Effect of ultraviolet radiation on acetylcholinesterase activity in freshwater copepods.

We analyzed the effects of UV radiation (UVR) effects on acetylcholinesterase (AChE) activity in two calanoid copepods, Boeckella gibbosa and Parabroteas sarsi that inhabit Patagonian shallow lakes. We studied the effect of experimental UVR (UV-B and UV-A) exposure on AChE activity in relation to basal antioxidant capacities of both copepods. Our experiments showed that UVR can effectively depress AChE activity, although with differences between species. In both copepods AChE was affected by UV-B, whereas UV-A only affected AChE in B. gibbosa. Both copepods also differed in body elemental composition (C:N:P), photoprotecting compound content (carotenoids and mycosporine-like amino acids) and enzymatic antioxidant capacity (glutathione S-transferase [GST]). Our results suggest that when exposed to UVR, AChE activity would depend more on the antioxidant capacity (GST) and P availability for enzyme synthesis than on the photoprotective compounds.

Radiomodulatory effect of liposome encapsulated AK-2123 on tumor in mice exposed to hepatocarcinogen.

An attempt was made to evaluate the whole body gamma-radiation effect on tumor in the presence of free and liposome encapsulated AK-2123, a hypoxic cell radiosensitizer that has widely been used in combination with a number of cancer therapies such as thermotherapy, chemotherapy and radiotherapy. Entrapment efficiency of AK-2123 into liposome was determined by LASER Raman spectroscopy. Cancer induction in mice was carried out by repeated exposure of N-nitrosodiethylamine (DEN) in combination with partial hepatectomy. Parameters such as marker enzymes activities (GGT and AChE), rates of nucleic acid synthesis, viability modification factor and the histology of liver tissues monitored, supported the induction of cancer in liver. In addition, the effect of free as well as liposome encapsulated AK-2123 on haemopoietic parameters were also studied. It was observed that AK-2123 after incorporation into liposome afforded more efficient radiomodulatory effects than that of free AK-2123 as determined by the above-mentioned parameters. Neither free AK-2123 nor liposome encapsulated AK-2123 showed any detectable toxic effects on the mice. Thus, it is seen that treatment of cancer with a combination of radiation, a radiomodifier and a drug delivery system, opens a wide scope for exploitation for the improvement of existing cancer therapies.

Structural and kinetic effects of mobile phone microwaves on acetylcholinesterase activity.

The present study provides evidence that "in vitro" simple exposure of an aqueous solution of electric eel acetylcholinesterase (EeAChE; EC 3.1.1.7.) to cellular phone emission alters its enzymatic activity. This paper demonstrates, by combining different experimental techniques, that radio frequency (RF) radiations irreversibly affect the structural and biochemical characteristics of an important CNS enzyme. These results were obtained by using a commercial cellular phone to reproduce the reality of the human exposition. This experimental procedure provided surprising effects collected practically without experimental errors because they were obtained comparing native and irradiated sample of the same enzyme solution. Although these results cannot be used to conclude whether exposure to RF during the use of cellular phone can lead to any hazardous health effect, they may be a significant first step towards further verification of these effects on other "ex vivo" or "in vivo" biological systems.

[Morphological and cytogenetic disorders in rats following chronic exposure to elevated radiation]. / Morfologicheskie i tsitogeneticheskie narusheniia u krys, nakhodivshikhsia v usloviiakh povyshennogo radiatsionnogo fona na protiazhenii dlitel'nogo vremeni.

Object of the investigation were effects of elevated radiation background on specific body systems in two generations of rats that had been chronically exposed to radiation in the most badly polluted areas of the Kaluga district and eaten local food. Findings included some compensatory deviations in the white blood count, decrease in karyocytes and increase in chromosomal aberration rate in nucleus-containing cells from the femoral bone marrow in younger animals of several groups, morphologic and histochemical shifts in mucous membrane of the esophagus and the stomach, and morphofunctional changes in neurons and glia of the cerebral cortex. These changes are compensatory-adaptive by nature and indicative of destabilization of homeostasis in experimental animals.

Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes.

The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.

Specific chemical and structural damage to proteins produced by synchrotron radiation.

Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.

[The effect of local ionizing irradiation on the presence of acetylcholinesterase-positive nerve fibers in the spleen of rats]. / Vplyv lokálneho pôsobenia ionizacného ziarenia na prítomnost ACHE-pozitívnych nervových vlákien v slezine potkana.

