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Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl β-methoxyacrylate group and a strobilurin analogue bearing modified β-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4′ position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells. [ABSTRACT FROM AUTHOR]

Environmental temperature is a critical factor for all forms of life, and thermal tolerance defines the habitats utilized by a species. Moreover, the evolutionary tuning of thermal perception can also play a key role in habitat selection. Yet, the relative importance of thermal tolerance and perception in environmental adaptation remains poorly understood. Thermal conditions experienced by anuran tadpoles differ among species due to the variation in breeding seasons and water environments selected by parental frogs. In the present study, heat tolerance and avoidance temperatures were compared in tadpoles from five anuran species that spatially and temporally inhabit different thermal niches. These two parameters were positively correlated with each other and were consistent with the thermal conditions of habitats. The species difference in avoidance temperature was 2.6 times larger than that in heat tolerance, suggesting the importance of heat avoidance responses in habitat selection. In addition, the avoidance temperature increased after warm acclimation, especially in the species frequently exposed to heat in their habitats. Characterization of the heat-sensing transient receptor potential ankyrin 1 (TRPA1) ion channel revealed an amphibian-specific alternatively spliced variant containing a single valine insertion relative to the canonical alternative spliced variant of TRPA1, and this novel variant altered the response to thermal stimuli. The two alternatively spliced variants of TRPA1 exhibited different thermal responses in a species-specific manner, which are likely to be associated with a difference in avoidance temperatures among species. Together, our findings suggest that the functional change in TRPA1 plays a crucial role in thermal adaptation processes. [ABSTRACT FROM AUTHOR]

We characterized ATP-induced changes in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and membrane current in cultured rat myenteric neurons using ratiometric [Ca.sup.2+] imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [[[Ca.sup.2+]].sub.i] responses to high [K.sup.+] and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [[[Ca.sup.2+]].sub.i] that was greatly decreased by the removal of extracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.o]). The amplitude of the [[[Ca.sup.2+]].sub.i] response to ATP was reduced by half in the presence of voltage-dependent [Ca.sup.2+] channel blockers. In [[[Ca.sup.2+]].sub.o]-free solution, ATP produced a small transient rise in [[Ca.sup.2+].sub.i] similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular [Na.sup.+] concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [[Ca.sup.2+].sub.i] was ATP [greater than or equal to] adenosine 5'-O-3-triphosphate [greater than or equal to] CTP [greater or equal to] 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. [Ca.sup.2+] and [Mg.sup.2+] suppressed the ATP-induced current, and [Zn.sup.2+], [Cu.sup.2+], and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2[X.sub.2] receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2[X.sub.2] receptors, resulting in a [[[Ca.sup.2+].sub.i] increase dependent on [[[Ca.sup.2+]].sub.o] in rat myenteric neurons. A small part of the ATP-induced [[[Ca.sup.2+]].sub.i] increase may be also mediated via a P2Y receptor-related mechanism. enteric neuron; fura-2; patch clamp; purinoceptor

The fortimicin-A (FTM-A) resistance genes (fmr) in five FTM-producing actinomycetes were isolated and characterized and compared to the fmr genes of other actinomycetes. The results showed that the different fmr genes could be classified into two types based on the resistance profiles to aminoglycoside antibiotics. DNA hybridization analysis confirmed this classification based on resistance profiles.

The neuraminidase gene (nedA) from Micromonospora viridifaciens was cloned and sequenced. The gene was cloned by using pIJ702 as a vector and expressed constitutively in the heterologous host Streptomyces lividans. An open reading frame (ORF) of 1,941 bp encoded a 68-kDa protein. Thededuced amino acid sequence had five Asp boxes found in other bacterial neuraminidases. Another ORF, termed nedR, was found in the region upstream of nedA. The deduced amino acid sequence showed some similarity with the Tn3 repressor.

