State-Dependent Block of Na<Superscript>+</Superscript> Channels by Articaine Via the Local Anesthetic Receptor

Articaine is widely used as a local anesthetic (LA) in dentistry, but little is known regarding its blocking actions on Na⁺ channels. We therefore examined the state-dependent block of articaine first in rat skeletal muscle rNav1.4 Na⁺ channels expressed in Hek293t cells. Articaine exhibited a weak block of resting rNav1.4 Na⁺ channels at −140 mV with a 50% inhibitory concentration (IC₅₀) of 378 ± 26 μM (n = 5). The affinity was higher for inactivated Na⁺ channels measured at −70 mV with an IC₅₀ value of 40.6 ± 2.7 μM (n = 5). The open-channel block by articaine was measured using inactivation-deficient rNav1.4 Na⁺ channels with an IC₅₀ value of 15.8 ± 1.5 μM (n = 5). Receptor mapping demonstrated that articaine interacted strongly with a D4S6 phenylalanine residue, which is known to form a part of the LA receptor. Thus the block of rNav1.4 Na⁺ channels by articaine is via the conserved LA receptor in a highly state-dependent manner, with a ranking order of open (23.9×) > inactivated (9.3×) > resting (1×) state. Finally, the open-channel block by articaine was likewise measured in inactivation-deficient hNav1.7 and rNav1.8 Na⁺ channels, with IC₅₀ values of 8.8 ± 0.1 and 22.0 ± 0.5 μM, respectively (n = 5), indicating that the high-affinity open-channel block by articaine is indeed preserved in neuronal Na⁺ channel isoforms.     

Gamma processing of Arabic bread for immune system-compromised cancer patients.

Arabic bread prepared from local Saudi flour contained a total of up to 10(4) organisms per g. Most of these were bacterial spores that survived the baking process (1.3 X 10(2) to 3.5 X 10(3] and a small number of yeasts and molds (10 to 40 cells per g). The organisms in Arabic bread appear to be harmless to healthy individuals. However, for immune system-compromised cancer patients and bone marrow transplant recipients, it is prudent to irradiate the bread to reduce microbial contamination. The decimal reduction doses (10% survival) for the most radiation-resistant organisms (spore formers) in bread were 0.11 to 0.15 Mrad. Accordingly, 0.6 Mrad was sufficient to reduce the number of spores in Arabic bread by a factor of 10,000, i.e., to less than 1/g. This treatment constitutes radiation pasteurization (radicidation), and to this extent, provides a margin of microbiological safety. Sensory evaluation by the nine-point hedonic scale showed no detectable loss of organoleptic quality of bread up to 0.6 Mrad, while irradiation to 2.5 Mrad induced unacceptable organoleptic changes.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

Stent implantation in acute myocardial infarction using a heparin-coated stent: a pilot study as a preamble to a randomized trial comparing balloon angioplasty and stenting

Preliminary experience with primary stenting in myocardial infarction has suggested a greater benefit in clinical outcome than has been obtained with direct balloon angioplasty. However, subacute thrombosis (SAT) remains a limitation for this new mode of therapy. In the BENESTENT II Pilot and main trials, the incidence of SAT with the heparin-coated Palmaz-Schatz stent was only 0.15%. Therefore, as a preamble to a large randomized trial, the feasibility and safety of the use of the Heparin-Coated Palmaz-Schatz trade mark Stent in Acute Myocardial Infarction (AMI) was tested in 101 patients enrolled between April and September 1996 in 18 clinical centres. In 101 stent-eligible AMI patients, as dictated by protocol, a heparin-coated stent was implanted. The primary objectives were to determine the in-hospital incidence of major adverse cardiac events (MACE: death, MI, target lesion revascularization) and bleeding complications, while the secondary objectives were the procedural success rate and the MACE, the restenosis and reocclusion rates at 6.5 months. Stent implantation (n 3 129 stents) was successful in 97 patients of the 101 who were included in this trial. During their hospital stay, two patients died and no patient experienced re-infarction, ischaemia prompting re-PTCA or CABG. Four patients suffered a bleeding complication, three major and one minor, of whom three required surgical repair. At 210 days follow-up, 81% of the patients were event free. At 6.5 months restenosis was documented in 18% of the 88 patients who underwent follow-up angiography, including three total occlusions. The results, both with respect to QCA and the occurrence of MACE, compare favourably with studies using elective stenting in both stable and unstable angina patients. As a result of this pilot study, a large randomized trial comparing direct balloon angioplasty with direct stenting in 900 patients with AMI was initiated in December 1996.     

