velocity with intracoronary wires at the left main stem, left anterior descending and circumflex arteries, and aorta in 14 subjects after a normal coronary arteriogram. Then, in 10 subjects, serial measurements were made along the aorta before and after intracoronary isosorbide dinitrate. Wave speed was derived by two methods in the aorta: 1) the two-site distance/time method (foot-to-foot delay of pressure waveforms) and 2) a new single-point method using simultaneous pressure and velocity measurements. Coronary wave speed was derived by the single-point method. Wave speed derived by the two methods correlated well (r = 0.72, P < 0.05). Coronary wave speed correlated with aortic wave speed (r = 0.72, P = 0.002). After nitrate administration, coronary wave speed fell by 43%: from 16.4 m/s (95% confidence interval 12.6-20.1) to 9.3 m/s (95% confidence interval 6.5-12.0, P < 0.001). This single-point method allows determination of wave speed in the human coronary artery. Aortic wave speed is correlated to coronary wave speed. Finally, this technique detects the prompt fall in coronary artery wave speed with isosorbide dinitrate.     

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gα_i. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.     

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml(-1), respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml(-1) SDS and 0.3 mg l(-1) CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml(-1) SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.     

Mimulus finds centromeres in the driver's seat

Sandler and Novitski first pointed out in 1957 that chromosomes could selfishly exploit meiotic asymmetries to maximize their own transmission, in a process termed 'meiotic drive'. However, since then, only post-meiotic processes of non-Mendelian inheritance have received serious scientific attention in studies of transmission distortion. A recent study by Fishman and Willis puts the focus squarely back on meiotic drive. They found completely biased transmission of a centromere-linked locus from an outcrossing Mimulus species over that from an inbred species, providing the first direct evidence that centromeres can act as general, powerful meiotic drivers. This study suggests that, although difficult to detect experimentally, female meiotic drive is a major evolutionary force in nature.     

In vivo up-regulation of brain-derived neurotrophic factor in specific brain areas by chronic exposure to Delta-tetrahydrocannabinol

Cannabinoids are widely abused drugs. Here we show that chronic administration of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the active psychotropic agent in marijuana and hashish, at 1.5 mg per kg per day intraperitoneally for 7 days, increases the expression, at both mRNA and protein levels, of brain-derived neurotrophic factor (BDNF), in specific rat brain areas, notably in those involved in reward and addiction. Real-time PCR revealed a 10-fold up-regulation of BDNF mRNA in the nucleus accumbens (NAc) upon chronic Delta(9)-THC treatment, but there was no change at 3 or 24 h after a single injection. Smaller increases in mRNA levels were found in the ventral tegmental area (VTA), medial prefrontal cortex and paraventricular nucleus (PVN). Immunohistochemistry showed large increases in BDNF-stained cells in the NAc (5.5-fold), posterior VTA (4-fold) and PVN (1.7-fold), but no change was observed in the anterior VTA, hippocampus or dorsal striatum. Altogether, our study indicates that chronic exposure to Delta(9)-THC up-regulates BDNF in specific brain areas involved with reward, and provides evidence for different BDNF expression in the anterior and posterior VTA. Moreover, BDNF is known to modulate synaptic plasticity and adaptive processes underlying learning and memory, leading to long-term functional and structural modification of synaptic connections. We suggest that Delta(9)-THC up-regulation of BDNF expression has an important role in inducing the neuroadaptive processes taking place upon exposure to cannabinoids.     

A simple method for characterising syndactyly in clinical practice

Non-syndromic syndactyly is a heterogeneous group of limb malformations involving webbing of fingers and/or toes. There are at least nine non-syndromic types described in the literature. For the clinician and the genetic counsellor not having gathered experience with this malformation, it is rather tedious to identify the correct subtype for the patient's phenotype. We therefore present a protocol for clinical use, which visualises the malformation in a graphical way and thereby simplifies typing. In addition, this protocol provides a simple documentation system for reporting clinical data for new syndactyly families. It might encourage clinicians to report families that are still unclassifed and thus, helping to extend and improve the existing classification system.     

TLR9 expression is related to immune activation but is impaired in individuals with chronic immune activation

Millions of individuals in developing countries are infected with helminths and other chronic infectious diseases, such as HIV-1, which lead to persistent immune activation and unbalanced immune state. We have suggested that the capacity of chronically immune activated individuals to protect themselves, cope with infections, and mount protective immunity following vaccination, is highly impaired. Here we examined the expression of toll-like receptor 9 (TLR9), as an essential component in the recognition of immunostimulating bacterial CpG-DNA motifs, in different subsets of human peripheral blood mononuclear cells (PBMC) obtained from chronically immune activated and non-activated individuals. TLR9 expression was correlated to immune cell activation and was upregulated following phytohemagglutinin or anti-CD3 activation. PBMC obtained from chronically immune activated individuals had a different overall pattern of TLR9 expression, including reduced upregulation of this receptor following additional immune activation, and diminished responsiveness to CpG-DNA stimulation, in comparison to non-activated individuals. These differences may partly account for the reduced capacity of chronically immune activated individuals to mount effective immune responses and strengthen the notion that the host immune background should be considered in the design and trial of potential adjuvants and vaccines.