Effects of fixatives on stable isotopes of fish muscle tissue: implications for trophic studies on preserved specimens.

Isotopic ecology has been widely used to understand spatial connectivity and trophic interactions in marine systems. However, its potential for monitoring an ecosystem's health and function has been hampered by the lack of consistent sample storage and long-term studies. Preserved specimens from museum collections are a valuable source of tissue for analyses from ancient and pre-modern times, but isotopic signatures are known to be affected by commonly used fixatives. The aim of the present study was to understand the effects of fixatives on isotopic signatures of bulk tissue (δ13 Cm and δ15 Nm ) and amino acids (δ13 CAA and δ15 NAA ) of fish muscle and to provide correction equations for the isotopic shifts. Two specimens of each: blue cod (Parapercis colias), blue warehou (Seriolella brama), and king salmon (Oncorhynchus tschawytscha) were sampled at five locations along their dorsal musculature, at four time periods: (1) fresh, (2) after 1 month preserved in formalin, and after (3) 3 and (4) 12 months fixed in either ethanol or isopropanol. Lipid content was positively correlated with C:N ratio (r² = 0.83) and had a significant effect on δ13 C after treatments, but not on δ15 N. C:N ratio (for δ13 Cm ) and percent N (for δ15 Nm ) from preserved specimens contributed to the most parsimonious mixed models, which explained 79% of the variation due to fixation and preservation for δ13 C and 81% for δ15 N. δ13 CAA were generally not affected by fixatives and preservatives, while most δ15 NAA showed different signatures between treatments. δ15 NAA variations did not affect the magnitude of differences between amino acids, allowing scientists to retrieve ecological information (e.g., trophic level) independently of time under preservation. Corrections were applied to the raw data of the experiment, highlighting the importance of δ13 Cm and δ15 Nm correction when fish muscle tissues from wet collections are compared to fresh samples. Our results make it possible to retrieve δ13 Cm , δ15 Nm , δ13 CAA , and δ15 NAA from museum specimens and can be applied to some of the fundamental questions in ecology, such as trophic baseline shifts and changes in community's food web structure through time.

Discrimination of honey of different floral origins by a combination of various chemical parameters.

Honey is a high value food commodity with recognized nutraceutical properties. A primary driver of the value of honey is its floral origin. The feasibility of applying multivariate data analysis to various chemical parameters for the discrimination of honeys was explored. This approach was applied to four authentic honeys with different floral origins (rata, kamahi, clover and manuka) obtained from producers in New Zealand. Results from elemental profiling, stable isotope analysis, metabolomics (UPLC-QToF MS), and NIR, FT-IR, and Raman spectroscopic fingerprinting were analyzed. Orthogonal partial least square discriminant analysis (OPLS-DA) was used to determine which technique or combination of techniques provided the best classification and prediction abilities. Good prediction values were achieved using metabolite data (for all four honeys, Q(2)=0.52; for manuka and clover, Q(2)=0.76) and the trace element/isotopic data (for manuka and clover, Q(2)=0.65), while the other chemical parameters showed promise when combined (for manuka and clover, Q(2)=0.43).

Sea ice microbial production supports Ross Sea benthic communities: influence of a small but stable subsidy.

Diversity in guilds of primary producers enhances temporal stability in provision of organic matter to consumers. In the Antarctic ecosystem, where temporal variability in phytoplankton production is high, sea ice contains a diatom and microbial community (SIMCO) that represents a pool of organic matter that is seasonally more consistent, although of relatively small magnitude. The fate of organic material produced by SIMCO in Antarctica is largely unknown but may represent an important link between sea ice dynamics and secondary production in nearshore food webs. We used whole tissue and compound-specific stable isotope analysis of consumers to test whether the sea ice microbial community is an important source of organic matter supporting nearshore communities in the Ross Sea. We found distinct gradients in delta13C and delta15N of SIMCO corresponding to differences in inorganic carbon and nitrogen acquisition among sites with different sea ice extent and persistence. Mass balance analysis of a suite of consumers demonstrated large fluxes of SIMCO into the nearshore food web, ranging from 5% to 100% of organic matter supplied to benthic species, and 0-10% of organic matter to upper water column or pelagic inhabitants. A delta13C analysis of nine fatty acids including two key biomarkers for diatoms, eicosapentaenoic acid (EPA, 20:5omega3), and docosahexaenoic acid (DHA, 22:6omega3), confirmed these patterns. We observed clear patterns in delta13C of fatty acids that are enriched in 13C for species that acquire a large fraction of their nutrition from SIMCO. These data demonstrate the key role of SIMCO in ecosystem functioning in Antarctica and strong linkages between sea ice extent and nearshore secondary productivity. While SIMCO provides a stabilizing subsidy of organic matter, changes to sea ice coverage associated with climate change would directly affect secondary production and stability of benthic food webs in Antarctica.

