Radioprotective effect of 2-mercaptopropionyl glycine on radiation-induced lipid peroxidation and enzyme release in erythrocytes

gamma-Irradiation of erythrocyte suspensions resulted in lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and from erythrocytes of high and low concentrations was observed at 480 and 320 Gy, respectively. This implied that the extent of enzyme release is likely to be masked when only a single dose of radiation is used, unless it happens to be an optimum dose. MPG mediated inhibition of lipid peroxidation and enzyme release indicated that lipid peroxidation may induce the breakdown of the phosphatidylinositol/enzyme interaction. Further, the enzyme damage was assigned to the direct and indirect effects of radiation on the enzyme in situ.     

Synthesis and photochemistry of photolabile N-glycine derivatives and effects of one on the glycine receptor.

Three photolabile precursors of glycine containing a photosensitive 2-nitrobenzyl moiety attached to the amino group have been synthesized. When exposed to ultraviolet radiation between 308 and 350 nm, the compounds photolyze to release glycine, an important inhibitory neurotransmitter in the central nervous system. The identification of glycine as a photolysis product was determined by two different methods: separation of the photolyzed sample by thin-layer chromatography followed by a reaction with ninhydrin, and recognition of derivatized glycine using the Waters pico-tag method in conjunction with high-performance liquid chromatography. The photolysis of these compounds at 22 degrees C has been investigated, and the rate of decay of a transient intermediate in the reaction, which is assumed to reflect product release, has been measured. For N-(alpha-carboxy-2-nitrobenzyl)glycine this decay rate was found to be 940 s-1 at pH 6.8 and 600 s-1 at pH 7.5. Additionally, this compound was found to exhibit biological activity upon photolysis; cultured mouse spinal cord cells containing neuronal glycine receptors were used to detect the glycine liberation. The approach adopted here is useful in demonstrating the utility of photolabile precursors of neurotransmitters that have the protecting group linked to the neurotransmitter through the amino group. The rapid photolysis of such compounds to release free neurotransmitter is valuable in gaining access to chemical kinetic studies of neurotransmitter receptors. Previously, such studies have been limited because the available methods for neurotransmitter delivery did not give a sufficiently high time resolution.     

An inquiry into the selective protection of glycine during the radiolysis of glycine-alanine mixtures in aqueous solutions and its implications to the preservation of optically active amino acids in the early earth

Akaboshi et al. (1990) has found an unexpected protection of the achiral amino acid, glycine, towards ionizing radiation at the expense of the selective destruction of the chiral amino acids, alanine and aspartic acid. The present work examines the mechanism of this protection for the case of alanine. We have developed a computer model for the radiolysis of glycine, alanine and glycine-alanine mixtures in aqueous solution. It is established that this protection is due in part to the reaction of the α-radical of glycine with alanine to regenerate a more stable α-radical, according to the following reaction, $$ \cdot CH(NH_3^ + )CO_2^ - + CH_3 CH(NH_3^ + )CO_2^ - \to CH_2 (NH_3^ + )CO_2^ - + CH_3 \dot C(NH_3^ + )CO_2^ -$$ The rate constant of this reaction was estimated to be ≤10⁴M^-1s^-1. The implications for this selective protection of glycine are considered for a hypothetical case in which there would be an enrichment of about 10% ofL-alanine in the primitive ocean and taking the glycine/alanine ratios obtained in CH₄-and CO₂- dominated atmospheres using electric discharge experiments. It is predicted that alanine would be rapidly destroyed and radioracemized in spite of the fact that the concentration of alanine is equal or significantly lower than that of glycine. Assuming that chiral amino acids were a prerequisite for the origin of life, it can be deduced that life could have appeared in a relatively short period of time unless there was a constant supply of optical amino acids from extraterrestrial sources.     