Changes in the distribution of ACHE-positive nerve fibres in the spleen of the rat after local irradiation have been studied by light microscopy using direct tiocholin method of El-Badawi and Schenk (1967). ACHE-positive nerve fibers enter the spleen in the vicinity of splenic artery branches and they are gradually distributed in form of perivascular plexuses in the white pulp to the periarterial lymphatic sheath (PALS). One dose of local irradiation of the head of the rat causes changes in the presence and distribution of the ACHE-positive nerve fibres in the spleen of the rat. After an intense decrease or the absence of these fibres during the period of the first days after irradiation, there is an evident restoration of ACHE-positive nerve fibres in the white pulp of the organ. The distribution and topography of these nerve structures is the same as in non-irradiated rats on the 35th day after irradiation. During the following days, there is another evident decrease in ACHE-positive nerve fibres in all described localizations of the spleen. Our findings suggest that the indirect effect of irradiation can affect some changes in innervation of the organ which was not exposed to its direct influence. (Fig. 3, Ref. 13.)

Effects of X-irradiation on nerve growth factor in the developing mouse brain.

The involvement of neurotrophins after radiation injury during brain development were studied in pregnant mice (C 57/B1) exposed on gestation day 15 to X-ray doses of 0.02-2 Gy. Nerve growth factor protein (NGF) and different cholinergic markers were investigated on postnatal day 1 (P1) and day 21 (P21); in situ hybridization with brain-derived neurotrophic factor (BDNF) and trkC (receptor serving to bind neurotrophin-3) probes was investigated on P21 in cortex, hippocampus, septum and cerebellum. The level of NGF protein was increased in irradiated forebrain on P1 in a dose-related manner. However, on P21 the NGF protein dropped down below the control levels in irradiated hippocampus and cerebellum. The response of acetylcholine esterase (AChE) activity in cerebellum at P21 was correlated with the changes in the amount of NGF. The intensity of cell labelling with trkC probe decreased after irradiation in the region of the hippocampus at P21, especially in dentate gyrus. The expression of BDNF mRNA was increased at P21 by low doses of irradiation (0.02-1 Gy) but was decreased by a high dose (2 Gy) in the same area. Thus, the radiation induced an alteration of neurotrophins, and the changes varied depending on the dose or time after irradiation. Such alterations in the pattern of growth factor production may modulate the response of cells to radiation. Furthermore, NGF protein levels and the expression of BDNF and trkC mRNA were affected by radiation doses as low as 0.02 Gy, indicating that during development the neurotrophins and their receptors are very sensitive to radiation.

Brain fixation for acetylcholine measurements.

Brain fixation using a commercially available microwave oven (power output: 750 W) has been investigated as a means for enzyme inactivation preventing post-mortem changes in brain acetylcholine (ACh) and choline (Ch) levels. Rats and mice were decapitated, and the severed heads immediately irradiated for 5.5 and 3 s, respectively, resulting in a complete inactivation of brain acetylcholine esterase (AChE) and choline acetyltransferase (ChAT). The ACh and Ch contents measured in various brain regions of rat and mouse were: (in rat) striatum 60.5 and 32.4 nmol/g, hippocampus 20.4 and 30.9 nmol/g, cortex 24.2 and 19.6 nmol/g; (in mouse) striatum 70 and 47.2 nmol/g, hippocampus 22. 1 and 30.2 nmol/g, cortex 22.9 and 27.9 nmol/g. These values were found in accordance with those reported in the literature by irradiating whole animals in instruments of higher power capabilities. Thus, the procedure described in the present work may be a simple and valuable means of brain fixation for neurochemical studies of brain ACh in small animal species.

Effect of environmental conditions on radiation target size analyses.

Target size determinations from radiation inactivation of proteins is dependent on the physical and chemical environment of the sample during radiation exposure. Effects of temperature and physical state have already been described. Buffers, the effects of protein concentration, and the addition of small molecules are examined for several enzymes. Phosphate buffer is found to have major effects on the rate of inactivation of certain, but not all, proteins. The amount of protein in irradiated samples is significant for all enzymes studied; the nature of the specific protein used is unimportant. Neither sucrose nor other glycitols could substitute for protein in target size determinations. Certain small molecules, especially cysteamine, were effective in sparing the need for high protein levels in radiation inactivation studies of four enzyme systems.

Influence of a stationary magnetic field on acetylcholinesterase in murine bone marrow cells.

A thirty-minute exposure of mice to a homogeneous stationary magnetic field (SMF) of 1.4 Tesla at either 27 degrees C or 37 degrees C body temperature causes an inhibition of about 20 per cent of acetylcholinesterase (AChE, E.C. 3.11.7) in murine bone marrow cells (BMC) after 3.5 and 2 h, respectively, at the two aforementioned body temperatures. The extent of enzyme inhibition is independent of ambient temperature, but dependent on the time after exposure. This initial inhibition of AChE activity is followed by a limited recovery which is dependent upon the temperature during exposure to the SMF and remains incomplete even 15 h afterwards. We describe here certain enzymologic properties of AChE in BMC as well as inhibition studies with diisopropylfluorophosphate (DFP) to differentiate between AChE and nonspecific cholinesterases.

Effect of amplitude-modulated radio frequency radiation on cholinergic system of developing rats.

