Tritiated Glycine Research Articles

Synthesis of Deuterated or Tritiated Glycine and Its Methyl Ester

Heating glycine (Gly) and methyl glycinate (GlyOCH3) supported on 5% Pd/C or 5% Pt/C in a deuterium or tritium gas atmosphere gave the isotope-labeled products. The experiments were carried out at 180°C for 10 min. The deuterium atom inclusion under these conditions averaged up to 1.8 atoms per molecule for Gly and up to 1.0 atom per molecule for GlyOCH3. The reaction with tritium gas gave labeled products with a specific radioactivity of 27–31 Ci/mmol for Gly and 18 Ci/mmol for GlyOCH3.

Tissue-specific incorporation and genotoxicity of different forms of tritium in the marine mussel, Mytilus edulis

Marine mussels (Mytilus edulis) were exposed to seawater spiked with tritiated water (HTO) at a dose rate of 122 and 79 μGy h−1 for 7 and 14 days, respectively, and tritiated glycine (T-Gly) at a dose rate of 4.9 μGy h−1 over 7 days. This was followed by depuration in clean seawater for 21 days. Tissues (foot, gills, digestive gland, mantle, adductor muscle and byssus) and DNA extracts from tissues were analysed for their tritium activity concentrations. All tissues demonstrated bio-accumulation of tritium from HTO and T-Gly. Tritium from T-Gly showed increased incorporation into DNA compared to HTO. About 90% of the initial activity from HTO was depurated within one day, whereas T-Gly was depurated relatively slowly, indicating that tritium may be bound with different affinities in tissues. Both forms of tritium caused a significant induction of micronuclei in the haemocytes of mussels. Our findings identify significant differential impacts on Mytilus edulis of the two chemical forms of tritium and emphasise the need for a separate classification and control of releases of tritiated compounds, to adequately protect the marine ecosystem.

Heterogeneity of glutathione synthesis and secretion in the proximal tubule of the rabbit.

This study was designed to examine the synthesis and possible secretion of glutathione (GSH) in the S1, S2, and S3 segments of the rabbit proximal tubule. GSH synthesis and secretion rates were measured in the three segments of the proximal tubule, using the isolated perfused renal tubule technique. Tritiated (3H) glycine was perfused into segments and synthesized [3H]GSH (3H on the glycine residue) was measured in the bathing solution, collectate, and tubule extract. In the S1 segments, GSH was synthesized at the rate of 8.65 +/- 0.88 fmol.min-1.mm-1 tubule length and preferentially secreted into the lumen at the rate of 7.28 +/- 0.74 fmol.min-1.mm-1. The difference between synthesis and secretion appeared in the bathing solution. The S2 segment synthesized GSH at the rate of 3.88 +/- 0.82 and secreted GSH at the rate of 2.78 +/- 0.57 fmol.min-1.mm-1. GSH synthesis and secretion rates in the S3 segment were 5.45 +/- 1.19 and 4.22 +/- 1.16 fmol.min-1.mm-1, respectively. Cellular concentrations of [3H]GSH increased along the length of the proximal tubule, with the highest concentrations in the S3 segment. The respective GSH cellular concentrations in the S1, S2, and S3 segments were 35.89 +/- 10.51, 49.65 +/- 9.32, and 116.90 +/- 15.76 microM. These findings indicate that there is heterogeneity of GSH synthesis along the proximal tubule and that synthesized GSH is secreted preferentially into the lumen.

Effect of magnesium sulfate on excitatory amino acid receptors in the rat brain: I. N-Methyl-d-aspartate receptor channel complex

OBJECTIVE: Our purpose was to determine the effect of peripherally administered magnesium sulfate on the N-methyl-D-aspartate receptor channel complex in the rat central nervous system. STUDY DESIGN: Six rats were injected intraperitoneally with 270 mg/kg magnesium sulfate, followed by 27 mg/kg every 20 minutes for 4 hours. Controls (n = 6) received saline solution. Six rats received intraperitoneal injections of magnesium sulfate (270 mg/kg) every 4 hours for 24 hours and 6 received saline solution. Six rats received intraperitoneal magnesium sulfate (270 mg/kg) every 12 hours for 2 weeks and 6 received saline solution. Rats were subsequently perfused and killed and their brains dissected and frozen. Cryostate sections were taken, labeled in vitro by one of three ligands for autoradiography assay, and mounted on tritium-sensitive film for 4 weeks. The ligands were tritiated glutamate agonist, N-methyl-D-aspartate binding site; tritiated glycine agonist, glycine binding site; and tritiated MK-801 noncompetitive antagonist, channel site. Optical density measurements of binding of 11 brain regions on each section were performed with an image analyzing system. RESULTS: N-methyl-D-aspartate receptor binding in the hippocampus was higher than in all other brain regions in all three experiments. Systemic administration of magnesium sulfate for 24 hours resulted in reduced tritiated glutamate binding, whereas long-term administration (2 weeks) resulted in significantly decreased tritiated glycine binding in all brain regions sampled. Binding of tritiated MK-801 was significantly increased in both short- and intermediate-term administration of magnesium sulfate. CONCLUSIONS: These data suggest that short-term magnesium sulfate administration results in increased inhibition of the ion channel. This effect is also continued with prolonged treatment, along with decreased sensitivity of the N-methyl-D-aspartate receptor channel complex to its agonists glutamate and glycine. This proposed time-dependent, twofold effect may provide insight into the mechanisms of magnesium sulfate's central anticonvulsant effect. (Am J Obstet Gynecol 1996;175:575-81.)

