Search

Sustainability standards and certification serve to differentiate and provide market recognition to goods produced in accordance with social and environmental good practices, typically including practices to protect biodiversity. Such standards have seen rapid growth, including in tropical agricultural commodities such as cocoa, coffee, palm oil, soybeans, and tea. Given the role of sustainability standards in influencing land use in hotspots of biodiversity, deforestation, and agricultural intensification, much could be gained from efforts to evaluate and increase the conservation payoff of these schemes. To this end, we devised a systematic approach for monitoring and evaluating the conservation impacts of agricultural sustainability standards and for using the resulting evidence to improve the effectiveness of such standards over time. The approach is oriented around a set of hypotheses and corresponding research questions about how sustainability standards are predicted to deliver conservation benefits. These questions are addressed through data from multiple sources, including basic common information from certification audits; field monitoring of environmental outcomes at a sample of certified sites; and rigorous impact assessment research based on experimental or quasi-experimental methods. Integration of these sources can generate time-series data that are comparable across sites and regions and provide detailed portraits of the effects of sustainability standards. To implement this approach, we propose new collaborations between the conservation research community and the sustainability standards community to develop common indicators and monitoring protocols, foster data sharing and synthesis, and link research and practice more effectively. As the role of sustainability standards in tropical land-use governance continues to evolve, robust evidence on the factors contributing to effectiveness can help to ensure that such standards are designed and implemented to maximize benefits for biodiversity conservation., (© 2014 Society for Conservation Biology.)

Sympathetic ganglia are primarily composed of noradrenergic neurons and satellite glial cells. Although both cell types originate from neural crest cells, the identities of the progenitor populations at intermediate stages of the differentiation process remain to be established. Here we report on the identification in vivo of glial and neuronal progenitor cells in postnatal sympathetic ganglia, by using mouse superior cervical ganglia as a model system. There are significant levels of cellular proliferation in mouse superior cervical ganglia during the first 18 days after birth. A majority of the proliferating cells express both nestin and brain lipid-binding protein (BLBP). Bromodeoxyuridine (BrdU) fate-tracing experiments demonstrate that these nestin and BLBP double-positive cells represent a population of glial progenitors for sympathetic satellite cells. The glial differentiation process is characterized by a marked downregulation of nestin and upregulation of S100, with no significant changes in the levels of BLBP expression. We also identify a small number of proliferating cells that express nestin and tyrosine hydroxylase, a key enzyme of catecholamine biosynthesis that defines sympathetic noradrenergic neurons. Together, these results establish nestin as a common marker for sympathetic neuronal and glial progenitor cells and delineate the cellular basis for the generation and maturation of sympathetic satellite cells., ((c) 2008 Wiley-Liss, Inc.)

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.

The current study provides biochemical and functional evidence that the targeting of protein kinase A (PKA) to sites of localized Ca(2+) release confers rapid, specific phosphoregulation of Ca(2+) signaling in pancreatic acinar cells. Regulatory control of Ca(2+) release by PKA-dependent phosphorylation of inositol 1,4, 5-trisphosphate (InsP(3)) receptors was investigated by monitoring Ca(2+) dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PKA activation. Ca(2+) dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca(2+)-activated Cl(-) current. Photolysis of low concentrations of caged InsP(3) evoked Cl(-) currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, PKA activators had no significant inhibitory effect on the activation of Cl(-) current evoked by uncaging Ca(2+) or by the photolytic release of higher concentrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca(2+) release. Further evidence for the targeting of PKA to the sites of Ca(2+) mobilization was revealed using immunocytochemical methods demonstrating that the R(IIbeta) subunit of PKA was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type I or type II InsP(3) receptors. Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more "CCK-like" by raising cAMP levels. Our data provide a simple mechanism by which distinct oscillatory Ca(2+) patterns can be shaped.

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

The present study expands the contemporary view of mitochondria as important participants in cellular Ca(2+) dynamics and provides evidence that mitochondria regulate the supply of release-competent secretory granules. Using pharmacological probes to inhibit mitochondrial Ca(2+) import, the ability of mitochondria to modulate secretory activity in single, patch-clamped bovine chromaffin cells was examined by simultaneously monitoring rapid changes in membrane surface area (DeltaC(m)) and cytosolic Ca(2+) levels ([Ca(2+)](c)). Repetitive step depolarizations or action potential waveforms were found to raise the [Ca(2+)](c) of chromaffin cells into the 1 microM to tens of micromolar range. Inhibiting mitochondria by treatment with carbonyl cyanide p-(trifuoro-methoxy)phenylhydrazone, antimycin-oligomycin, or ruthenium red revealed that mitochondria are a prominent component for the clearance of Ca(2+) that entered via voltage-activated Ca(2+) channels. Disruption of cellular Ca(2+) homeostasis by poisoning mitochondria enhanced the secretory responsiveness of chromaffin cells by increasing the amplitude of the transient rise and the time course of recovery to baseline of the evoked Delta[Ca(2+)](c). The enhancement of the secretory response was represented by significant deviation of the Ca(2+)-exocytosis relationship from a standard relationship that equates Ca(2+) influx and DeltaC(m). Thus, mitochondria would play a critical role in the control of secretory activity in chromaffin cells that undergo tonic or repetitive depolarizing activity, likely by limiting the Ca(2+)-dependent activation of specific proteins that recruit or prime secretory granules for exocytosis.