The photolytic degradation and oxidation of organic compounds under simulated Martian conditions

Summary Cosmochemical considerations suggest various potential sources for the accumulation of organic matter on Mars. However the Viking Molecular Analysis did not indicate any indigenous organic compounds on the surface of Mars. Their disappearance from the top layer is most likely caused by the combined action of the high solar radiation flux and various oxidizing species in the Martian atmosphere and regolith. In this study the stability of several organic substances and a sample of the Murchison meteorite was tested under simulated Martian conditions. After adsorption on powdered quartz, samples of adenine, glycine and naphthalene were irradiated with UV light at various oxygen concentrations and exposure times. In the absence of oxygen, adenine and glycine appeared stable over the given irradiation period, whereas a definite loss was observed in the case of naphthalene, as well as in the volatilizable and pyrozable content of the Murchison meteorite. The presence of oxygen during UV exposure caused a significant increase in the degradation rate of all samples. It is likely that similar processes have led to the destruction of organic materials on the surface of Mars.     

条件必需氨基酸对放射损伤大鼠血浆蛋白的影响

观察条件必需氨基酸对放射损伤大鼠血浆蛋白的影响。以雄性Wistar大鼠 6 9只 ,普通饲料喂养 6天 ,随机分成A、B、C、D四组 ,A、B、C组各 2 0只、D组 9只。A、B、C三组动物均接受 6 .5Gy的γ线全身照射 ,D组动物不照射。照射后在A组饲料中添加 3%精氨酸 +5 %牛磺酸 +1%谷氨酰胺 ;B组饲料中添加与A组等氮量的甘氨酸 (10 .91% ) ;C组及D组仍喂普通饲料。照射后 7及 14天测血浆总蛋白、白蛋白、前白蛋白、纤维结合蛋白。结果显示A组在饲料摄入量、体重及血浆总蛋白、白蛋白、前白蛋白、纤维结合蛋白水平等方面均优于B组及C组。提示饲料中增加条件必需氨基酸含量可以改善进食 ,促进体重恢复及蛋白质合成 ,进而提高受照大鼠应激能力     

The biochemical basis for the conjugation of bile acids with either glycine or taurine

All animals, except for the placental mammals, conjugate their bile acids exclusively with taurine. However, in certain of the placental mammals, glycine conjugates are also found. The basis for the appearance of glycine conjugation among the placental mammals was investigated. The reaction of choloyl-CoA with glycine and taurine, as catalysed by the soluble fraction from guinea-pig liver, had a high affinity for taurine and a poor affinity for glycine. The predominant synthesis of glycine conjugates in the guinea pig can be related to the fact that guinea-pig liver contains an unusually low concentration of taurine and a high concentration of glycine. Rabbits make exclusively glycine conjugates and their livers also contain low concentrations of taurine. However, the biochemical basis for their glycine conjugation is more straightforward than in the guinea pig in that the soluble fraction from rabbit liver has a high affinity for glycine and a poor affinity for taurine. Alternative-substrate-inhibition studies with glycine and taurine in soluble fractions from guinea-pig and rabbit liver revealed that glycine and taurine were mutually inhibitory. This suggests that there is only one enzyme for glycine and taurine conjugation in these tissues. The soluble fractions from bovine liver and human liver also made both glycine and taurine conjugates and evidence is presented that suggests that there is only one enzyme in these tissues too. Even the rat, which excretes mostly taurine conjugates, could make both glycine and taurine conjugates in vitro. However, in contrast with all of the placental mammals studied, the supernatant fraction from liver of the chicken, and other non-mammals, could not make glycine conjugates even in the presence of very high concentrations of glycine.     

Nucleophilic addition of glycine esters to Gd@C82

The first investigation of nucleophilic addition of glycine esters to Gd@C₈₂ is reported and hydroxyl was found to compete with glycine esters in the reaction. The multiple adducts containing either mere glycine esters or both glycine esters and hydroxyl group were identified by MALDI TOF mass spectrometry and characterized with UV-Vis-NIR spectrometry. Because glycine ethyl ester is more soluble than glycine methyl ester in alcohol and toluene, as many as eight glycine ethyl ester groups can be added to the metallofullerene cage while the maximal number for glycine methyl ester groups is only four.     