Mapping and QTL analysis of horticultural traits in a narrow cross in cucumber (Cucumis sativus L.) using random-amplified polymorphic DNA markers

An 80-point genetic map [77 random-amplified polymorphic DNAs (RAPD), F (female sex expression), de (determinate), and ll (little leaf)] was constructed from a narrow cross in cucumber using the determinate, gynoecious, standard-sized leaf line G421 and the indeterminate, monoecious, little leaf line H-19. The map defined nine linkage groups and spanned ca. 600 cM with an average distance between markers of 8.4 ± 9.4 cM. The RAPD loci BC-551 and BC-592 were found to flank ll at 3.4 and 12.2 cM, respectively. The locus OP-L18-2 was linked (16 cM) to de, and the F locus was flanked by markers at 44 and 31 cM. One-hundred F3 families were used to identify quantitative trait loci (QTL) for sex expression, main stem length, number of lateral branches, days to anthesis, fruit number and weight, fruit length and diameter, and fruit length: diameter ratio in two replicated test locations (Wisconsin and Georgia). QTL on linkage group B explained major portions (R2 = ca. 2 to 74%) of the variation observed for sex expression, main stem length, lateral branch number, and fruit diameter (LOD = 2.1 to 29.8). Although ca. 62 to 74% of the variation for sex expression was associated with a putative QTL spanning the F locus (OP-AJ-2 to F and F to de), other regions (three) of the genome were important for the determination of sex in the F3 families examined depending upon environment. The number of genomic regions affecting main stem length (five) and number of lateral branches (three) coincided with expectations as determined by calculations of minimum number of genes in previous studies. Evaluation of QTL associated with several fruit number determinants of early, first-harvest yield demonstrating additive genetic variance (i.e., sex expression, main stem length, and number of laterals) suggests that marker-assisted selection may have utility for the development of determinate, multiple lateral branching germplasm suited for once-over mechanical harvesting in this population.     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. II. Effects of cyclic nucleotides and protaglandins

Substances known to alter cyclic nucleotide levels in cells were applied to the isolated toad retina and effects on rod electrical and adaptive behavior were studied. The retina was continually superfused in control ringer’s or ringer’s containing one or a combination of drugs, and rod activity was recorded intracellularly. Superfusion with cGMP, Bu(2)GMP, isobutylmethylxanthine (IBMX; a phosphodiesterase inhibitor), or PGF(2α) (a prostaglandin) caused effects in rods that closely match those observed when extracellular Ca(2+) levels were lowered. For example, short exposures (up to 6 min) of the retina to these substances caused depolarization of the membrane potential, increase in response amplitudes, and some changes in waveform; but under dark-adapted or partially light-adapted conditions receptor sensitivity was virtually unaffected. That is, the position of the V-log I curve on the intensity axis was determined by the prevailing light level, not by drug level. These drugs, like lowered extracellular Ca(2+), also decreased the period of receptor saturation after a bright-adapting flash, resulting in an acceleration of the onset of membrane and sensitivity recovery during dark adaptation.

Long-term (6-15 min) exposure of a dark-adapted retina to 5 mM IBMX or a combination of IBMX and cGMP caused a loss of response amplitude and a desensitization of the rods that was similar to that observed in rods after a long-term low Ca(2+) (10(-9)M) treatment. Application of high (3.2 mM) Ca(2+) to the retina blocked the effects of applied Bu(2)cGMP. PGE(1) superfusion mimicked the effects of increasing extracellular Ca(2+). The results show that increased cGMP and lowered Ca(2+) produce similar alterations in the electrical activity of rods. These findings suggest that Ca(2+) and cGMP are interrelated messengers. We speculate that low Ca(2+) may lead to increased intracellular cGMP, and/or that applied cGMP, and/or that applied cGMP may lower cytosol Ca(2+), perhaps by stimulating Ca(2+)- ATPase pumps in the outer segment.

     

Neuronostatin encoded by the somatostatin gene regulates neuronal, cardiovascular, and metabolic functions

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide.     

State-dependent calcium mobilization by urotensin-II in cultured human endothelial cells

Human endothelial cells express urotensin-II (U-II) as well as its receptor GPR14. Using microfluorimetric techniques, the effect of human U-II on cytosolic Ca2+ concentrations [Ca2+]i in cultured human aortic endothelial cells (HAECs) loaded with Fura-2 was evaluated in static or flow conditions. Under the static state, U-II (100 nM) abolished spontaneous Ca2+ oscillations, which occurred in a population of cultured HAEC. Similarly, U-II reduced thrombin-, but not ATP-induced calcium responses, suggesting that the peptide does not alter the Gq/11/IP3 pathway; rather, it modifies the coupling between protease-activated receptors and Gq/11/IP3. Under the flow condition, U-II (1, 10 and 100 nM) produced a dose-dependent increase in [Ca2+]i, which was subjected to desensitization. The result demonstrates a state-dependent effect of U-II in cultured HAEC, which may explain the variable responses to U-II under different experimental conditions.     

Genetic conflict outweighs heterogametic incompatibility in the mouse hybrid zone?

Background  

The Mus musculus musculus/M. m. domesticus contact zone in Europe is characterised by sharp frequency discontinuities for sex chromosome markers at the centre of wider clines in allozyme frequencies.