Muscle glycogen oxidation during prolonged exercise measured with oral [13C]glucose: comparison with changes in muscle glycogen content.

Plasma glucose and muscle glycogen oxidation during prolonged exercise [75-min at 48 and 76% maximal O(2) uptake (Vo(2 max))] were measured in eight well-trained male subjects [Vo(2 max) = 4.50 l/min (SD 0.63)] using a simplified tracer technique in which a small amount of glucose highly enriched in (13)C was ingested: plasma glucose oxidation was computed from (13)C/(12)C in plasma glucose (which was stable beginning at minute 30 and minute 15 during exercise at 48 and 76% Vo(2 max), respectively) and (13)CO(2) production, and muscle glycogen oxidation was estimated by subtracting plasma glucose oxidation from total carbohydrate oxidation. Consistent data from the literature suggest that this small dose of exogenous glucose does not modify muscle glycogen oxidation and has little effect, if any, on plasma glucose oxidation. The percent contributions of plasma glucose and muscle glycogen oxidation to the energy yield at 48% Vo(2 max) [15.1% (SD 3.8) and 45.9% (SD 5.8)] and at 76% Vo(2 max) [15.4% (SD 3.6) and 59.8% (SD 9.2)] were well in line with data previously reported for similar work loads and exercise durations using conventional tracer techniques. The significant reduction in glycogen concentration measured from pre- and postexercise vastus lateralis muscle biopsies paralleled muscle glycogen oxidation calculated using the tracer technique and was larger at 76% than at 48% Vo(2 max). However, the correlation coefficients between these two estimates of muscle glycogen utilization were not different from zero at each of the two work loads. The simplified tracer technique used in the present experiment appears to be a valid alternative approach to the traditional tracer techniques for computing plasma glucose and muscle glycogen oxidation during prolonged exercise.

A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization.

Changes in iron supply to oceanic plankton are thought to have a significant effect on concentrations of atmospheric carbon dioxide by altering rates of carbon sequestration, a theory known as the 'iron hypothesis'. For this reason, it is important to understand the response of pelagic biota to increased iron supply. Here we report the results of a mesoscale iron fertilization experiment in the polar Southern Ocean, where the potential to sequester iron-elevated algal carbon is probably greatest. Increased iron supply led to elevated phytoplankton biomass and rates of photosynthesis in surface waters, causing a large drawdown of carbon dioxide and macronutrients, and elevated dimethyl sulphide levels after 13 days. This drawdown was mostly due to the proliferation of diatom stocks. But downward export of biogenic carbon was not increased. Moreover, satellite observations of this massive bloom 30 days later, suggest that a sufficient proportion of the added iron was retained in surface waters. Our findings demonstrate that iron supply controls phytoplankton growth and community composition during summer in these polar Southern Ocean waters, but the fate of algal carbon remains unknown and depends on the interplay between the processes controlling export, remineralisation and timescales of water mass subduction.

Effects of bis(2-chloroethyl)sulfide on ATP receptor-mediated responses of the rat vas deferens: possible relationship to cytotoxicity.