Selective decomposition of either enantiomer or aspartic acid irradiated with60Co-γ-rays in the mixed aqueous solution with D- or L-alanine

Aqueous solutions of various amino acids were irradiated with⁶⁰Co--rays, and subsequently the remaining amino acids were analyzed using HPLC. The D₃₇ for the 1 mM glycine and alanine solutions were 1.95×10⁴ and 1.48×10⁴ Gy, respectively. However, when the mixed solutions of glycine and alanine (each in 0.5 mM) were irradiated under the identical condition, the D₃₇ for the glycine decomposition increased to 3.56×10⁴ Gy, while that for alanine decreased to 0.65×10⁴ Gy. A similar phenomenon was observed also in the case of the mixed solutions of aspartic acid and alanine. Namely, aspartic acid was protected from the attack of radiation by the presence of alanine in the solutions. The most interesting finding in this combination experiment is that, when D,L-aspartic acid was irradiated in the presence of L-alanine, the radiation-sensitivity of L-aspartic acid decreased selectively and vice versa. Namely, the asymmetric field induced in the solutions by adding D- or L-alanine might affect the radiodecomposition rate of either aspartic acid. Addition of glycine to D,L-aspartic acid did not bring about the asymmetric decomposition. It seems that some interaction between these amino acid molecules resulted in this effect.     

Covalent structure of fibrinopeptides from buffaloes breeding in Italy.

The primary structure of fibrinopeptides A and B from buffaloes breeding in Italy has been determined with a view to establishing whether this animal is an autochthonous species or is the result of recent radiation mutation. Some differences exist at the morphologic and physiologic levels between the Indian buffalo and that breeding in Italy, But they do not allow a clear evolutionary line to be traced between these species. The amino acid sequences, if compared with homologous sequences of the Indian buffalo, show one difference in the most variable region of fibrino-peptides A, in particular the substitution of a serine residue by a glycine residue in position 8. This difference supports the hypothesis of the autochthonous origin of the Italian buffalo.     

Activity coefficients of the electrolyte and the amino acid in water + NaNO(3) + glycine and water + NaCl + DL-methionine systems at 298.15 K

The activity coefficients at 298.15 K of glycine in water + NaNO(3) + glycine system and dl-methionine in water + NaCl + dl-methionine system are reported. The measurements were performed in an electrochemical cell with two ion selective electrodes, a cation and an anion ion selective electrode, each versus a double junction reference electrode. The concentrations of the electrolytes and the amino acids studied covered up to 1.0 molality electrolyte, 2.4 molality glycine and 0.2 molality dl-methionine. The results of the activity coefficients of glycine are compared with the activity coefficients of glycine in water + NaCl + glycine and water + KCl + glycine systems, obtained from the previous studies. The results show that the nature of both the cation and the anion of an electrolyte have significant effects on the activity coefficient of glycine in aqueous electrolyte solutions. The results also show that there are attractive interactions between the molecules of glycine and NaNO(3) and repulsive interactions between the molecules of dl-methionine and NaCl.     

A new photolabile precursor of glycine with improved properties: A tool for chemical kinetic investigations of the glycine receptor

The synthesis and characterization of a new photolabile precursor of glycine (caged glycine) is described. The alpha-carboxyl group of glycine is covalently coupled to the alpha-carboxy-2-nitrobenzyl (alphaCNB) protecting group. Photolysis of the caged glycine with UV light produces free glycine. At 308 nm, the compound photolyzes with a quantum yield of 0.38. The absorption spectrum and the pH dependence of a transient absorption produced after laser-flash illumination are typical for aci-nitro intermediates of alphaCNB-protected compounds. The time constant for the major component of the aci-nitro intermediate decay ( approximately 84% of the total aci-nitro absorbance) was determined to be 7 micros at physiological pH. A minor component ( approximately 16%) decays with a rate constant of 170 micros. The compound does not activate or inhibit the alpha(1)-homomeric glycine receptor transiently expressed in HEK293 cells. After photolysis with a 10 ns pulse of 325 nm laser light, the glycine released from the caged compound activates glycine-mediated whole-cell currents in the same cells. The rise of these currents can be measured in a time-resolved fashion and occurs on a millisecond to sub-millisecond time scale. It can be described with a single-exponential function over >85% of the total current. The rate constant of the current rise is about 2 orders of magnitude slower than the rate constant of caged glycine photolysis. Thermal hydrolysis of the alphaCNB-caged glycine takes place with a half-life of 15.6 h at physiological pH. The new caged glycine is the first in a series of photoprotected glycine derivatives that has the required properties for use with chemical kinetic methods for investigation of glycine-activated cell surface receptors. Photolysis is rapid and efficient with respect to the receptor reactions to be studied; hydrolysis in aqueous solution is sufficiently slow, and the compound is biologically inert. It will, therefore, be a useful tool for investigation of the processes leading to channel opening of glycine receptor channels and the effects of mutations of the glycine receptor and of inhibitors on these processes.     

Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine

Glycine betaine is an osmoprotectant found in many organisms, including bacteria and higher plants. The bacterium Escherichia coli produces glycine betaine by a two-step pathway where choline dehydrogenase (CDH), encoded by betA, oxidizes choline to betaine aldehyde which is further oxidized to glycine betaine by the same enzyme. The second step, conversion of betaine aldehyde into glycine betaine, can also be performed by the second enzyme in the pathway, betaine aldehyde dehydrogenase (BADH), encoded by betB. Transformation of tobacco (Nicotiana tabacum), a species not accumulating glycine betaine, with the E. coli genes for glycine betaine biosynthesis, resulted in transgenic plants accumulating glycine betaine. Plants producing CDH were found to accumulate glycine betaine as did F1 progeny from crosses between CDH- and BADH-producing lines. Plants producing both CDH and BADH generally accumulated higher amounts of glycine betaine than plants producing CDH alone, as determined by 1H NMR analysis. Transgenic tobacco lines accumulating glycine betaine exhibited increased tolerance to salt stress as measured by biomass production of greenhouse-grown intact plants. Furthermore, experiments conducted with leaf discs from glycine betaine-accumulating plants indicated enhanced recovery from photoinhibition caused by high light and salt stress as well as improved tolerance to photoinhibition under low temperature conditions. In conclusion, introduction of glycine betaine production into tobacco is associated with increased stress tolerance probably partly due to improved protection of the photosynthetic apparatus.     

Modulation of motoneuron N-methyl-D-aspartate receptors by the inhibitory neurotransmitter glycine.

Previous studies in the central nervous system have shown that glycine is a co-agonist with glutamate at central N-methyl-D-aspartate receptors (NMDA-Rs). However, there is considerable controversy as to whether the glycine site on NMDA-Rs is saturated. If this site were not saturated then glycine released from glycinergic synaptic terminals might 'spill-over' and activate NMDA-Rs. Since motoneurons have both NMDA and glycine synapses these neurons present an optimal substrate for testing whether the glycine binding site of NMDA-Rs is activated by transmitter released from glycine synaptic terminals. Using an in vitro brainstem slice preparation we report on initial experiments to investigate whether the glycine binding site of NMDA-Rs is saturated in motoneurons. Specifically, we investigated the question of whether the response of neonatal rat hypoglossal motoneurons (HMs) to a brief application of NMDA is enhanced by the presence of exogenous glycine. We found that exogenously applied glycine (1 mM) enhanced the NMDA activated membrane current. We conclude that in brainstem slices the glycine site at motoneuronal NMDA-Rs is not saturated, and that synaptically-released glycine may modulate NMDA-Rs mediated responses.     

Electrophilic levuglandin E2-protein adducts bind glycine: a model for protein crosslinking

Prevention by glycine of protein crosslinking which accompanies binding of levuglandin E2 (LGE2) is shown to involve binding of glycine with the protein-LGE2 adduct. With ovalbumin, the LGE2 adduct initially binds nearly 2 equivalents of glycine, but the capacity to bind glycine decreases with time reflecting a competition, inter alia, with crosslinking.     

