HERBICIDE PHOSPHINOTHRICIN CAUSES DIRECT STIMULATION HORMESIS

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 μM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoen- zymes is proposed. The mechanism is in concurrence with all experimental and literature data

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-las), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with p/ values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a p/ value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing, patterns revealed the induction of one isoform of SOD with a p/ of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alflalfa tissues

Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight

Genetically encoded calcium indicators (GECIs) produce unprecedentedly large signals that have enabled routine optical recording of single neuron activity in vivo in rodent brain. Genetically encoded voltage indicators (GEVIs) offer a more direct measure of neuronal electrical status, however the signal-to-noise characteristics and signal polarity of the probes developed to date have precluded routine use in vivo. We applied directed evolution to target modulable areas of the fluorescent protein in GEVI ArcLight to create the first GFP-based GEVI (Marina) that exhibits a Δ<i>F</i>/Δ<i>V</i> with a positive slope relationship. We found that only three rounds of site-directed mutagenesis produced a family of “brightening” GEVIs with voltage sensitivities comparable to that seen in the parent probe ArcLight. This shift in signal polarity is an essential first step to producing voltage indicators with signal-to-noise characteristics comparable to GECIs to support widespread use in vivo

A Fluorescent, Genetically-Encoded Voltage Probe Capable of Resolving Action Potentials

<div><p>There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1–S4) of <em>Ciona intestinalis</em> voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ∼1–2 ms) than any currently published fluorescent protein-based voltage probe. ElectricPk can follow the rise and fall of neuronal action potentials with a modest decrease in fluorescence intensity (∼0.7% ΔF/F). The probe has a nearly linear fluorescence/membrane potential response to both hyperpolarizing and depolarizing steps. This is the first probe based on CiVSP that captures the rapid movements of the voltage sensor, suggesting that voltage probes designed with circularly permuted fluorescent proteins may have some advantages.</p> </div

Linear ΔF/ΔV response of ElectricPK.

<p>A) Fluorescence response (upper traces) of HEK293 cells transiently expressing ElectricPk to depolarizing and hyperpolarizing voltage steps (−170 to +30 mV from a −70 mV holding potential, lower traces). B) ΔF/ΔV curve of ElectricPk derived from data presented in A.</p

Detection of action potentials in hippocampal neurons in vitro using ElectricPK.

<p>A) Wide field image of an in vitro hippocampal neuron expressing ElectricPk. Bar = 15 µm. B) Single (light red trace) and averaged (red trace) optical response to action potentials evoked in the neuron seen in (A) taken using wide field microscopy and a RedShirtImaging NeuroCCD camera. The red trace is an average of 32 action potentials. All responses captured at 2000 fps. C) Fluorescence change (light red trace-unfiltered, red trace-filtered) to a single train of evoked action potentials recorded from an in vitro hippocampal neuron expressing ElectricPk. Lower black trace is the voltage recording made from the patch electrode. All fluorescence traces are bleach subtracted and where indicated, low pass filtered (Bessel) at 350 Hz.</p

The protein sequence of the various fusion sites of cpEGFP with CiVSP described in this report.

<p>From top: CiVSP sequence includes S4 domain (bold cyan), linker domain (cyan and black) and phosphatase domain (red). The fusion sites and linker sequences of VSFP 2.1, 3.1 (17, 18) and Mermaid (4) probes. The ten different fusion sites and linkers described in this report (pLB1.x-10.x; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043454#pone-0043454-g001" target="_blank">Figure 1</a>). The fusion site is identified by the last CiVSP amino acid present in the probe (bold red). In all cases, this residue is followed by a five amino acid linker (purple) and the cpEGFP (green). The amino acid at the beginning of the cpEGFP depends on the hole size and position of the cpEGFP (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043454#pone-0043454-g001" target="_blank">Figure 1</a>).</p