Kinetic Evaluation of Photosensitivity in Bi-Stable Variants of Chimeric Channelrhodopsins

Channelrhodopsin-1 and 2 (ChR1 and ChR2) form cation channels that are gated by light through an unknown mechanism. We tested the DC-gate hypothesis that C167 and D195 are involved in the stabilization of the cation-permeable state of ChRWR/C1C2 which consists of TM1-5 of ChR1 and TM6-7 of ChR2 and ChRFR which consists of TM1-2 of ChR1 and TM3-7 of ChR2. The cation permeable state of each ChRWR and ChRFR was markedly prolonged in the order of several tens of seconds when either C167 or D195 position was mutated to alanine (A). Therefore, the DC-gate function was conserved among these chimeric ChRs. We next investigated the kinetic properties of the ON/OFF response of these bi-stable ChR mutants as they are important in designing the photostimulation protocols for the optogenetic manipulation of neuronal activities. The turning-on rate constant of each photocurrent followed a linear relationship to 0–0.12 mWmm⁻² of blue LED light or to 0–0.33 mWmm⁻² of cyan LED light. Each photocurrent of bi-stable ChR was shut off to the non-conducting state by yellow or orange LED light in a manner dependent on the irradiance. As the magnitude of the photocurrent was mostly determined by the turning-on rate constant and the irradiation time, the minimal irradiance that effectively evoked an action potential (threshold irradiance) was decreased with time only if the neuron, which expresses bi-stable ChRs, has a certain large membrane time constant (eg. τ_m > 20 ms). On the other hand, in another group of neurons, the threshold irradiance was not dependent on the irradiation time. Based on these quantitative data, we would propose that these bi-stable ChRs would be most suitable for enhancing the intrinsic activity of excitatory pyramidal neurons at a minimal magnitude of irradiance.     

In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata)

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 μM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.     

Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca²⁺ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca²⁺-sensor, the Ca²⁺ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca²⁺ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca²⁺. It is suggested that the photo-activation of ChRFR facilitated the release of Ca²⁺ from intracellular Ca²⁺ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.     

Investigation of the insulin-like properties of zinc(II) complexes of 3-hydroxy-4-pyridinones: identification of a compound with glucose lowering effect in STZ-induced type I diabetic animals

Results from an investigation in an in vivo model of STZ-induced diabetic rats demonstrate that compound bis(1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate)zinc(II), Zn(dmpp)₂, significantly lowers the blood glucose levels of individuals, thus showing evidence of glucose lowering activity.The compound was selected from a set of eight zinc(II) complexes of 3-hydroxy-4-pyridinones with diverse lipophilicity that were prepared and characterized in our laboratory. Assessment of insulin-like activity of the complexes was firstly performed in vitro by measuring the inhibition of FFA release in isolated rat adipocytes. The results indicate that compounds bis(2-methyl-3-hydroxy-4-pyridinonate)zinc(II), Zn(mpp)₂ and Zn(dmpp)₂ display significantly higher activity than that of the respective positive control thus suggesting its selection for in vivo tests.Safety evaluation of the active zinc(II) compounds was performed in freshly isolated rat hepatocytes. The results support that cell viability is not significantly different from the control set after 1 and 2 h of incubation with both zinc(II) complexes.     

Structure-based design, synthesis, and nonalcoholic steatohepatitis (NASH)-preventive effect of phenylpropanoic acid peroxisome proliferator-activated receptor (PPAR) α-selective agonists

A series of α-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) α-selective agonists, based on our PPARα/δ dual agonist 3 as a lead compound. Structure-activity relationship studies clearly indicated that the steric bulkiness and position of the distal hydrophobic tail part are critical for PPARα agonistic activity and PPARα selectivity, as had been predicted from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic steatohepatitis (NASH) in an animal model.     

A new type of orally active anti-diabetic Zn(II)-dithiocarbamate complex

In order to find orally active Zn(II) complexes that can treat diabetes mellitus (DM) at low doses, four new Zn(II)-dithiocarbamate complexes with Zn(II)-sulfur coordination bonds were prepared and their in vitro insulinomimetic activity and in vivo anti-diabetic ability were evaluated. Among the Zn(II)-dithiocarbamate complexes, the bis(pyrrolidine-N-dithiocarbamate)zinc(II) (Zn(pdc)(2)) complex was found to be the most effective in terms of inhibiting free fatty acid-release and enhancing glucose-uptake in adipocytes. After oral administration of the Zn(pdc)(2) complex to KK-A(y) mice with obesity and type 2 DM, we observed that the high blood glucose levels in the mice were lowered from approximately 500 mg/dL to 350 mg/dL within 6 days, and the effect was maintained during the administration period. Also, indicators of insulin resistance such as serum insulin, leptin, and triglyceride levels were also reduced compared with those in untreated mice. Moreover, the Zn(pdc)(2) complex improved not only the hypertension in the mice, but also the adiponectin level in the serum. On the basis of the results, the Zn(pdc)(2) complex is proposed to improve hyperglycemia and insulin resistance in type 2 DM animals on daily oral administrations.