Transient Increase In Fluorescence Research Articles

Sources of Calcium at Connexin 36 Gap Junctions in the Retina.

Synaptic plasticity is a fundamental feature of the CNS that controls the magnitude of signal transmission between communicating cells. Many electrical synapses exhibit substantial plasticity that modulates the degree of coupling within groups of neurons, alters the fidelity of signal transmission, or even reconfigures functional circuits. In several known examples, such plasticity depends on calcium and is associated with neuronal activity. Calcium-driven signaling is known to promote potentiation of electrical synapses in fish Mauthner cells, mammalian retinal AII amacrine cells, and inferior olive neurons, and to promote depression in thalamic reticular neurons. To measure local calcium dynamics in situ, we developed a transgenic mouse expressing a GCaMP calcium biosensor fused to Connexin 36 (Cx36) at electrical synapses. We examined the sources of calcium for activity-dependent plasticity in retina slices using confocal or Super-Resolution Radial Fluctuations imaging. More than half of Cx36-GCaMP gap junctions responded to puffs of glutamate with transient increases in fluorescence. The responses were strongly dependent on NMDA receptors, in keeping with known activity-dependent signaling in some amacrine cells. We also found that some responses depended on the activity of voltage-gated calcium channels, representing a previously unrecognized source of calcium to control retinal electrical synaptic plasticity. The high prevalence of calcium signals at electrical synapses in response to glutamate application indicates that a large fraction of electrical synapses has the potential to be regulated by neuronal activity. This provides a means to tune circuit connectivity dynamically based on local activity.

Real-time imaging of hydrogen peroxide dynamics in vegetative and pathogenic hyphae of Fusarium graminearum.

Balanced dynamics of reactive oxygen species in the phytopathogenic fungus Fusarium graminearum play key roles for development and infection. To monitor those dynamics, ratiometric analysis using the novel hydrogen peroxide (H2O2) sensitive fluorescent indicator protein HyPer-2 was established for the first time in phytopathogenic fungi. H2O2 changes the excitation spectrum of HyPer-2 with an excitation maximum at 405 nm for the reduced and 488 nm for the oxidized state, facilitating ratiometric readouts with maximum emission at 516 nm. HyPer-2 analyses were performed using a microtiter fluorometer and confocal laser scanning microscopy (CLSM). Addition of external H2O2 to mycelia caused a steep and transient increase in fluorescence excited at 488 nm. This can be reversed by the addition of the reducing agent dithiothreitol. HyPer-2 in F. graminearum is highly sensitive and specific to H2O2 even in tiny amounts. Hyperosmotic treatment elicited a transient internal H2O2 burst. Hence, HyPer-2 is suitable to monitor the intracellular redox balance. Using CLSM, developmental processes like nuclear division, tip growth, septation, and infection structure development were analyzed. The latter two processes imply marked accumulations of intracellular H2O2. Taken together, HyPer-2 is a valuable and reliable tool for the analysis of environmental conditions, cellular development, and pathogenicity.

Optogenetic measurement of presynaptic calcium transients using conditional genetically encoded calcium indicator expression in dopaminergic neurons.

Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.

Characterization of activity-dependent changes in flavoprotein fluorescence in cerebellar slices from juvenile rats

Flavoprotein autofluorescence signals attributed to neuronal metabolism have been used to assess synaptic function. Here, we characterized flavoprotein autofluorescence responses in the molecular layer of rat cerebellar slices. High frequency stimulation elicited a transient fluorescence increase (peak phase) that was followed by a longer-lasting fluorescence decrease (valley phase). The peak phase was restricted to the molecular layer, whereas the valley phase extended into the Purkinje cell layer and a portion of the granule cell layer. Responses were abolished by either the Na+ channel antagonist, tetrodotoxin, or a combination of the AMPA receptor antagonists, NBQX and GIKI-53655, and were also reduced by a flavoprotein inhibitor (diphenyleneiodonium). These findings are consistent with responses being mediated by an increase in mitochondrial activity triggered by increased energy demands evoked by AMPA receptor-mediated synaptic transmission. The GABAA receptor antagonist picrotoxin did not significantly influence evoked responses. Likewise, exogenous application of ethanol, at concentrations known to increase GABAA receptor-mediated synaptic transmission at Purkinje cells, did not modify peak responses. These observations indicate that flavoprotein autofluorescence imaging could be useful to assess the coupling between glutamatergic synaptic transmission and neuronal metabolism in cerebellar slices.

