Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases.

As diffusible second messengers, cyclic nucleoside monophosphates (cNMPs) relay and amplify molecular signals in myriad cellular pathways. The triggering of downstream physiological responses often requires defined cNMP gradients in time and space, generated through the concerted action of nucleotidyl cyclases and phosphodiesterases (PDEs). In an approach denoted optogenetics, sensory photoreceptors serve as genetically encoded, light-responsive actuators to enable the noninvasive, reversible, and spatiotemporally precise control of manifold cellular processes, including cNMP metabolism. Although nature provides efficient photoactivated nucleotidyl cyclases, light-responsive PDEs are scarce. Through modular recombination of a bacteriophytochrome photosensor and the effector of human PDE2A, we previously generated the light-activated, cNMP-specific PDE LAPD. By pursuing parallel design strategies, we here report a suite of derivative PDEs with enhanced amplitude and reversibility of photoactivation. Opposite to LAPD, far-red light completely reverts prior activation by red light in several PDEs. These improved PDEs thus complement photoactivated nucleotidyl cyclases and extend the sensitivity of optogenetics to red and far-red light. More generally, our study informs future efforts directed at designing bacteriophytochrome photoreceptors.

How to control cyclic nucleotide signaling by light.

Optogenetics allows to non-invasively manipulate cellular functions with spatio-temporal precision by combining genetic engineering with the control of protein function by light. Since the discovery of channelrhodopsin has pioneered the field, the optogenetic toolkit has been ever expanding and allows now not only to control neuronal activity by light, but rather a multitude of other cellular functions. One important application that has been established in recent years is the light-dependent control of second messenger signaling. The optogenetic toolkit now allows to control cyclic nucleotide-dependent signaling by light in vitro and in vivo.

[Second messenger systems and protein kinase activity in rat thymus and liver in the early stages of radiation injury]. / Sistemy vtorichnykh messendzherov i aktivnost' proteinkinaz v timuse i pecheni krys na rannikh étapakh luchevogo porazheniia.

The effect of ionizing radiation (7, 76 Gr) on the content of cyclic nucleotides and Ca2+ ions from rat thymus and liver has been established: it was increased 15, 30 min and 4.6 h and decreased 2, 12 h and 24 h after the irradiation. Changes in activity of cyclic nucleotide-dependent protein kinases and Ca2+, phospholipid-dependent protein kinase correlated with the contents of secondary messengers in the irradiated rats (30 min and 4 h after irradiation) are shown.

[The individual postradiation reactions of cyclic nucleotides in the lymphoid cells of the rat spleen]. / Individual'nye postradiatsionnye reaktsii tsiklicheskikh nukleotidov v limfoidnykh kletkakh selezenki krys.

X irradiation (4.3 Gy) of rats was shown to cause different reactions of the cyclic nucleotide system in differently reactive animals which was determined by the neutrophilic response in the peripheral blood after three-hour immobilization. Changes in the cyclic nucleotide content and in the activity of adenylate cyclase were found to occur in both hyper- and hyporeactive animals 24 h after irradiation. In hyperreactive animals, however, these changes were more pronounced: a relative ability of adenylate cyclase to be activated in hyperreactive animals made 30%, and 52% in hyporeactive animals. The cAMP content in lymphocytes of the spleen of hyporeactive rats was twice as high as that in hyperreactive animals.

[The content of cyclic nucleotides, free cytoplasmic Ca2+ and malondialdehyde in the splenic lymphocytes and thymocytes of rats under the action of moderate doses of radiation]. / Soderzhanie tsiklicheskikh nukleotidov, svobodnogo tsitoplazmaticheskogo Ca2+ i malonovogo dial'degida v limfotsitakh selezenki i timotsitakh krys pri deistvii nevysokikh doz radiatsii.

It has been shown that the malonic dialdehyde (MDA) content in spleen lymphocytes of rats increases after whole-body X irradiation with a dose of 0.5 Gy to reach the maximum level in 24 h. Simultaneously, the concentration of cGMP and free cytosol Ca2+ increases. With a dose of 1 Gy MDA content of cells increases 6 h following irradiation. The maximum drop of the release of viable lymphocytes from the spleen and thymus, observed 24 h and 3 days after irradiation respectively, coincides with the appearance of the second peak of the MDA content. The level of cGMP remains decreased throughout the period of about 6 days. The onset of lymphocyte repopulation in the spleen on day 6 coincides with the decrease in the MDA level and increase in the cytosol Ca2+ concentration.

[Biogenic amines and cyclic nucleotides in the laser therapy of long-term nonhealing stomach ulcers]. / Biogennye aminy i tsiklicheskie nukleotidy pri lazeroterapii dlitel'no ne zazhivaiushchikh iazv zheludka.