Extracellular ATP is a broad-spectrum cytotoxic agent that produces effects via cell surface P2 purinoceptors. The ligand-gated P2X purinoceptor subtype has very high sequence homology with the RP-2 gene, which encodes for apoptosis. The P2X RNA found in rat vas deferens is expressed preferentially by apoptotic thymocytes. P2X purinoceptor-mediated phasic (twitch) motor responses of the isolated rat vas deferens to neurogenic or exogenous ATP were rapidly, specifically and irreversibly potentiated by bis(2-chloroethyl)sulfide (HD 10-100 microM). Both untreated and HD-potentiated neurogenic responses were Ca++ dependent, blocked in the absence of Ca++ plus 0.1 mM EGTA, by the neuronal Ca++ channel blocker omega-conotoxin-MVIIC (3 microM), by the P2 purinoceptor antagonist suramin (100 microM) and by tetrodotoxin (100 nM). HD also potentiated the effects of ATP on isolated guinea pig taenia caecum, where the nucleotide acts at G protein-coupled P2Y purinoceptor subtypes to cause relaxation. HD failed to inhibit the metabolism of ATP by ecto-ATPase in vas deferens or to cause the release of endogenous ATP. Potentiation of the twitch response to electric field stimulation by HD was attenuated or eliminated in tissues excised from rats previously challenged with topically applied HD, suggesting that HD absorbed into the systemic circulation had already effected maximal potentiation of ATP responses before in vitro testing. The physiological consequences of HD-induced potentiation of the extracellular actions of ATP are discussed in relation to apoptosis and necrosis.

A role for Q type Ca2+ channels in neurotransmission in the rat urinary bladder.

1. In isolated bladder strips of the rat, a substantial component (46%) of the Ca(2+)-dependent contractile response to electrical field stimulation (5 Hz) was resistant to combined block of both N and P type Ca2+ channels by omega-conotoxin-GVIA (300 nM) and omega-agatoxin-IVA (100 nM) respectively. 2. The resistant portion (non-N, non-P) was sensitive to omega-conotoxin-MVIIC (3 microM), which in addition to N and P also blocks Q type channels at this concentration. omega-Conotoxin-MVIIC administered alone, inhibited the neurogenic response to the same degree as that observed in the combined presence of omega-agatoxin-IVA, omega-conotoxin-GVIA and omega-conotoxin-MVIIC. 3. omega-Agatoxin-IVA (100 nM), a concentration that fully inhibits P type channels, had a negligible effect on the neurogenic response. Following blockade of N type Ca2+ channels with omega-conotoxin-GVIA (300 nM), omega-agatoxin-IVA (3 microM) (a concentration well above that used to block P channels, inhibits Q type channels, but spares N type channels), inhibited the residual response to the same degree as omega-conotoxin-MVIIC alone. 4. Results suggest that neurotransmission in rat urinary bladder is supported by both N and Q type Ca2+ channels.

Effect of omega-agatoxin-IVA on autonomic neurotransmission.

omega-Agatoxin-IVA, a peptide from the venom of the funnel-web spider Agelenopsis aperta and a P type Ca2+ channel inhibitor, was examined for effects on responses to nerve stimulation in isolated autonomic neuroeffector preparations from the rabbit, guinea-pig and rat. Ca(2+)-dependent, tetrodotoxin sensitive, noradrenergic excitatory responses of rabbit pulmonary artery, rat vas deferens, and anococcygeus muscles, and cholinergic guinea-pig myenteric plexus preparations (all highly sensitive to the N type Ca2+ channel inhibitor omega-conotoxin-GVIA) were unaffected by omega-agatoxin-IVA (100 nM). Similarly, the neurogenic response of rat bladder, which has cholinergic, and non-adrenergic non-cholinergic (NANC) excitatory components, and the NANC inhibitory response of rat jejunum (atropine 0.5 microM- and guanethidine 5.0 microM-treated), which are partially sensitive and insensitive to omega-conotoxin-GVIA, respectively, were unaffected by omega-agatoxin-IVA (100 nM). Neurogenic NANC inhibitory responses of the guinea-pig taenia caecum, and rat anococcygeus muscles (atropine- and guanethidine-treated, and tone raised with prostaglandin F2 alpha), were also insensitive to omega-agatoxin-IVA. These results suggest that P type Ca2+ channels, if present, play an insignificant role in supplying the Ca2+ necessary for neurotransmitter release in the peripheral autonomic nervous system.