Detection of dihydropyridine- and voltage-sensitive intracellular Ca2+ signals in normal human parathyroid cells

We recently showed dihydropyridine- and voltage-sensitive Ca(2+) entry in cultured parathyroid cells from patients with secondary hyperparathyroidism. To determine whether normal parathyroid cells have a similar extracellular Ca(2+) entry system, cells were isolated from normal (non-hyperplastic) human parathyroid glands. Fluorescence signals related to the cytoplasmic Ca(2+) concentration ([Ca(2+)]I) were examined in these cells. Cells loaded with fluo-3/AM showed a transient increase in fluorescence (Ca(2+) transient) following a 10-s exposure to a 150mM K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was reduced by dihydropyridine antagonists or 0.5mM Cd(2+), but enhanced by FPL-64176, an L-type Ca(2+)-channel agonist. Ca(2+) transients induced by the 10-s exposure to 3.0mM extracellular Ca(2+) ([Ca(2+)]o) were also inhibited by dihydropyridine antagonists or 0.5mM Cd(2+). These results provide the first evidence that normal human parathyroid cells express a dihydropyridine-sensitive Ca(2+) entry system that may be involved in the [Ca(2+)]o-induced change in [Ca(2+)]I. This system might provide a compensatory pathway for negative feedback regulation of parathyroid hormone secretion under physiological conditions.

In vivo Photometry during Movement Changes Our Understanding of the Direct and Indirect Pathways

The role of the direct and indirect pathways in movement and their dysfunction in the pathophysiology of most movement disorders is difficult to grasp for clinicians. The current model whereby the direct pathway has a role akin to the accelerator when driving and the indirect to the brake makes sense at a basic level for many disorders of movement. However, some clinical phenomenology such as hemiballismus are difficult to reconcile with the current model. Cui et al. (1), using an elegant experimental paradigm employing photometric methods in vivo in a mouse model have observed the moving brain at work and have attempted to disentangle the complex network of spiny projection neurons involved in the direct and indirect pathways. The authors reflect on the prevailing model whereby two distinct projection circuits composed of spiny projection neurons in the striatum act in an opposing fashion to either promote movement (direct pathway) or inhibit movement (indirect pathway). The anatomical location of these pathways is the striatum. Surmeier in an accompanying editorial further identifies the striatum as the seat of “past experiences” and when “asked” by cortex what way to proceed, the current model which he identifies as intrinsically binary sends “go” or “no-go” instructions to cortex, via the thalamus (2). Cui et al. reflect on the fact that despite increased understanding of basal ganglia circuitry, we still do not understand the physiological relationship and interactions between these pathways. To do so we need to observe the activity of the direct and indirect pathway spiny projection neurons in “behaving” animal models (1). However, it is difficult to distinguish direct and indirect spiny projection neuron firing using electrophysiological recordings and antidromic stimulation is unreliable and difficult due to “cross talk” between axons of both pathways (1). The authors highlight that optogenetic technology in vivo facilitates the identification of specific cell types and the monitoring of activity of specific cell types (1). Limitations of this technology include the requirement for head restraint, which limits the study of structures involved in movement and the technology does not penetrate to subcortical structures therein limiting the ability to study the basal ganglia (1). Cui et al. developed an experimental paradigm utilizing in vivo photometry to optically record neural activity within the striatum during free movement in a mouse model (1). They implanted a fiber-optic probe to deliver laser pulses and record emitted photons in the dorsal striatum over time. The optical indicator was a genetically encoded calcium indicator (GECI), selectively expressed in the direct pathway spiny projection neurons by injection with a recombinant viral vector to produce D1-Cre mice (2). A similar technique was used to selectively express the indicator in the indirect pathway spiny projection neurons of A2A-Cre mice (1). Focusing specifically on their examination of the relationship between activity in the direct and indirect pathway spiny projection neurons during movement, the authors measured fluorescence whilst mice performed a lever-pressing task. They demonstrated time-locked increases in fluorescence during the initiation of the task with a similar temporal profile in both the direct and indirect pathways in all of the mice (1). They identified transient increases in fluorescence throughout the completion of different tasks and in addition during the monitoring sessions, mice were exposed to a series of active states and periods of inactivity (1). Using the classical direct and indirect pathway model it would be logical to hypothesize that during the active periods there should be relatively more transients identified in the direct pathway spiny projection neurons compared to the indirect and during periods of inactivity there should be more transients identified in the indirect spiny projection neurons compared to the direct (1). Cui et al. identified increased activity in both direct and indirect pathways during movement and silence of both pathways during periods of inactivity. These results question the accelerator-brake metaphor applied to our current understanding of the interaction between the direct and indirect pathways. Further analysis of self-paced movement initiation toward the contralateral side demonstrated co-activation of both the direct and indirect pathways and furthermore activity in both pathways preceded movement initiation. Activity in both pathways predicted the occurrence of specific movements within 500 ms of transient initiation. In the accompanying editorial it is suggested that rather than giving sequential “go” or “no-go” recommendations, the striatum is simultaneously making recommendations about “what to do and what not to do” (2). Through the interrogation of the direct and indirect pathways in vivo and during movement, this study builds upon and changes our current understanding of the function of the basal ganglia. Whilst the results challenge the current concepts used to explain movement, further work is required including lesional studies to translate this theory into the clinical environment in order to explain the clinical phenomenology we see at the bedside.

