Pflügers Archiv: European Journal of Physiology, Jun 1, 2008
Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK ... more Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK Ca channel alpha1-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside-out, excised membrane patches. Activation by CO was concentration dependent (EC50 approximately 20 microM), rapid, reversible, and evoked a shift in the V 0.5 of -20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca2+]i in the physiological range (100-1,000 nM). Importantly, CO "super-stimulated" BK Ca activity even in saturating [Ca2+]i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9-S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9-S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK Ca channel by redox-independent changes in gating.
... Strains of the wood-decay basidiomycetes T. versicolor (CvD2), S. gausapatum (Sg1), H. fascic... more ... Strains of the wood-decay basidiomycetes T. versicolor (CvD2), S. gausapatum (Sg1), H. fasciculare (HfGTWV2) and B. adusta (Bk1), from Cardiff University culture collection, were maintained on 0.5 % (w/v) malt agar (MA; 5 g Munton & Fison spray malt, 15 g Lab M agar no. ...
Competition between mycelia of saprotrophic cord-forming basidiomycetes occurs both within dead w... more Competition between mycelia of saprotrophic cord-forming basidiomycetes occurs both within dead woody resources and in the soil-litter interface, and involves a variety of antagonistic mechanisms including the production of volatile organic compounds (VOCs). The antagonistic potential of VOC profiles from interactions in wood blocks and in soil microcosms was assessed using shared headspace experiments, and the profile of VOCs emitted over the course of interactions elucidated using solid phase microextraction (SPME) with gas chromatography-mass spectrometry (GC–MS). Quantitative and qualitative changes in VOC production occurred in interactions compared to self-pairing controls, with different VOC profiles from fungi growing in wood blocks compared to soil trays. There were both stimulatory and inhibitory effects of VOCs on target mycelial extension rate, hyphal coverage and fractal dimension. VOC-mediated effects were greater in self-pairing controls compared to interactions, and differed depending on the substratum in which the VOC-producing fungi were growing.
The mycelia of two wood decay basidiomycete fungi were grown opposing each other across a 1-micro... more The mycelia of two wood decay basidiomycete fungi were grown opposing each other across a 1-microm pore membrane supported on the surface of malt broth, contained within a sealable reaction vessel. Production of volatiles during the time course of interaction was followed by collecting head space samples by solid phase microextraction (100 microm polydimethylsiloxane fiber) on five occasions over 25 d following coinoculation of the fungi: 1, 3 (i.e., immediately prior to mycelial contact), 9 (1-2 d after initiation of pigment production by Resinicium bicolor), 17, and 25 d. Ten volatiles were produced during interactions that were not detected in single species controls. In general, most (18) fungal volatiles were sesquiterpenes eluted between 12.5 and 21 min, with a further two eluted at 29.1 and 33.9 min; a benzoic acid methyl ester, a benzyl alcohol, and a quinolinium type compound with a distinctive fragmentation pattern at m/z 203, 204, 206, and 207 were also identified; three volatiles with m/z maxima of 163, 159, and 206-208, respectively, remained unidentified. The results are discussed in relation to possible ecological roles of volatiles.
Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin ... more Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations confirmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence.
Nutrient-specific foraging is the ecological theory that generalist consumers select food resourc... more Nutrient-specific foraging is the ecological theory that generalist consumers select food resources based on their nutritional content. While laboratory experiments support this, it has not yet been demonstrated in invertebrate predators in the field. We combined dietary metabarcoding with prey abundance and macronutrient content data to analyze nutrient-specific foraging in the field. Spider nutrient intake and prey choice deviated from random. Through a novel nutrient-based taxonomy and null modelling, we reveal a stable average macronutrient intake and disproportionate foraging for different macronutrients by individual spiders. This aligns with the expectation that individual spiders at different stages of nutrient balancing will be biased toward prey rich in different nutrients. This finding suggests that spiders are redressing nutritional deficits to obtain a target nutrient intake, as expected of nutrient-specific foraging. This evidence for nutrient-specific foraging under field conditions significantly extends our understanding beyond lab-based behavioral assays to resolve complex real-world systems.
Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin ... more Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations confirmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence.