Pharmacological identification of a novel Ca2+ channel in chicken brain synaptosomes.

Ca2+ influx was measured in rat and chicken brain synaptosomes in the presence of a number of pharmacological tools which have recently been used to define voltage-sensitive Ca(2+)-channel (VSCC) types. In chicken brain synaptosomes. VSCCs which, because of their sensitivity to inhibition by omega-conotoxin (omega-CgTx), are thought to be exclusively N-type, the P-type VSCC polyamine inhibitor FTX (from Agelenopsis aperta venom; 1 microliters/ml), its synthetic analogue, sFTX (1-5 mM) and the polypeptides AgaIVA (IC50 0.29 microM) and omega-CgTx MVIIC (IC50 0.0022 microM) inhibited 70-100% of the measurable K+ stimulated Ca2+ influx. The prototypical N-channel VSCC inhibitor omega-CgTx GVIA (IC50 0.014 microM), Cd2+ (50 microM) and diluted venom from Hololena curta (1:2,500) also caused complete or almost complete, inhibition of Ca2+ influx. In comparable studies using rat brain synaptosomes, sFTX (1-10 mM) caused a dose-dependent reduction of Ca2+ influx, while FTX (1 microliters/ml) and AgaIVA (IC50 0.02 microM) completely inhibited Ca2+ influx. Similar to the findings in chicken synaptosomes, Cd2+ (50 microM) and H. curta (1:2,500 dilution) both inhibited K+ stimulated influx by > 80% whereas omega-CgTx (1 microM) only caused a maximum 25% inhibition. Both sFTX and its congener spermine, inhibited [125I]omega-CgTx binding to rat and chicken synaptosomal membranes. These results strongly implicate P-type channels as the major VSCC in rat brain. The results also clearly demonstrate a heretofore unrecognized, novel, FTX/AgaIVA/omega-CgTx GVIA/omega-CgTx MVIIC-sensitive VSCC in chicken brain.

Identification of noradrenaline in venom from the funnel-web spider Hololena curta.

Venom from the funnel-web spider Hololena curta was added to the Krebs-Henseleit solution bathing isolated ring preparations of rat thoracic aorta, suspended in water-jacketed organ baths, for the purpose of tension recordings. Hololena curta venom at dilutions of 1:100,000 to 1:1000 caused a marked vasoconstriction, which was completely inhibited by the alpha 1 adrenoceptor antagonist prazosin (1 microm). The vasoconstriction appears to be due to the direct effects on alpha 1 adrenoceptors of a venom constituent, which we have identified using HPLC/ECD as the catecholamine, noradrenaline.

Evaluation of Guamanian dental caries preventive programs after 13 years.

OBJECTIVE: To address the high prevalence of dental caries in Guamanian children, a school-based fluoride mouthrinse program, a clinic-based pit and fissure sealant program, and community water fluoridation were phased in over a 13-year period. The purpose of this paper is to evaluate the impact of these programs on the prevalence of dental caries. METHODS: Cross-sectional samples representative of schoolchildren in grades 1 through 8 were selected in 1976, 1979, 1984, 1986, and 1989. These years include the starting years for each of the three interventions and varying follow-up periods for each. About 1,000 children in each of the survey years were examined to derive DMF surface scores. RESULTS: After eight years of fluoride mouthrinsing, DMFS scores were reduced by 25.4 percent in 6-14-year-olds. With two additional years of fluoride mouthrinsing and with pit and fissure sealants, overall DMFS scores declined an additional 44.4 percent. In 1989, three years after community water fluoridation was initiated on the island and continuation of the other two programs, there was a further decline in overall DMFS scores of 34.5 percent. Over the entire study period--during which there were 13 years of fluoride mouthrinsing in the schools, five years of sealant application, and three years of community water fluoridation--DMFS scores declined 72.8 percent overall (5.14 surfaces per child) and 71.9, 71.0, and 78.8 percent for occlusal, buccal-lingual, and proximal surfaces, respectively. CONCLUSIONS: An intensified preventive dentistry program introduced on an island with high caries prevalence twice that of the US mainland was successful, and contributed to a reduction in the prevalence of caries to a level equivalent to that of the United States at the end of the study period.