We examined the effect of long-term exposure to radio frequency radiation 147 MHz and its sub-harmonics 73.5 and 36.75 MHz amplitude modulated at 16 and 76 Hz (30-35 days, 3 h per day) on cholinergic systems in developing rat brain. A significant decrease in acetylcholine esterase activity was found in exposed rats as compared to the control. Decrease in acetylcholine esterase (AChE) activity was independent of carrier wave frequencies. A short-term exposure did not have any significant effect on AChE activity.

A 30 kDa functional size for the erythrocyte water channel determined in situ by radiation inactivation.

The functional unit size of the water channel in rabbit erythrocytes was assessed using target size analysis following radiation inactivation. Using Radiochromic nylon dosimetry, accurate values of accumulated dose yielded an absolute target analysis, leading to direct determination of molecular size. The erythrocyte water channel functional size was shown to be 30 kDa, and is identical to the size found in rat renal proximal tubule brush border membranes (1), suggesting close homology of these two water channels. The result suggests that the 28 kDa channel-like intrinsic protein (CHIP28) recently isolated from human erythrocytes and proximal tubule (2), which is believed to form water channels of oligomeric construction may have a functional water channel activity in monomeric form.

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.

A wavelength dependent mechanism for rose bengal-sensitized photoinhibition of red cell acetylcholinesterase.

A 2-fold enhancement in the efficiency of rose bengal-photosensitized inhibition of red cell acetylcholinesterase activity was observed upon excitation of the dye in the ultraviolet (UV) (313 nm) compared to irradiation in the visible (514 or 550 nm). The measurements of efficiency of photosensitized enzyme inhibition were based on the effect produced when the same number of photons are absorbed by rose bengal (RB) at each wavelength. The mechanism for this unexpected enhancement of RB photosensitization upon UV excitation was investigated. The yield of singlet oxygen (O2(1 delta g], detected by time-resolved luminescence at 1270 nm, was independent of excitation wavelength for RB. Radicals were produced upon irradiation of RB at 313 nm but not at 514 nm as detected by bleaching of N,N-dimethylnitrosoaniline (RNO). Irradiation of RB at 313 nm but not at 514 nm appeared to cause homolytic cleavage of carbon-iodine bonds in the dye because iodine radicals, I, detected as I2 were produced with a quantum yield of 0.0041 +/- 0.0005 upon excitation in the UV. Photolysis of I2 in the presence of RNO caused bleaching of the RNO absorption at 440 nm, apparently resulting from reaction of I with RNO. Thus, the enhanced photosensitization upon UV excitation of RB is attributed to formation of I and/or RB. These results indicate that radicals, produced with low relative yield but having high reactivity compared to O2(1 delta g), can contribute to photosensitized enzyme inhibition and may represent an alternative mechanism for photodynamic therapy.

The apparent target size of rat brain benzodiazepine receptor, acetylcholinesterase, and pyruvate kinase is highly influenced by experimental conditions.

Radiation inactivation is a method to determine the apparent target size of molecules. In this report we examined whether radiation inactivation of various enzymes and brain receptors is influenced by the preparation of samples preceding irradiation. The apparent target sizes of endogenous acetylcholinesterase and pyruvate kinase from rat brain and from rabbit muscle and benzodiazepine receptor from rat brain were investigated in some detail. In addition the target sizes of alcohol dehydrogenase (from yeast and horse liver), beta-galactosidase (from Escherichia coli), lactate dehydrogenase (endogenous from rat brain), and 5-HT2 receptors, acetylcholine muscarine receptors, and [35S] butyl bicyclophosphorothionate tertiary binding sites from rat brain were determined. The results show that apparent target sizes are highly influenced by the procedure applied for sample preparation before irradiation. The data indicate that irradiation of frozen whole tissue as opposed to lyophilized tissue or frozen tissue homogenates will estimate the smallest and most relevant functional target size of a receptor or an enzyme.

[Brain metabolism of sarcoma 45-bearing rats undergoing radiation and the effect of hyperthermia on the tumor]. / Nekotorye storony metabolizma mozga krys-opukholenositelei sarkomy 45 pri radiatsionnom i gipertermicheskom vozdeistviiakh na opukhol'.

Activity of hexokinase and acetylcholinesterase and pyridoxal co-enzyme content of brain subcellular fractions were studied in rats, bearing sarcoma 45, after local exposure of the tumor to 20 Gy X-radiation and microwave hyperthermia. The carbohydrate metabolism was sharply inhibited while the pyridoxal coenzyme content and acetylcholinesterase activity increased.

[Catecholamine content and acetylcholinesterase activity in the brain of rats exposed to lasers]. / Soderzhanie katekholaminov i aktivnost' atsetilkholinésterazy v golovnom mozge krys pri lazernom vozdeistvii.

The catecholamine content was quantitated and acetylcholinesterase activity determined in rat brain locally exposed to laser radiation. It was shown that the adrenaline/noradrenaline ratio changed in the tissues under study and the cholinergic system was involved in the abscopal effect of laser radiation.