A dietary source of retinoid or carotenoid has been shown to be necessary for the biosynthesis of functional visual pigment in flies. In the present study, the larvae or adults of Drosophila melanogaster were administered specific carotenoid-containing diets and high performance liquid chromatography was used to identify and quantify the carotenoids in extracts of wild type and ninaD visual mutant flies. When beta-carotene was fed to larvae, wild type flies were shown to hydroxylate this molecule and to accumulate zeaxanthin and a small amount of beta-cryptoxanthin. Zeaxanthin content was found to increase throughout development and was a major carotenoid peak detected in the adult fly. Carotenoids were twice as effective at mediating zeaxanthin accumulation when provided to larvae versus adults. In the ninaD mutant, zeaxanthin content was shown to be specifically and significantly altered compared to wild type, and was ineffective at mediating visual pigment synthesis when provided to both larval and adult mutant flies. It is proposed that zeaxanthin is the larval storage form for subsequent visual pigment chromophore biosynthesis during pupation, that zeaxanthin or beta-crytoxanthin is the immediate precursor for light-independent chromophore synthesis in the adult, and that the ninaD mutant is defective in this pathway.

Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance (Cm) measurements and by the membrane fluorescent dye FM1-43. When measured with the Cm method, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3. 5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s-1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of Cm changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.

Whole-cell patch-clamp recordings were performed together with time-resolved measurements of membrane capacitance (Cm) in nerve terminals acutely dissociated from neurohypophysis of adult rats to investigate modulation of Ca2+ currents and secretion by activation of opioid receptors. Bath superfusion of the kappa-opioid agonists U69,593 (0.3-1 microM), dynorphin A (1 microM), or U50,488H (1-3 microM) reversibly suppressed the peak amplitude of Ca2+ currents 32. 7 +/- 2.7% (in 41 of 56 terminals), 37.4 +/- 5.3% (in 5 of 8 terminals), and 33.5 +/- 8.1% (in 5 of 10 terminals), respectively. In contrast, tests in 11 terminals revealed no effect of the mu-opioid agonist [D-Pen2,5]-enkephalin (1-3 microM; n = 7) or of the delta-agonist Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (1 microM; n = 4) on Ca2+ currents. Three components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nicardipine, and omega-conotoxin MVIIC: N-, L-, and P/Q-type current, respectively. Administration of U69,593 inhibited N-type current in these nerve terminals on average 32%, whereas L-type current was reduced 64%, and P/Q-type current was inhibited 28%. Monitoring of changes in Cm in response to brief depolarizing steps revealed that the kappa-opioid-induced reductions in N-, L-, or P/Q-type currents were accompanied by attenuations in two kinetically distinct components of Ca2+-dependent exocytotic release. These data provide strong evidence of a functional linkage between blockade of Ca2+ influx through voltage-dependent Ca2+ channels and inhibitory modulation of release by presynaptic opioid receptors in mammalian central nerve endings.

While the presence of post-synaptic NMDA receptors in the CNS is well-established, the present study addressed the question of whether NMDA receptors may also be present on secretory nerve endings. Using microspectrofluorometry of fura-2 loaded isolated neurohypophysial nerve endings of the rat, we found that both glutamate (EC50 = 50 microM) and NMDA (EC50 = 30 microM) induced a rapid rise in (Ca2+]i. These responses were glycine-dependent and abolished by 1 mM Mg2+, 1 microM dizocilpine, and removal of extracellular Ca2+. Responses were not significantly affected by treatment with Ca2+ channel blockers or 10 microM CNQX.

Although glutamate is the predominant excitatory amino acid in the vertebrate central nervous system (CNS) where it affects a variety of physiological processes and pathophysiological states, the role that glutamate receptors may play outside the CNS has not been clearly established. In the present study, the effects of N-methyl-D-aspartate (NMDA), alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) kainate, and metabotropic glutamate receptor agonists and antagonists were investigated on neuroendocrine melanotropes of the rat pars intermedia using single-cell dual-wavelength microfluorometry and the Ca(2+)-sensitive probe, fura-2, to monitor changes in [Ca2+]i. Glutamate induced a rapid, concentration-dependent rise in [Ca2+]i with an EC50 of 24 microM that was Mg(2+)-sensitive and dependent on the presence of extracellular Ca2+. NMDA increased [Ca2+]i in a glycine-dependent manner with an EC50 of 83 microM that was blocked by 1 microM MK-801 and 1 mM Mg2+. The non-NMDA receptor agonists kainate, AMPA, and quisqualate increased [Ca2+]i with an EC50 of 124, 5 and 8 microM, respectively. Responses to kainic acid were blocked by 10 microM CNQX and were shown to be sensitive to Mg2+ and dihydropyridine. AMPA stimulation was the most potent, and glutamate stimulation was the most efficacious at mediating increases in [Ca2+]i. The metabotropic receptor-specific agonist, trans-ACPD, failed to induce a change in [Ca2+]i. The glutamate-induced Ca2+ influx was about half of that elicited by a 50 mM K(+)-induced membrane depolarization and activation of voltage-sensitive Ca2+ channels. These results demonstrate the presence of glutamate receptors on rat melanotropes and suggest that glutamate receptors in the intermediate lobe of the pituitary may provide the excitatory counterbalance to the well-described secretoinhibiting input via dopamine and gamma-aminobutyric acid receptors.