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.

Retinal Glial (Müller) Cells: Sensing and Responding to Tissue Stretch

To test whether Müller glial cells sense, and respond to, mechanical tension in the retina. A device was designed to stretch the retina at right angles to its surface, across retinal layers. Pieces of retina were mounted between two hollow tubes, and uniaxial force was applied to the tissue using a micrometer-stepping motor. Müller cells were selectively stained with the fluorescent, calcium-sensitive dye X-Rhod-1 and were monitored in real time during retinal stretch in vitro. Immunohistochemistry was used to study protein levels and activation of intracellular pathways in stretched retinas. Müller cells responded acutely with transient increases in fluorescence during stretch, indicative of increased intracellular calcium levels. All the Müller cells elongated uniformly, and there was no apparent difference between retinal layers in resistance against mechanical deformation. After stretch, Müller cells showed fast activation of extracellular signal-regulated kinase (after 15 minutes), upregulation of transcription factor c-Fos (after 1 hour), and basic fibroblast growth factor (after 3 hours). No changes in intermediate filament protein expression were observed in Müller cells up to 3 hours after stretch. A novel technique was developed for real-time monitoring of Müller cells during retinal stretch, which allowed the identification of Müller cells as a mechanoresponsive cell type. Mechanical stress triggers molecular responses in Müller cells that could prevent retinal damage.

Dihydropyridine‐ and voltage‐sensitive Ca2+ entry in human parathyroid cells

Patch-clamp and fluorescence measurements of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) were performed to directly detect extracellular Ca(2+) entry into cultured parathyroid cells from patients with secondary hyperparathyroidism. Cells loaded with fluo-3 AM or fluo-4 AM showed a transient increase in fluorescence (Ca(2+) transient) following 10 s exposure to 150 mm K(+) solution in the presence of millimolar concentrations of external Ca(2+). The Ca(2+) transient was completely inactivated after 30-40 s exposure to the high-K(+) solution, was reduced by dihydropyridine antagonists and was enhanced by FPL-64176, an L-type Ca(2+) channel agonist. The electrophysiological and pharmacological properties of the whole-cell Ca(2+) and Ba(2+) currents were similar to those of L-type Ca(2+) channels. The Ca(2+) transients induced by 10 s exposure to 3.0 mm extracellular Ca(2+) concentration ([Ca(2+)](o)) were inhibited by dihydropyridine antagonists and were partly inactivated following 30-40 s exposure to the high-K(+) solution. These results demonstrate, for the first time, that human parathyroid cells express L-type-like Ca(2+) channels that are possibly involved in the [Ca(2+)](o)-induced change in [Ca(2+)](i). This Ca(2+) entry system might provide a compensatory pathway for the negative feedback regulation of parathyroid hormone secretion, especially in hyperplastic conditions in which the Ca(2+)-sensing receptor is poorly expressed.