Evidence of mammalian Ca2+ channel inhibitors in venom of the spider Plectreurys tristis.

Plectreurys tristis venom inhibited K(+)-stimulated Ca2+ influx in a concentration-dependent manner in rat (0.5-4.0 micrograms venom protein/ml) and chicken (1.0-64.0 micrograms venom protein/ml) brain synaptosomes. In contrast to Hololena curta venom or omega conotoxin GVlA which both show selectivity for avian synaptosomes, inhibition of Ca2+ influx by the venom appeared to be relatively selective for rat synaptosomes. Plectreurys tristis venom also inhibited K(+)-evoked release of [3H](-)-noradrenaline from labeled rat cortical synaptosomes. Responses to electric field stimulation of the sympathetically innervated rat vas deferens in vitro were inhibited by Plectreurys tristis venom at dilutions similar to those which inhibited Ca2+ influx in synaptosomes. Inhibition persisted following washout of the venom. K(+)-evoked contractions of rat aortic rings were relaxed by the dihydropyridine antagonist (-)-202-791, but not by Plectreurys tristis venom, thus precluding an effect on K(+)-depolarized smooth muscle L-type channels. Contractions to exogenous (-)-noradrenaline in rat aorta were not inhibited by Plectreurys tristis venom, ruling out an effect on alpha 1-adrenergic receptors, and further suggesting a prejunctional site of action. The results suggest that this venom inhibits N-type Ca2+ channels, as well as unclassified Ca2+ channels, which are neither N- nor L-type.

Pharmacological characterization of voltage-sensitive Ca2+ channels in autonomic nerves.

Hololena curta venom a potent inhibitor of voltage sensitive Ca2+ channels and neurotransmitter release in mammalian brain, and synthetic funnel web spider toxin an inhibitor of P channels, were examined for their activity on autonomic nerves. Hololena curta (0.5 to 5.0 micrograms venom protein/ml) potently inhibited motor responses of the cholinergic guinea pig ileum myenteric plexus and the adrenergic rat anococcygeus muscle. Synthetic funnel web spider toxin was inactive at concentrations up to 100 microM. Hololena curta inhibited K+, and electrically evoked release of tritium from labeled superfused tissues. Furthermore, K(+)-contracted rat aorta was not relaxed by Hololena curta thereby precluding effects of Hololena curta on postjunctional L type smooth muscle Ca2+ channels. The pattern of effects of Hololena curta on peripheral autonomic nerves was similar to the N channel inhibitor omega-conotoxin GVIA. These results suggest that Hololena curta venom constituents block Ca2+ channels in peripheral autonomic nerves. The study failed to establish the presence of functional P type Ca2+ channels on these peripheral autonomic nerves and further suggests that N type channels may be exclusively responsible for supplying the Ca2+ necessary for neurotransmitter release in these nerves.

Inhibition of a dihydropyridine, omega-conotoxin insensitive Ca2+ channel in rat synaptosomes by venom of the spider Hololena curta.

Inhibition of the N and L type Ca2+ channels with omega conotoxin GVIA (omega-CgTx) together with the dihydropyridine (-)-202-791 produces slight reduction (congruent to 25%) of K(+)-evoked Ca2+ influx in mammalian synaptosomes. These results and others suggest the existence of a third high threshold voltage sensitive calcium channel (VSCC) responsible for the majority of influx. Venom from the funnel web spider Hololena curta potently and persistently inhibited Ca2+ influx in rat cortical synaptosomes (IC50 1:10,000 or 4.21 micrograms/venom protein/ml of synaptosomes). Also Ca2+ influx in cerebellar synaptosomes was inhibited in a similar manner. K(+)-evoked tritium release from synaptosomes labeled with [3H]noradrenaline was inhibited by Hololena venom (congruent to 60% reduction at 10 micrograms/venom protein). Inhibition of Ca2+ influx by venom was unaffected by combined omega-CgTx and (-)-202-791 pretreatment (both 1 microM). Hololena venom and its active constituent should provide useful tools to investigate the role of this novel Ca2+ channel in neuronal function.