Pharmacokinetics and cerebral distribution of glycine administered to rats

High doses of glycine have been reported to improve negative schizophrenic symptoms, suggesting that ingested glycine activates glutamatergic transmission via N-methyl-d-aspartate (NMDA) receptors. However, the pharmacokinetics of administered glycine in the brain has not been evaluated. In the present study, the time- and dose-dependent distributions of administered glycine were investigated from a pharmacokinetic viewpoint. Whole-body autoradiography of radiolabeled glycine was performed, and time–concentration curves for glycine and serine in plasma, cerebrospinal fluid (CSF), and brain tissues were obtained. Furthermore, pharmacokinetic parameters were calculated. For a more detailed analysis, the amount of glycine uptake in the brain was evaluated using the brain uptake index method. Radiolabeled glycine was distributed among periventricular organs in the brain. Oral administration of 2?g/kg of glycine significantly elevated the CSF glycine concentration above the ED₅₀ value for NMDA receptors. The glycine levels in CSF were 100 times lower than those in plasma. Glycine levels were elevated in brain tissue, but with a slower time-course than in CSF. Serine, a major metabolite of glycine, was elevated in plasma, CSF, and brain tissue. Glycine uptake in brain tissue increased in a dose-dependent manner. Time–concentration curves revealed that glycine was most likely transported via the blood–CSF barrier and activated NMDA receptors adjacent to the ventricles. The pharmacokinetic analysis and the brain uptake index for glycine suggested that glycine was transported into brain tissue by passive diffusion. These results provide further insight into the potential therapeutic applications of glycine.     

Transport Is the Primary Determinant of Glycine Content in Retinal Neurons

Abstract: This study demonstrates that in mammalian and nonmammalian species it is possible to deplete selectively and reversibly retinal glycinergic neurons of their content of glycine by exposure to sarcosine, a competitive inhibitor of glycine transporter 1 (glyt-1). This observation was used as a tool to test the hypothesis that uptake of glycine rather than de novo synthesis is the main determinant of glycine content in retinal neurons. Isolated retinae were depleted of immunocytochemically detectable pools of glycine. Thereafter retinae were exposed either to physiological medium containing glycine or to medium lacking glycine but containing precursors for the synthesis of glycine. Retinae exposed to glycine-containing medium rapidly recovered their content of glycine, whereas retinae exposed to medium lacking glycine but containing serine, a substrate for synthesis of glycine, showed only a slow recovery of immunoreactivity for glycine in a few amacrine cells. These data indicate that uptake of glycine is the primary determinant of glycine content in most retinal glycinergic neurons. The origins of the extracellular pools of glycine remain to be identified; however, it is suggested that such glycine may be derived from the vitreous humor and that in turn this glycine may be derived from the peripheral circulation.     

Dietary glycine blunts lung inflammatory cell influx following acute endotoxin

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-alpha (TNF-alpha) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-alpha levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.     

Coumarin-caged glycine that can be photolyzed within 3 microseconds by visible light

The synthesis and characterization of a new photolabile precursor of glycine (coumarin-caged glycine) are reported. The new compound is suitable for rapid chemical kinetic investigations of the membrane-bound neurotransmitter receptor activated by glycine. Unlike previously used caging groups for glycine, this precursor can be photolyzed rapidly and efficiently in the visible wavelength region. This allows the use of a relatively inexpensive light source. The alpha-carboxyl group of glycine was covalently coupled to the 7-(diethylamino)coumarin (DECM) caging group. The caged compound has a major absorption band with a maximum at 390 nm (epsilon390 = 13,900 M-1 cm-1). Photolysis was performed at wavelengths of >or=400 nm (epsilon400 = 12,400 M-1 cm-1). Under physiological conditions, DECM-caged glycine is water soluble and stable. In the visible wavelength region, it photolyzes rapidly to release glycine with a half-life of approximately 2.5 micrometers and a quantum yield of 0.12 +/- 0.01. The experimental results demonstrated that neither DECM-caged glycine nor its byproduct inhibits or activates human alpha1 glycine receptors expressed on the surface of HEK 293 cells.     

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.