Fast calcium responses along endothelium of arteriolar networks during blood flow

To study endothelium‐specific calcium signaling in vivo we utilized the superfused (pH 7.4; 34 °C) cremaster muscle preparation in anesthetized male transgenic mice expressing the calcium sensor GCaMP2 under endogenous transcriptional regulation of connexin40 (Cx40). Endothelial calcium waves have been characterized as a transient fluorescence increase initiated by ACh that travels bidirectionally along arterioles at 116 µm/s and decays within 1 mm. Here we report a novel "fast calcium response (FCR)" that travels at mm/s along arteriolar networks in the direction of convection during blood flow. To determine whether FCR was blood flow‐dependent, an arteriole (diameter, 30‐50 µm) was occluded upstream or downstream from the site of abluminal ACh microiontophoresis (1 µm pipette tip; 500 ms, 500 nA). The FCR was eliminated when blood flow ceased (n=4), whereas removing the occluder restored the FCR along with blood flow. Redirecting flow into a side branch also redirected the FCR. These findings demonstrate that focal release of a vasoactive substance adjacent to an arteriole can activate entire networks of arteriolar endothelial cells through gaining access to the flow stream. Support: NIH HL041026, AR048523.

Monitoring The Activation Of Rhodopsin By The Transient Fluorescence Of Fluorescently Labeled Helix 8

The transient changes of the fluorescence of bovine rhodopsin in ROS membranes selectively labeled with Alexa594 at cysteine 316 in helix 8 were followed in time from 1 μs to 10 s after flash excitation of the photoreceptor. A large light-induced transient fluorescence increase was observed with time constants in the ms- range at pH6. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state MII (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6), the sequence of events is: Schiff base deprotonation, structural change, proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Monitoring the Conformational Changes of Photoactivated Rhodopsin from Μicroseconds to Seconds by Transient Fluorescence Spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.

Highlighting Synaptic Communication in the Enteric Nervous System

Highlighting Synaptic Communication in the Enteric Nervous System

Folding Kinetics of Staphylococcal Nuclease Studied by Tryptophan Engineering and Rapid Mixing Methods

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.

Visualization of Rab3A dissociation during exocytosis: A study by total internal reflection microscopy

Rab3A is a small G protein in the Rab3 subfamily, and is thought to act at late stage of exocytosis. However, the detailed mechanism of its action is not completely understood. To study the role of Rab3A in exocytosis, we used a total internal reflection fluorescence microscope to examine the fluorescence changes of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles in PC12 cells upon stimulation. The fluorescence of EGFP-Rab3A-labeled and NPY-EGFP-labeled vesicles decreased while showing different patterns. The NPY-EGFP-labeled vesicles that exocytosed showed a transient fluorescence increase before NPY-EGFP fluorescence disappearance, which represents fusion and NPY release. This transient increase was diminished in cells that co-expressed the GDP-bound Rab3A mutant. The fluorescence of EGFP-Rab3A-labeled vesicles dispersed before disappearance, which represents the dissociation of Rab3A from the vesicles. The dispersion was not found in GTP-bound Rab3A mutant-labeled vesicles. Interestingly, EGFP-Rab3A F59S, a mutant unable to bind rabphilin, dissociates slower from the vesicles than wild type Rab3A and caused a slower release of NPY-EGFP. The results provide direct evidence to support the hypothesis that GTP hydrolysis and rabphilin are involved in Rab3A dissociation from the vesicles and the occurrence of exocytosis.

Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat*1

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC50 of capsaicin and OLDA on 45Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 μM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 μM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca2+ transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6–9 °C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.

Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC 50 of capsaicin and OLDA on 45Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 μM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 μM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca 2+ transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6–9 °C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.

PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis.

