Near-infrared light responsive synthetic c-di-GMP module for optogenetic applications.

Enormous potential of cell-based therapeutics is hindered by the lack of effective means to control genetically engineered cells in mammalian tissues. Here, we describe a synthetic module for remote photocontrol of engineered cells that can be adapted for such applications. The module involves photoactivated synthesis of cyclic dimeric GMP (c-di-GMP), a stable small molecule that is not produced by higher eukaryotes and therefore is suitable for orthogonal regulation. The key component of the photocontrol module is an engineered bacteriophytochrome diguanylate cyclase, which synthesizes c-di-GMP from GTP in a light-dependent manner. Bacteriophytochromes are particularly attractive photoreceptors because they respond to light in the near-infrared window of the spectrum, where absorption by mammalian tissues is minimal, and also because their chromophore, biliverdin IXα, is naturally available in mammalian cells. The second component of the photocontrol module, a c-di-GMP phosphodiesterase, maintains near-zero background levels of c-di-GMP in the absence of light, which enhances the photodynamic range of c-di-GMP concentrations. In the E. coli model used in this study, the intracellular c-di-GMP levels could be upregulated by light by >50-fold. Various c-di-GMP-responsive proteins and riboswitches identified in bacteria can be linked downstream of the c-di-GMP-mediated photocontrol module for orthogonal regulation of biological activities in mammals as well as in other organisms lacking c-di-GMP signaling. Here, we linked the photocontrol module to a gene expression output via a c-di-GMP-responsive transcription factor and achieved a 40-fold photoactivation of gene expression.

Upon the photostability of 8-nitro-cGMP and its caging as a 7-dimethylaminocoumarinyl ester.

8-Nitro-cGMP was recently discovered as a second messenger of nitric oxide. We describe here the synthesis and properties of DMACM-modified 8-nitro-cGMP for photochemical uncaging. Owing to the limited photostability of 8-nitro-cGMP care must be taken, but the photorelease of the intact product was readily feasible. Unexpectedly, 8-nitro-cGMP decays under formation of 8-nitrosoguanine when irradiated with light.

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron.

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.

A comparison between the distinct inward currents activated in rat cultured dorsal root ganglion neurones by intracellular flash photolysis of two forms of caged cyclic guanosine monophosphate.

Whole cell inward currents activated by intracellular photorelease of cyclic guanosine monophosphate (cGMP) were investigated in cultured dorsal root ganglion (DRG) neurones. The actions of two distinct types of caged cGMP (NPE-caged cGMP and a highly water-soluble caged cGMP) were compared. Rapidly activating inward currents were evoked by cGMP in a subpopulation (12.5%) of neurones and these currents may be due to activity of cyclic nucleotide-gated channels. In contrast in 52% of DRG neurones intracellular photorelease of cGMP activated a delayed Ca(2+)-dependent inward current through the generation of cyclic ADPribose and mobilisation of Ca(2+) from ryanodine sensitive intracellular stores. Similar delayed inward currents were activated by both caged compounds but only NPE-caged cGMP evoked rapidly activating currents. Cyclic GMP appears to increase excitability in some DRG neurones by diverse mechanisms.

Blood irradiation by He-Ne laser induces a decrease in platelet responses to physiological agonists and an increase in platelet cyclic GMP.

The effect of He-Ne laser irradiation on platelet adhesion, activation and aggregation was investigated. Citrated whole blood was irradiated in vitro by He-Ne laser (632.8 nm, 7 mW) and then subjected to shear stress (1300 s-1) on subendothelial extracellular matrix (ECM)-coated plates. Laser irradiation was followed by a decrease in platelet adhesion and aggregation on ECM under flow conditions in a time exposure-dependent manner (by 30-40%). The inhibiting effect of laser light on platelets was detectable up to 1 h after the termination of irradiation. Laser irradiation of either platelet-rich plasma, gel-filtered platelets, platelet-poor plasma, or packed blood cells followed by whole blood reconstitution revealed a marked decrease in platelet deposition on ECM only in the cases of platelet-rich plasma or gel filtered platelets. In conventional aggregometry, laser-treated platelet-rich plasma demonstrated a diminished platelet response to both thrombin receptor-activating peptide (TRAP), converting a two-wave aggregation curve to reversible, and to the protein kinase C activator PMA (by 45%). In flow cytometry analysis, irradiated platelets presented lower fibrinogen binding and P-selectin expression in response to TRAP. Laser irradiation had no additional inhibitory effect on dibutyryl cGMP- and dibutyryl cAMP-pretreated platelets. A 50% increase in cGMP level was observed in laser-treated gel filtered platelets, both in the presence and in absence of the phosphodiesterase inhibitor, isobuthylmethylxanthine. The results suggest that guanylate cyclase is one of the primary mediators of the laser effect on platelet function.