Teraoka, Hiroki, Yutaka Maruyama, Kazushige Takehana, Toshihiko Iwanaga, Takeo Hiraga, Shoichi Fujita, and Toshio Ohta. [Ca.sup.2+] signaling in porcine duodenal glands by muscarinic receptor activation. Am J Physiol Gastrointest Liver Physiol 280: G729-G737, 2001.--The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [[[Ca.sup.2+]].sub.i], and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the [M.sub.3] subtype far exceeded that for either [M.sub.1] or [M.sub.2] as revealed by real-time quantitative PCR and in situ hybridization. The rise in [[[Ca.sup.2+]].sub.i] evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular [Ca.sup.2+] deprivation. Caffeine had no effect on [[[Ca.sup.2+]].sub.i]. ACh elicited [Ca.sup.2+]-dependent [K.sup.+] currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of [M.sub.3] receptors linked to [Ca.sup.2+] release in porcine duodenal glands. Brunner's gland; calcium-induced calcium release; inositol 1,4,5-trisphosphate-induced calcium release; mucus; secretion

Transient receptor potential ankyrin 1 (TRPA1) is known as one of the nociceptors expressed in sensory neurons. It also plays a role in non-neural cells in inflammatory sites. However, the regulatory mechanisms for the reactivity of TRPA1 in these cells under inflammatory conditions are not clear. To clarify these mechanisms, we examined the effects of inflammatory cytokines (interleukin [IL]-1α, IL-1β and tumor necrosis factor α [TNFα]) on TRPA1 reactivity and expression in the endogenously TRPA1-expressing lung tumor cell line A549. Treatment with IL-1α, but not IL-1β or TNFα, increased the number of cells responding to allyl isothiocyanate, a TRPA1 agonist, in a dose- and time-dependent manner. The IL-1α-induced increase of TRPA1 responsiveness was inhibited by an extracellular-regulated kinase (Erk) inhibitor (PD98059) but not by inhibitors of c -Jun kinase, p38 mitogen-activated protein kinase or phosphatidylinositol-3 kinase. Phosphorylation of Erk gradually increased at 24 h after its transient induction in cells treated with IL-1α. IL-1α increased the TRPA1 levels on biotinylated cell surface proteins. These results suggest that IL-1α enhances the translocation of TRPA1 to the plasma membrane via the activation of Erk in A549. TRPA1 may have a pathophysiological role in non-neural lung cells under inflammatory conditions. [ABSTRACT FROM AUTHOR]

Summary Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity. [ABSTRACT FROM AUTHOR]

Background: Cadmium (Cd) is an environmental pollutant and acute exposure to it causes symptoms related to pain and inflammation in the airway and gastrointestinal tract, but the underlying mechanisms are still unclear. TRPA1 is a nonselective cation channel expressed in sensory neurons and acts as a nociceptive receptor. Some metal ions such as Ca, Mg, Ba and Zn are reported to modulate TRPA1 channel activity. In the present study, we investigated the effect of Cd on cultured mouse dorsal root ganglion neurons and a heterologous expression system to analyze the effect of Cd at the molecular level. In addition, we examined whether Cd caused acute pain in vivo. Results: In wild-type mouse sensory neurons, Cd evoked an elevation of the intracellular Ca concentration ([Ca2+]i) that was inhibited by external Ca removal and TRPA1 blockers. Most of the Cd-sensitive neurons were also sensitive to cinnamaldehyde (a TRPA1 agonist) and [Ca2+]i responses to Cd were absent in TRPA1(-/-) mouse neurons. Heterologous expression of TRPA1 mutant channels that were less sensitive to Zn showed attenuation of Cd sensitivity. Intracellular Cd imaging revealed that Cd entered sensory neurons through TRPA1. The stimulatory effects of Cd were confirmed in TRPA1-expressing rat pancreatic cancer cells (RIN-14B). Intraplantar injection of Cd induced pain-related behaviors that were largely attenuated in TRPA1(-/-) mice. Conclusions: Cd excites sensory neurons via activation of TRPA1 and causes acute pain, the mechanism of which may be similar to that of Zn. The present results indicate that TRPA1 is involved in the nociceptive or inflammatory effects of Cd. [ABSTRACT FROM AUTHOR]