Plastid gene expression is regulated by a variety of nuclear genes. We have isolated Arabidopsis thaliana proton gradient regulation 3 (pgr3) mutants, which display aberrant chlorophyll fluorescence because of defects in chloroplast gene expression. High chlorophyll fluorescence (HCF) because of a reduced level of the cytochrome b6/f complex was observed in two alleles, pgr3-1 and pgr3-2 but not in pgr3-3. In contrast, a transient increase in fluorescence after turning off the actinic light, which was ascribed to chloroplast NADPH dehydrogenase (NDH) activity, was impaired in pgr3-1 and pgr3-3 but not in pgr3-2. Both phenotypes were complemented by the introduction of a single gene, PGR3, encoding a protein containing 27 pentatrico-peptide repeat (PPR) motifs. PPR motifs are present in proteins functioning in the post-transcriptional regulation of organellar gene expression. The conserved threonine in the motif was substituted by isoleucine in the 15th and 12th PPR motifs in pgr3-1 and pgr3-2, respectively, and the conserved leucine by phenylalanine in the final incomplete motif of pgr3-3. We consider that the different domains of the PPR repeats in PGR3 might have different functions in conferring RNA stability and probably allowing translation as well as recognizing at least two distinct target RNAs.

Characterization of Superoxide-producing Sites in Isolated Brain Mitochondria

Mitochondrial respiratory chain complexes I and III have been shown to produce superoxide but the exact contribution and localization of individual sites have remained unclear. We approached this question investigating the effects of oxygen, substrates, inhibitors, and of the NAD+/NADH redox couple on H2O2 and superoxide production of isolated mitochondria from rat and human brain. Although rat brain mitochondria in the presence of glutamate+malate alone do generate only small amounts of H2O2 (0.04 +/- 0.02 nmol H2O2/min/mg), a substantial production is observed after the addition of the complex I inhibitor rotenone (0.68 +/- 0.25 nmol H2O2/min/mg) or in the presence of the respiratory substrate succinate alone (0.80 +/- 0.27 nmol H2O2/min/mg). The maximal rate of H2O2 generation by respiratory chain complex III observed in the presence of antimycin A was considerably lower (0.14 +/- 0.07 nmol H2O2/min/mg). Similar observations were made for mitochondria isolated from human parahippocampal gyrus. This is an indication that most of the superoxide radicals are produced at complex I and that high rates of production of reactive oxygen species are features of respiratory chain-inhibited mitochondria and of reversed electron flow, respectively. We determined the redox potential of the superoxide production site at complex I to be equal to -295 mV. This and the sensitivity to inhibitors suggest that the site of superoxide generation at complex I is most likely the flavine mononucleotide moiety. Because short-term incubation of rat brain mitochondria with H2O2 induced increased H2O2 production at this site we propose that reactive oxygen species can activate a self-accelerating vicious cycle causing mitochondrial damage and neuronal cell death.

Fluorescence detection of plant extracts that affect neuronal voltage-gated Ca 2+ channels

Structurally novel compounds able to block voltage-gated Ca 2+ channels (VGCCs) are currently being sought for the development of new drugs directed at neurological disorders. Fluorescence techniques have recently been developed to facilitate the analysis of VGCC blockers in a multi-well format. By utilising the small cell lung carcinoma cell line, NCI-H146, we were able to detect changes in intracellular Ca 2+ concentration ([Ca 2+] i) using a fluorescence microplate reader. NCI-H146 cells have characteristics resembling those of neuronal cells and express multiple VGCC subtypes, including those of the L-, N- and P-type. We found that K +-depolarisation of fluo-3 loaded NCI-H146 cells causes a rapid and transient increase in fluorescence, which was readily detected in a 96-well plate. Extracts of Australian plants, including those used traditionally as headache or pain treatments, were tested in this study to identify those affecting Ca 2+ influx following membrane depolarisation of NCI-H146 cells. We found that E. bignoniiflora, A. symphyocarpa and E. vespertilio caused dose-dependent inhibition of K +-depolarised Ca 2+ influx, with IC 50 values calculated to be 234, 548 and 209 μg/ml, respectively. This data suggests an effect of these extracts on the function of VGCCs in these cells. Furthermore, we found similar effects using a fluorescence laser imaging plate reader (FLIPR) that allows simultaneous measurement of real-time fluorescence in a multi-well plate. Our results indicate that the dichloromethane extract of E. bignoniiflora and the methanolic extract of E. vespertilio show considerable promise as antagonists of neuronal VGCCs. Further analysis is required to characterise the function of the bioactive constituents in these extracts and determine their selectivity on VGCC subtypes.