Dose-related effect of irradiation on circadian rhythm of cellular messengers in neural and immune system of mouse.

OBJECTIVE: To explore toxic effect of gamma-irradiation on circadian rhythms of cAMP and cGMP in signal pathway between the central nuclei and peripheral lymphocyte. METHODS: 60Co gamma-ray of 0.5 Gy and 5.0 Gy was applied to groups of mice at each circadian timepoint of a day. 24 hours after the irradiation, the pineal and suprachiasmatic nuclei (SCN) were removed, and the T lymphocytes in blood were separated by a step-centrifugal and nylon-filter treatment, for measurements of cAMP and cGMP by using the RIA procedure. The results were expressed as circadian rhythms of the cAMP/cGMP ratio derived from the Cosinor analysis. RESULTS: The daily average cAMP/cGMP ratio in cells of SCN, pineal, serum and lymphocyte changed according to the dose of irradiation at which the mice were exposed. The high dose of 5 Gy increased the ratio, and the low dose of 0.5 Gy decreased the ratio. There was a significant difference of cAMP/cGMP levels between the irradiated and the control groups, as well as between CT00:00 and CT12:00 of a day. In addition, the peak phases of the cAMP/cGMP ratio at the low-dose groups appeared in advance from about 2 to 8 hours, whereas those at the high-dose groups were delayed from about 3 to 6 hours in comparison to that of the normal mice. CONCLUSION: Toxic effect of gamma-irradiation on the cAMP/cGMP circadian rhythm in the central nuclei and peripheral lymphocyte could be characterized by a low-dose hormesis.

An unusual cGMP pathway underlying depolarizing light response of the vertebrate parietal-eye photoreceptor.

All cellular signaling pathways currently known to elevate cGMP involve the activation of a guanylyl cyclase to synthesize cGMP. Here we describe an exception to this rule. In the vertebrate parietal eye, the photoreceptors depolarize to light under dark-adapted conditions, unlike rods and cones but like most invertebrate photoreceptors. We report that the signaling pathway for this response involves a rise in intracellular cGMP resulting from an inhibition of the phosphodiesterase that hydrolyzes cGMP. Furthermore, this phosphodiesterase is driven by an active G protein in darkness. These results indicate an antagonistic control of the phosphodiesterase by two G proteins, analogous to the Gs/Gi control of adenylyl cyclase. Our findings demonstrate an unusual phototransduction mechanism and at the same time indicate that signaling involving cyclic nucleotides is more elaborate than previously known.

[Post-radiation disturbances in the cyclic nucleotide system in rat spleen and thymus lymphocytes]. / Postradiatsionnye narusheniia v sisteme tsiklicheskikh nukeltidov limfotsitov selezenki i timusa krys.

The changes in cAMP and cGMP content in rat spleen and thymus lymphocytes after irradiation in doses of 0.5 and 1 Gy, are determined, indicating to significant disturbances in the system of cyclic nucleotides. Radiation affected the functioning of enzymes both of synthesis and hydrolysis of cAMP and cGMP in spleen lymphocytes. The activity of adenylate cyclase and guanylate cyclase did not change in thymocytes after the exposure, while the activity of phosphodiesterase of cyclic nucleotides slightly increased.

[The role of the sex hormones in regulating the spermatogenesis process in different strains of mice irradiated at low doses of gamma quanta]. / Rol' polovykh gormonov v reguliatsii protsessa spermatogeneza u myshei raznykh linii, obluchennykh v malykh dozakh gamma-kvantami.

Intact BALB/c and CBA mice distinct in the total number of germ cells and testosterone level in blood plasma were exposed to doses of 0.1 and 0.25 Gy. Being tested 4-8 days after irradiation the BALB/c mice display compensatory-protective reaction which promote the maintenance of the germ cell number by active division of all spermatogonia types including the reserve ones. The CBA mice use the reserve later and only when the cells have reduced their proliferation activity or died. Testosterone plays a significant role in the process as the increase in its concentration stimulates proliferation activity and promotes mitosis block.

Role of magnesium as radiosensitizer in the in vitro radiolysis of aqueous solutions of guanosine-5'-monophosphate disodium.