Abstract: The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. The β3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in β3-null mice to determine the role of the β3 subunit in catecholamine release from the adrenal chromaffin system. Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The β3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in β3-null mice, while patch–clamp recordings showed a significant reduction in Ca2+-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production. In conclusion, our results indicate the importance of the β3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells. [Copyright &y& Elsevier]

Transient receptor potential V1 (TRPV1) is a nonselective cation channel expressed in nociceptors and activated by capsaicin. TRPV1 detects diverse stimuli, including acid, heat, and endogenous vanilloids, and functions as a molecular integrator of pain perception. Herein we demonstrate a novel regulatory role of extracellular Na+ ([Na+]o) on TRPV1 function. Inhuman embryonic kidney 293 cells expressing porcine TRPV1, low [Na+]o evoked increases of [Ca2+]i, that were suppressed by TRPV1 antagonists and facilitated responses to capsaicin, protons, heat, and an endovanilloid. [Na+]o removal simultaneously elicited a [Ca2+]i increase and outward-rectified current with a reversal potential similar to those of capsaicin. Neutralization of the two acidic residues which confer the proton sensitivity to TRPV1 resulted in a reduction of low [Na+]o-induced responses. In primary culture of porcine sensory neurons, the removal of [Na+]o produced a [Ca2+]i increase and current responses only in the cells responding to capsaicin. Low [Na+]o evoked a [Ca2+]i increase in sensory neurons of wild type mice, but not TRPV1-null mice, and in human embryonic kidney 293 cells expressing human TRPV1. The present results suggest that [Na+]o negatively regulates the gating and polymodal sensitization of the TRPV1 channel. [Na+]o surrounding several proton-sensitive sites on the extracellular side of the pore-forming loop of the TRPV1 channel may play an important role as a brake to suppress the excessive activity of this channel under physiological conditions. [ABSTRACT FROM AUTHOR]

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP ⩾ adenosine 5'-O-3-triphosphate ⩾ CTP ⩾ 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the endogenous control through vesicular contents of voltage-dependent Ca2+ channels (VDCCs) in cultured porcine adrenal chromaffin cells. To examine paracrine regulation of VDCCs, catecholamine release was monitored amperometrically together with patch-clamp recording under culture conditions at different cell densities. A depolarizing pulse evoked Ca2+- (ICa) and Ba2+-currents (IBa) in Ca2+- and Ba2+-containing solutions, respectively. In cells cultured at high density, stop-flow of the external solution decreased the IBa concomitant with a sustained increase of amperometric current (Iamp), but not in cells at low density, suggesting the endogenous modulation of VDCCs in a paracine fashion. The degree of the prepulse facilitation was similar regardless of the flow condition. Application of noradrenaline (NA), ATP, methionine–enkephalin (ENK) or protons decreased IBa. The extent of the prepulse facilitation of the endogenous VDCC inhibition was similar to those induced by NA and ATP. GDPβS, pertussis toxin (PTX), blockers for α-adrenoceptors and P2-purinoceptors significantly reduced the endogenous VDCC inhibition. These results suggest that VDCCs are regulated by vesicular substances in a paracrine fashion, at least by noradrenaline and ATP, through activation of α-adrenoceptors and P2-purinoceptors, respectively, in porcine adrenal chromaffin cells. [Copyright &y& Elsevier]