The effect of neuropeptide Y on voltage-sensitive Ca2+ channels.

Neuropeptide Y (NPY) (50-1000 nM) failed to modify basal or K(+)-stimulated Ca2+ influx in cortical or hippocampal synaptosomes from rat brain, whereas the voltage-sensitive Ca2+ channel (VSCC) blocker Cd2+ (50 microM) caused major inhibition. In cortical synaptosomes from chicken brain NPY (1.0 microM) failed to modify, whereas omega-conotoxin GV1A (0.1 microM) markedly inhibited Ca2+ influx. NPY does not appear to modify synaptosomal Ca2+ influx, however it may still affect VSCCs spatially distinct or 'upstream' from the nerve terminals.

Pharmacological evidence for an omega-conotoxin, dihydropyridine-insensitive neuronal Ca2+ channel.

Inactivation of N-type voltage-sensitive Ca2+ channels (VSCC) with omega-conotoxin (omega-CgTx) in tissue obtained from chicken brain produces a concentration dependent (0.01-0.1 microM) inhibition of K(+)-stimulated Ca2+ influx (delta K+), the rise in [Ca2+]i and acetylcholine (ACh) release. In identical preparations from rat brain, Ca2+ influx and the rise in [Ca2+]i were only marginally affected by much higher (1-10 microM) concentrations of omega-CgTx. The release of ACh, however, was inhibited to the same degree with similar amounts of omega-CgTx as those used in chicken brain. An L-type VSCC inhibitor failed to affect any of these parameters alone, or to augment the effect of omega-CgTx. The results suggest that almost all the VSCC in chicken brain are of the N type and that these channels regulate neurotransmitter release. In rat brain, on the other hand, Ca2+ channels resistant to N- or L-type blockers account for almost 75% of the measurable Ca2+ influx and rise in [Ca2+]i. The conspicuous dissociation between the regulation of Ca2+ influx and ACh release demonstrated in rat brain by using omega-CgTx, suggest that neurotransmitter release is governed by only a small proportion of strategically located N-type, omega-CgTx sensitive, VSCC in the presynaptic terminal.

Evaluation of a school-based fluoride mouthrinsing and clinic-based sealant program on a non-fluoridated island.

For many years the children of Guam have experienced a high prevalence of dental caries. Surveys conducted on the fluoride-deficient island found that caries levels were double those of US mainland children. In 1976 a school-based fluoride mouthrinse program was initiated involving over 22,000 children in grades kindergarten through eight in weekly rinses with 0.2% neutral NaF. A clinic-based dental pit and fissure sealant program was added in 1984 to the fluoride mouthrinse program. Over 15,000 children participated annually in the sealant program where more than 75,000 teeth were sealed the first year. After 8 yr of fluoride mouthrinsing (1976-1984) mean DMFS scores were 1.79 surfaces per child lower compared to baseline, a decrease of 0.22 DMFS per child per year. During this period proximal DMFS scores decreased 61%, buccal-lingual surfaces 31%, and occlusal surfaces 7%. After 2 yr of fluoride mouthrinsing and sealant application combined overall DMFS scores decreased an additional 2.34 surfaces per child, a reduction of 1.17 DMFS per child per year. Most of this decline took place on pit and fissure surfaces. For the 10-yr period a reduction of 4.13 DMFS per child was seen--a decline from 7.06 DMF at baseline to 2.93 DMF surfaces per child in 1986. This long-term evaluation indicates that dental sealants when used in combination with fluoride mouthrinse were particularly effective in lowering the prevalence of dental caries. Schoolchildren in participating grades on Guam now have dental caries rates close to those of US schoolchildren.