A combination of Fourier Transform Infrared (FT-IR) spectroscopy and mass spectrometry (MS) was used to investigate the role of Mg2+ ions in gamma-radiolysis of aqueous solutions of guanosine-5'-monophosphate disodium salt. The FT-IR intensity changes together with the mass spectra suggest the interaction of the magnesium atom with the base guanine at the N7 site. The decomposition-limiting yield G(-5'-GMPNa2) in deaerated as well as in nitrous oxide saturated solutions, determined by measuring the absorbance at 253 nm, was found to be 0.6 and 1.2 respectively. Comparison of these values with the corresponding limiting yields of 0.3 and 0.6 that were found in the absence of Mg2+ ions shows the sensitizing role of this ion in the radiolysis of the mononucleotide. This in vitro behaviour may result in more effective local therapy of tumour by gamma-ray irradiation in the presence of Mg2+, due to the 100 per cent sensitization of the radiolysis reaction. It remains to be seen whether this is also true in vivo.

[The molecular mechanisms of exposure to ionizing radiation]. / Molekuliarnye mekhanizmy vozdeistviia ioniziruiushchikh izluchenii.

The article studies biochemical processes which occur in man's organism after the influence of ionizing radiation. The authors thoroughly describe the mechanisms of radioactively induced free radical reactions, radiation injuries of membranes, post-radiation disorders in cellular energy supply and cellular cyclatic system.

[Cyclic nucleotides in rat tissues under long-term irradiation]. / Tsiklicheskie nukleotidy v tkaniakh krys pri dlitel'nom radiatsionnom vozdeistvii.

Considerable changes in concentrations of cAMP and cGMP and in the CAMPOcGMP ratio were found in the thymus and blood plasma of rats subjected to long-term irradiation at cumulative doses of 10 to 20 Gy. This might be a manifestation of adaptation reactions that develop in response to the effect of ionizing radiation.

[The individual characteristics of the postradiation reaction of the cyclic nucleotides in rat thymocytes]. / Individual'nye osobennosti postradiatsionnoi reaktsii tsiklicheskikh nukleotidov timotsitov krys.

It was shown that X-irradiation of rats (4.3 Gy), which were preliminarily divided into two groups by the neutrophilic reaction in the peripheral blood to the effect of a three-hour immobilization, induced different reactions of the cyclic nucleotide system. Thus, in animals hyper-reactive to stress radiation injury to the above system was severe: relative reactivity made 22.8% of the initial value, and adenylate cyclase ability to respond to a hormonal stimulus was drastically inhibited. In hyporeactive animals, relative reactivity of adenylate cyclase system after irradiation made 47% throughout the same period of observation; inhibition of adenylate cyclase activity was also less pronounced.

The structure of the guanine cation: ESR/ENDOR of cyclic guanosine monophosphate single crystals after X irradiation at 10 K.

Following X irradiation of 3',5'-cyclic guanosine monophosphate single crystals at 10 K, several free radicals were trapped and detected by ESR/ENDOR/FSE spectroscopy. The two dominant species both have unpaired spin located on the guanine base. One is the product of net hydrogen atom loss from the exocyclic amino group. The spectroscopic characteristics of this resonance leave this assignment unambiguous. The experimental conditions make it likely that this species was formed by deprotonation of the guanine base cation. The nature of the other species is more uncertain. However, the evidence is consistent with the assignment that it is a net OH adduct to the C4 position of the base. Several species in which the unpaired spin was located on the sugar-phosphate region of the molecule were also observed. The mechanisms for the decay of the primary radicals, also leading to the well-known C8 hydrogen addition radical of the guanine base, are described and discussed.

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.

[The cyclic nucleotide content of the blood plasma in mice irradiated at different doses]. / Soderzhanie tsiklicheskikh nukleotidov v plazme krovi myshei, obluchennnykh v raznykh dozakh.

Cyclic nucleotide content of plasma was shown to increase after irradiation of mice within a wide range of doses. Compared was the dynamics of changes in the cyclic nucleotide content 24 hr following irradiation with supralethal doses.

[Cyclic nucleotides in a high molecular weight aminoacyl-tRNA synthetase complex from the liver of normal and irradiated rats]. / Tsyklichni nukleotydy u skladi vysokomolekyliarnogo kompleksu aminoatsyl-tRNK-syntetaz iz pechinky normal'nykh ta oprominenykh shchuriv.

The content of cAMP and cGMP in the composition of the high-molecular complex aminoacyl-tRNA-synthetase from the rat liver has been studied. The ionizing radiation decreases the level of the cyclic nucleotides in the composition of the complex. A problem on interregulation of the cyclic nucleotides and prostaglandins in the complex has been examined.

[The effect of ceruloplasmin on metabolic processes in the liver of mice during neutron exposure]. / Vliianie tseruloplazmina na metabolicheskie protsessy v pecheni myshei pri neitronnom vozdeistvii.

A single intraperitoneal administration of human ceruloplasmin to BALB/c mice 60 min before fast neutron irradiation normalizes lipid content and cAMP/cGMP index, and increases catalase activity in the liver of irradiated animals.