PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba²⁺ currents through voltage gated Ca²⁺ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca²⁺ channel blockers, volatile anesthetics blocked Ca²⁺ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca²⁺ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca²⁺ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.     

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

A taxonomic revision of Tyrini of the Oriental?region. V. Revision of the genus Lasinus Sharp, 1874 (Coleoptera,Staphylinidae, Pselaphinae)

The genus Lasinus Sharp, 1874 of the Pselaphodes complex of genera (Pselaphitae: Tyrini: Tyrina) is revised. The three so far known species, Lasinus mandarinus Raffray, 1890, Lasinus monticola Sawada, 1961 and Lasinus spinosus Sharp, 1874 are redescribed. Eight new species, Lasinus sinicus sp. n. from China, Lasinus mikado sp. n., Lasinus yamamotoi sp. n., Lasinus inexpectatus sp. n., Lasinus yakushimanus sp. n., Lasinus amamianus sp. n., Lasinus saoriae sp. n., and Lasinus okinawanus sp. n. from Japan, are described. And all species are illustrated. Lectotypes are designated for Lasinus mandarinus and Lasinus spinosus. An identification key to species of the genus Lasinus is provided.     

Topical application of Pya-AKH stimulates lipid mobilization and locomotion in the flightless bug, Pyrrhocoris apterus (L.) (Heteroptera)

Abstract Two different methods of applying Pya -AKH to long-winged (macropterous) females of the firebug, Pyrrhocoris apterus (Linnaeus) (Heteroptera) were compared: both injection and topical application increased the levels of lipids in the haemolymph and stimulated locomotor activity. Lipid mobilization was maximal when 10 pmol was applied by injection or 40–100 pmol by topical application, with the first significant responses occurring 1.5 h after injection and 2 h after topical application. The highest elevations of lipid concentration in the haemolymph were comparable between the treatments, i.e. 14.36 ± 3.59 mg/mL for injection and 14.43 ± 4.07 mg/mL for topical application. However, these maximal elevations were achieved at different times: 3 h after the injection and 7 h after the topical application.
Injection of 10 and 40 pmol of Pya -AKH stimulated locomotor activity with maximal activity 3 h later but, surprisingly, injection of 80 pmol showed no effect initially and than a slight inhibitory effect after 6–8 h.
Increased locomotor activity was found after topical application of Pya -AKH, but the response was lower than after injection and appeared later, 5–9 h after the hormone application. In addition, the greatest increase in walking activity required topical application of 300 pmol and was still less dramatic than the response to injection. The stimulatory effect of Pya -AKH on locomotion was positively correlated with its effect on lipid mobilization only for injection of the hormone. It is argued that a stress caused by injection could play a role in the appearance of the complex response to adipokinetic hormone.     

Wing morph-related physiological differences in adults of temperate population of Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae)

The reproductive and diapausing adult females of brachypterous morph and macropterous females with reproductive arrest of non-diapause type, originating from the laboratory cultures of Pyrrhocoris apterus, were studied for their feeding and drinking behaviour, digestive enzyme activities, and carbohydrate and lipid contents. The highest feeding and drinking activities were observed in reproductive brachypters, the lowest in macropters. Macropters also differed from brachypters by lower activities of gut lipase, peptidase and protease, lower concentration of haemolymph sugars, and lower weight of fat body, which probably reflects their low feeding activity. The total content of fat body lipids was also lower in macropters (0.6 mg) than in reproductive and diapausing brachypters (4.6 and 7.5 mg, respectively) on day 14. A very high amount of glycogen was found in the fat body of diapausing brachypters, 363 μg on day 14, as opposed to 15 and 80 μg in macropterous and reproductive brachypterous females, respectively. The obtained data indicate that the most important difference between macropterous and brachypterous females with different types of reproductive arrest consists of an enhanced mobilization of lipids for dispersal in macropters and accumulation of energetic reserves for hibernation in brachypters.     