IntroductionStrawberry fruit are highly valued for their aroma which develops during ripening. Ho... more IntroductionStrawberry fruit are highly valued for their aroma which develops during ripening. However, they have a short shelf-life. Low temperature storage is routinely used to extend shelf-life for transport and storage in the supply chain, however cold storage can also affect fruit aroma. Some fruit continue to ripen during chilled storage; however, strawberries are a non-climacteric fruit and hence ripening postharvest is limited. Although most strawberry fruit is sold whole, halved fruit is also used in ready to eat fresh fruit salads which are of increasing consumer demand and pose additional challenges to fresh fruit storage.MethodsTo better understand the effects of cold storage, volatilomic and transcriptomic analyses were applied to halved Fragaria x ananassa cv. Elsanta fruit stored at 4 or 8°C for up to 12 days over two growing seasons.Results and discussionThe volatile organic compound (VOC) profile differed between 4 or 8°C on most days of storage. Major differences w...
IntroductionPeach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch,... more IntroductionPeach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch, var nectarine), are characterized by a rapid deterioration at room temperature. Therefore, cold storage is widely used to delay fruit post-harvest ripening and extend fruit commercial life. Physiological disorders, collectively known as chilling injury, can develop typically after 3 weeks of low-temperature storage and affect fruit quality.MethodsA comparative transcriptomic analysis was performed to identify regulatory pathways that develop before chilling injury symptoms are detectable using next generation sequencing on the fruits of two contrasting cultivars, one peach (Sagittaria) and one nectarine, (Big Top), over 14 days of postharvest cold storage.ResultsThere was a progressive increase in the number of differentially expressed genes between time points (DEGs) in both cultivars. More (1264) time point DEGs were identified in ‘Big Top’ compared to ‘Sagittaria’ (746 DEGs). Both cu...
Pflügers Archiv: European Journal of Physiology, Jun 1, 2008
Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK ... more Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK Ca channel alpha1-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside-out, excised membrane patches. Activation by CO was concentration dependent (EC50 approximately 20 microM), rapid, reversible, and evoked a shift in the V 0.5 of -20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca2+]i in the physiological range (100-1,000 nM). Importantly, CO "super-stimulated" BK Ca activity even in saturating [Ca2+]i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9-S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9-S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK Ca channel by redox-independent changes in gating.
... Strains of the wood-decay basidiomycetes T. versicolor (CvD2), S. gausapatum (Sg1), H. fascic... more ... Strains of the wood-decay basidiomycetes T. versicolor (CvD2), S. gausapatum (Sg1), H. fasciculare (HfGTWV2) and B. adusta (Bk1), from Cardiff University culture collection, were maintained on 0.5 % (w/v) malt agar (MA; 5 g Munton & Fison spray malt, 15 g Lab M agar no. ...
Competition between mycelia of saprotrophic cord-forming basidiomycetes occurs both within dead w... more Competition between mycelia of saprotrophic cord-forming basidiomycetes occurs both within dead woody resources and in the soil-litter interface, and involves a variety of antagonistic mechanisms including the production of volatile organic compounds (VOCs). The antagonistic potential of VOC profiles from interactions in wood blocks and in soil microcosms was assessed using shared headspace experiments, and the profile of VOCs emitted over the course of interactions elucidated using solid phase microextraction (SPME) with gas chromatography-mass spectrometry (GC–MS). Quantitative and qualitative changes in VOC production occurred in interactions compared to self-pairing controls, with different VOC profiles from fungi growing in wood blocks compared to soil trays. There were both stimulatory and inhibitory effects of VOCs on target mycelial extension rate, hyphal coverage and fractal dimension. VOC-mediated effects were greater in self-pairing controls compared to interactions, and differed depending on the substratum in which the VOC-producing fungi were growing.
The mycelia of two wood decay basidiomycete fungi were grown opposing each other across a 1-micro... more The mycelia of two wood decay basidiomycete fungi were grown opposing each other across a 1-microm pore membrane supported on the surface of malt broth, contained within a sealable reaction vessel. Production of volatiles during the time course of interaction was followed by collecting head space samples by solid phase microextraction (100 microm polydimethylsiloxane fiber) on five occasions over 25 d following coinoculation of the fungi: 1, 3 (i.e., immediately prior to mycelial contact), 9 (1-2 d after initiation of pigment production by Resinicium bicolor), 17, and 25 d. Ten volatiles were produced during interactions that were not detected in single species controls. In general, most (18) fungal volatiles were sesquiterpenes eluted between 12.5 and 21 min, with a further two eluted at 29.1 and 33.9 min; a benzoic acid methyl ester, a benzyl alcohol, and a quinolinium type compound with a distinctive fragmentation pattern at m/z 203, 204, 206, and 207 were also identified; three volatiles with m/z maxima of 163, 159, and 206-208, respectively, remained unidentified. The results are discussed in relation to possible ecological roles of volatiles.
Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin ... more Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations confirmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence.
Nutrient-specific foraging is the ecological theory that generalist consumers select food resourc... more Nutrient-specific foraging is the ecological theory that generalist consumers select food resources based on their nutritional content. While laboratory experiments support this, it has not yet been demonstrated in invertebrate predators in the field. We combined dietary metabarcoding with prey abundance and macronutrient content data to analyze nutrient-specific foraging in the field. Spider nutrient intake and prey choice deviated from random. Through a novel nutrient-based taxonomy and null modelling, we reveal a stable average macronutrient intake and disproportionate foraging for different macronutrients by individual spiders. This aligns with the expectation that individual spiders at different stages of nutrient balancing will be biased toward prey rich in different nutrients. This finding suggests that spiders are redressing nutritional deficits to obtain a target nutrient intake, as expected of nutrient-specific foraging. This evidence for nutrient-specific foraging under field conditions significantly extends our understanding beyond lab-based behavioral assays to resolve complex real-world systems.
Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin ... more Immunofluorescence labeling on postembedded tissue in resin is a formidable task. Although resin components and stabilizers are a source of additional strong native fluorescence that overlaps with absorption and emission spectra of commonly used green fluorophores, the unfixed tissue is also subject to native fluorescence. For tissue embedded in resin, we hypothesized that initially removing the resin and subsequently quenching the native fluorescence from the sample could result in specific immunofluorescence signals. The hypothesis was tested on fixed tissue samples embedded in Technovit 9100 New. Deacrylated and rehydrated semithin sections from a variety of soft tissues were exposed to a quenching solution prior to immunolabeling. Cryostat sections from snap frozen tissue were also stained to assess whether all antigens investigated in fixed tissue were adequately detected. The secondary detection included antibodies conjugated with fluorescein isothiocyanate. The results were evaluated using conventional dark-field and confocal laser scanning microscopy. Both forms of microscopy confirmed the considerable lowering of the native fluorescence associated with the resin and fixed tissue samples with enhanced specific signal. The cryostat tissue sections using the same antibodies in equivalent concentrations confirmed labeling of the same cellular sites as those observed in the fixed tissue. This article describes a method for immunofluorescence labeling in Technovit 9100 New resin embedded tissue and suggests the likely chromogenic elements generating autofluorescence.
IntroductionStrawberry fruit are highly valued for their aroma which develops during ripening. Ho... more IntroductionStrawberry fruit are highly valued for their aroma which develops during ripening. However, they have a short shelf-life. Low temperature storage is routinely used to extend shelf-life for transport and storage in the supply chain, however cold storage can also affect fruit aroma. Some fruit continue to ripen during chilled storage; however, strawberries are a non-climacteric fruit and hence ripening postharvest is limited. Although most strawberry fruit is sold whole, halved fruit is also used in ready to eat fresh fruit salads which are of increasing consumer demand and pose additional challenges to fresh fruit storage.MethodsTo better understand the effects of cold storage, volatilomic and transcriptomic analyses were applied to halved Fragaria x ananassa cv. Elsanta fruit stored at 4 or 8°C for up to 12 days over two growing seasons.Results and discussionThe volatile organic compound (VOC) profile differed between 4 or 8°C on most days of storage. Major differences w...
IntroductionPeach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch,... more IntroductionPeach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch, var nectarine), are characterized by a rapid deterioration at room temperature. Therefore, cold storage is widely used to delay fruit post-harvest ripening and extend fruit commercial life. Physiological disorders, collectively known as chilling injury, can develop typically after 3 weeks of low-temperature storage and affect fruit quality.MethodsA comparative transcriptomic analysis was performed to identify regulatory pathways that develop before chilling injury symptoms are detectable using next generation sequencing on the fruits of two contrasting cultivars, one peach (Sagittaria) and one nectarine, (Big Top), over 14 days of postharvest cold storage.ResultsThere was a progressive increase in the number of differentially expressed genes between time points (DEGs) in both cultivars. More (1264) time point DEGs were identified in ‘Big Top’ compared to ‘Sagittaria’ (746 DEGs). Both cu...
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