A study is presented of the effect of copper vapors laser therapy on the content of biogenic amines--serotonin and histamine and the state of adenylcyclase (AC) system (content of cAMP, cGMP and AC activity) at the edge of the gastric ulcer. Direct effect of laser radiation (single dose 10--15 J.) produced a significant increase of serotonin, histamine, cAMP, AC activity and an insignificant increase of cGMP. Healing of the ulcerative defect after 5--6 laser therapy sessions was followed by a reduction of the content of serotonin, increase of histamine, cAMP and AC activity. The authors discuss the biostimulating effect of laser radiation by influencing the inflammatory-proliferative processes in the epitheliocytes in prolonged nonhealing gastric ulcers.

[The dynamic content of prostaglandins and cyclic nucleotides in the blood of irradiated dogs and monkeys]. / Dinamika soderzhanie prostaglandinov i tsiklicheskikh nukleotidov v krovi obluchennykh sobak i obez'ian.

In experiments with dogs and monkeys a study was made of the dynamics of the content of prostaglandins (PG) and cyclic nucleotides after gamma irradiation. In dogs irradiation with lethal doses (3.1 and 50 Gy) caused a short-term, evidently stress growth of cAMP, PGE and PGF2 alpha levels. At the height of radiation sickness PGF2 alpha and cGMP content decreased considerably. Irradiation of monkeys with a nonlethal dose of 3.2 Gy changed PGE and PGF2 alpha levels to a lesser extent, while concentrations of cyclic nucleotides varied but their ratio remained stable.

[The dynamics of the immunobiological effects in transcerebral microwave exposures]. / Dinamika immunobiologicheskikh éffektov pri transtserebral'nykh mikrovolnovykh vozdeistviiakh.

The trend in immunobiological responses to transcerebral action of decimeter microwaves was studied in intact rabbits. Transcerebral microwaves (120 mW/cm2) can alter functional activity of certain links of endocrine system due to possible activation of hypothalamus and hypophysis exposed to microwave electromagnetic field. Such exposure given in courses at early stages leads to an increase in the number of antibody-forming cells in the spleen, whereas total number of splenocytes is on the decrease.

[Excretion of cyclic nucleotides during prolonged gamma irradiation of rats]. / Eksrektsiia tsiklicheskikh nukleotidov pri dlitel'nom gamma-obluchenii krys.

Excretion of cGMP in urine was significantly increased and cAMP/cGMP ratio decreased in rats subjected to long-term repeated gamma irradiation with cumulative doses of 15 to 20 Gy.

[Changes in metabolism of the nervous system in exposure to ionizing radiation at supralethal doses]. / Izmeneniia metabolizma nervnoi sistemy pri deistvii ioniziruiushchei radiatsii v sverkhletal'nykh dozakh.

Some biochemical disorders in the animals' central nervous system mainly in brain have been analysed after the exposure to superlethal doses of ionizing radiation as well in a state of the so-called early transient incapacity. The metabolism of gamma-aminobutyric acid, ammonia, histamine, cyclic nucleotides, prostaglandins and other biologically active substances is compared. Their investigation as metabolic regulators and modulators for nerve tissue seems to be of particular importance for deciphering the molecular mechanisms of changes in the central nervous system functional state and for discovering the possibility of its maintaining at a given level of activity.

[Redistribution of bone marrow cells according to mitotic cycle phases after the action of agents altering the intracellular concentration of cyclic nucleotides]. / Pereraspredelenie kostnomozgovykh kletok po fazam mitoticheskogo tsikla posle deistviia agentov, izmeniaiushchikh vnutrikletochnuiu kontsentratsiiu tsiklicheskikh nukleotidov.

The method of flow cytofluorometry was used to determine the distribution of murine bone marrow cells along the phases of the cell cycle in normal mice (CBA X C57Bl) F1 after the whole body equal X-ray irradiation, after irradiation in combination with burn, after administration of imidazole, insulin and caffeine, and after the treatment of cells by acetylcholine. In non-irradiated mice insulin and caffeine induced an increased ratio of bone marrow cells in G0 and G1 and a reduced one in S phase. Imidazole increased the number of G2- and M-cells by 1.5 times only in regenerating bone marrow of irradiated mice. After X-ray irradiation of mice at a dose of 2.3 Gy, reducing the percentage of cells in S and increasing it in G2 and M phases, insulin, acetylcholine and caffeine were found to decrease the number of cells in G2 and M phases: besides, caffeine and acetylcholine increased the percentage of S-phase cells. The data obtained are discussed in terms of possibility of normalization of bone marrow cell proliferation broken after exposure to physical stress-factors.