Synthesis and biological evaluation of novel mono- and bivalent ASGP-R-targeted drug-conjugates

Asialoglycoprotein receptor (ASGP-R) is a promising biological target for drug delivery into hepatoma cells. Nevertheless, there are only few examples of small-molecule conjugates of ASGP-R selective ligand equipped by a therapeutic agent for the treatment of hepatocellular carcinoma (HCC). In the present work, we describe a convenient and versatile synthetic approach to novel mono- and multivalent drug-conjugates containing N-acetyl-2-deoxy-2-aminogalactopyranose and anticancer drug – paclitaxel (PTX). Several molecules have demonstrated high affinity towards ASGP-R and good stability under physiological conditions, significant in vitro anticancer activity comparable to PTX, as well as good internalization via ASGP-R-mediated endocytosis. Therefore, the conjugates with the highest potency can be regarded as a promising therapeutic option against HCC.     

Intracellular cAMP: the "switch" that triggers on "spontaneous transient outward currents" generation in freshly isolated myocytes from thoracic aorta

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called "silent" myocytes and myocytes with STOCs have been called "active." STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 microM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 +/- 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BK(Ca) channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be "switched" on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.     

Ontogeny of light response in the early life history of the red king crab Paralithodes camtschaticus (Anomura: Lithodidae)

Phototactic responses of light-adapted zoeae IV, glaucothoe, and first stage juveniles of the red king crab to three intensities of white light were quantitatively measured under laboratory conditions. All stages observed were photopositive to all light intensities tested, except for late glaucothoe (10 days since moulting) which did not respond to light stimuli. Phototactic response changed in the early life history of the red king crab. The extent of photopositive movement decreased after each metamorphosis. Peak phototactic response in zoea IV were observed at a light intensity of 1.9 × 10¹³ q cm^-2 s^-1, in early glaucothoe at 1.1 × 10¹⁰ q cm^-2 s^-1 and in juveniles at 1.3 × 10⁹ q cm^-2 s^-1. The data on behavioural responses to light may provide a better understanding of the early life history, survival and recruitment of the red king crab and assist the development of feasible methods and techniques for aquaculture of this species.     

Interactions in a tritrophic acarine predator-prey metapopulation system IV: effects of host plant condition on Tetranychus urticae (Acari: Tetranychidae)

Feeding by spider mites can cause severe injury to a host plant and lead to a decreasing per capita growth rate and an increasing per capita emigration rate. Such density-dependent responses to local conditions are important in a metapopulation context because they allow the herbivores to colonize new host plants and thereby prolong the time until regional (metapopulation) extinction. In order to include density-dependent responses of the two-spotted spider mite (Tetranychus urticae) in a realistic metapopulation model, a series of greenhouse experiments was conducted with the purpose to quantify how the condition of bean plants (Phaseolus vulgaris) influences the demographic parameters of T. urticae. Plant age per se reduced the growth rate of the spider mites only slightly, whereas the growth rate declined significantly as the plants were injured by the mites. The relationships between plant condition (expressed by the plant injury index D) and the birth and loss (death + emigration) rates of the mites were quantified so as to predict population growth as a function of D. Maximum per capita growth rate (r) was estimated to be c. 0.21 day⁻¹. The growth rate is expected to be negative when D exceeds 0.8. When mites were allowed to emigrate to neighbouring plants via bridges, the per capita emigration rate increased almost exponentially with D. The proportion of eggs in the population decreased with D while the numerical ratio between immatures to adults and the sex ratio did not change with D. Overall, immatures and adults constituted 74% and 26%, respectively, of the active mites and c. 46% of the adults were males. The bridges that connected a donor plant with the surrounding recipient plants were responsible for the majority of the emigrations from donor plants. Most mites stopped after having crossed a single bridge, but a few crossed two bridges while none crossed three bridges within 24 h. The significance of the results for biological control is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.