The involvement of mitochondrial Na+–Ca2+ exchange in Ca2+ responses to ATP was examined in rat pheochromocytoma (PC) 12 cells. Intracellular Ca2+ ([Ca2+]i) and Na+ concentrations ([Na+]i) were measured using fura-2 and SBFI, respectively. ATP caused concentration-dependent increases in [Ca2+]i and [Na+]i. High concentrations of ATP elicited a Ca2+ transient followed by a slow recovery of [Ca2+]i (a sustained phase) in 77% of PC12 cells. The sustained phase of Ca2+ response appeared only when the peak Ca2+ transient exceeded 500 nM. FCCP, a protonophore, greatly enhanced Ca2+ responses to ATP only in cells with the sustained phase but not without this phase. The sustained phase was decreased by clonazepam and CGP37157, mitochondrial Na+–Ca2+ exchange inhibitors, and extracellular Na+ removal but not by cyclosporin A, an inhibitor of permeability transition pores. The reintroduction of Na+ 3.5 min after ATP stimulation in the absence of Na+ caused Na+ concentration-dependent increases in [Ca2+]i and [Na+]i. The increase in [Na+]i was correlated with that in [Ca2+]i. FCCP caused a great increase in [Ca2+]i 4.5 min after ATP stimulation in the absence of extracellular Na+ but not in its presence, indicating that mitochondria retain Ca2+ in the absence of Na+. These results suggest that ATP causes a large increase in [Ca2+]i which was sequestered in mitochondria and that the sustained phase of Ca2+ response to ATP are mainly due to the release of mitochondrial Ca2+ through Na+–Ca2+ exchangers in PC12 cells. [Copyright &y& Elsevier]

Mammalian homologues of the Drosophila canonical transient receptor potential (trp) protein (TRPC) have been implicated to function as receptor-operated Ca2+ channels (ROCs) or store-operated Ca2+ channels (SOCs). To determine the role of TRPC5 protein in neural cells, TRPC5 was recombinantly expressed in rat pheochromocytoma cells (PC12) and changes in intracellular Ca2+ concentration ([Ca2+]i) and Na+ concentration ([Na+]i) were analyzed. TRPC1 and TRPC3 mRNAs were endogenously expressed in PC12 cells. In TRPC5-expressed cells (TRPC5-cells), the resting [Ca2+]i and [Na+]i were significantly higher than those in control cells. The [Ca2+]i increases induced by bradykinin and uridine 5′-triphosphate were significantly larger in TRPC5-cells. TRPC5 expression did not change in store-operated Ca2+ entry elicited by thapsigarigin. TRPC5-cells showed larger inward current and increase of [Na+]i in response to BK than control cells. These results suggest that TRPC5 channels expressed in PC12 cells function as ROCs activated by G-protein/phospholipase C coupled receptors, but not as SOCs. [Copyright &y& Elsevier]

We investigated the effects of dexamethasone, cortisol and aldosterone on responses to nicotine and muscarine in guinea-pig isolated adrenal medullary cells. Nicotine-induced inward currents were reversibly inhibited by these steroids in a dose-dependent and non-competitive manner. These steroids inhibited an increase in [Ca2+]i in response to nicotine but not muscarine. Muscarine-induced catecholamine secretion was inhibited by cortisol and aldosterone but not dexamethasone. Phorbol 12-myristate 13-acetate, a protein kinase C activator, caused catecholamine secretion which was inhibited by cortisol and aldosterone but not dexamethasone. These results suggest that catecholamine secretion induced by cholinoceptor stimulation is inhibited by steroids via two distinct mechanisms; one is the inhibition of nicotinic receptors, another is the inhibition of protein kinase C activation in guinea-pig adrenal medullary cells. [Copyright &y& Elsevier]

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H 2 O 2 and investigated the roles of protein kinase C δ (PKCδ) and Ca 2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H 2 O 2 caused the accumulation of p53, which was inhibited by rottlerin (a PKCδ inhibitor) but not by BAPTA-AM (an intracellular Ca 2+ chelator). PKCδ itself was activated through the phosphorylation at tyrosine residues. H 2 O 2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKCδ contributes to the accumulation of p53 and that Ca 2+ plays a role in downstream signals of p53 leading to apoptosis in H 2 O 2 -treated BAEC. [ABSTRACT FROM AUTHOR]

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55°C, respectively. The enzyme acted on 3β-hydroxysteroids such as cholesterol, pregnenolone, and β-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (Km=30 μM). [Copyright &y& Elsevier]