Development of glycinergic cells and puncta in nuclei of the superior olivary complex of the postnatal ferret

The distribution of glycine-immunopositive cells and axonal endings was studied in the adult and early postnatal ferret superior olive. As in other species, the most prominent glycine-immunopositive cell group in the adult ferret superior olive was the medial nucleus of the trapezoid body. Other darkly immunostained cells were present, although more scattered, in most periolivary regions, including the lateral and ventral trapezoid body nuclei. In the lateral superior olivary nuclei, glycine-immunopositive cells were intermingled with immunonegative cells. A comparable population of cells in the ipsilateral lateral superior olivary nucleus was retrogradely labeled in cases with unilateral injections of tritiated glycine in the inferior colliculus. Glycine-immunopositive puncta were widely distributed in the neuropil in most periolivary regions, including dense accumulations in the dorsomedial periolivary region and ventral and lateral nuclei of the trapezoid body. In the lateral and medial superior olivary nuclei, immunopositive puncta were distributed around the principal cells in characteristic perisomatic halos. In postnatal ferrets, immunopositive cell bodies were first observed by postnatal day 7 and were distributed in regions comparable to regions in the adult, with the exception that immunopositive cells in the lateral superior olivary nucleus did not appear until about postnatal day 28. There was diffuse staining in the neuropil in principal and periolivary nuclei by postnatal day 7. During the third postnatal week, the immunostaining in the neuropil began to take on a more granular appearance and immunopositive puncta could be seen by postnatal day 35. In the lateral and medial superior olivary nuclei, the earliest distribution of immunostaining in the neuropil was nonuniform, being greater in the high-frequency, medial, and ventral regions, respectively. The density gradient in these areas was gradually eliminated over the next 2 postnatal weeks as immunostained processes and endings appeared over greater portions of the nuclei.

Neutral Amino Acid Uptake by Microvillous and Basal Plasma Membrane Vesicles from Appropriate- and Small-for-Gestational Age Human Pregnancies

Fetal growth depends upon nutrient availabilty in the maternal circulation as well as appropriate transfer across the placenta via known transport mechanisms in the maternal- and fetal-facing plasma membranes of the syncytiotrophoblast. In this study, microvillous (maternal-facing) and basal (fetal-facing) plasma membrane vesicles were isolated from the placentas of appropriate-for-gestational age (AGA) and small-for-gestational age (SGA) human pregnancies and the uptakes of tritiated leucine, alanine, and glycine were determined. There was no significant difference in the uptakes of either alanine or leucine by the microvillous and basal vesicles from AGA and SGA pregnancies. In contrast, glycine uptake by both microvillous and basal membrane vesicles from SGA pregnancies was significantly less than vesicles from AGA pregnancies. Glycine is delivered to the umbilical circulation in amounts only minimally in excess of its accretion into protein resulting in a narrow margin of safety for fetal growth. The ...

1H and3H NMR study of tritiated glycine and alanine isotopic mixtures

A method has been developed for the analysis of deuterium water solutions of tritiated glycine and alanine complex isotopic mixtures by using high resolution3H (at 266.8 MHz) and1H (at 250.13 and 500.13 MHz) NMR spectra. Approaches have been worked out for the determination of quantitative composition of mixtures and spectral parameters.

Distribution of glycine receptor immunoreactivity in the spinal cord of the rat: cytochemical evidence for a differential glycinergic control of lamina I and V nociceptive neurons.

In this study we characterized the distribution of glycine receptor immunoreactivity in the spinal cord of the rat by using monoclonal antisera directed against the purified glycine receptor. There was dense, punctate glycine receptor immunoreactive staining in all regions of the gray matter ventral to the substantia gelatinosa. The densest staining was found in laminae III and IV of the dorsal horn. There were also distinct, tributarylike bands of punctate staining that extended well into the white matter of the lateral and ventral funiculi. The only consistent cell body staining was found in small neurons of the ventral horn. The labelled neurons were distributed among larger, unlabelled motoneurons. In general, the pattern of glycine receptor immunoreactivity was similar at all levels of the spinal cord and was comparable to that seen with binding of a tritiated glycine receptor antagonist, strychnine, to sections of rat spinal cord (Zarbin et al.: J. Neurosci. 1:532-547, '81). Two important exceptions, however, were observed. In contrast to the high levels of strychnine binding reported in the substantia gelatinosa, we found almost no glycine receptor immunoreactivity in laminae I and II of the superficial dorsal horn of the spinal cord or of the trigeminal nucleus caudalis. There was also a notable absence of antibody staining in the intermediolateral cell column of the thoracic cord. The presence of dense glycine receptor immunoreactivity in the region of lamina V and its absence in the superficial dorsal horn are discussed in terms of a possible differential glycinergic control of nociceptive neurons of laminae I and V.

52] Purification of 5-aminolevulinate synthase

Publisher Summary 5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the formation of 5-aminolevulinic acid from glycine and succinyl- CoA. The enzyme has been purified from a wide variety of sources, but the higher levels in R. spheroides make this organism the choice for isolation of the larger amounts required for biosynthetic studies on tetrapyrroles. In the assay of the enzyme, 5-Aminolevulinic acid, produced enzymatically, is first condensed with acetyl acetone and the resulting pyrrole is estimated by reaction with modified Ehrlich's reagent. The enzyme 5-aminolevulinate synthase is purified by a procedure which incorporates some stages from the original method of Warnick and Burnham. All operations are carried out at 0–4°. The purification steps of the enzyme includes: disruption of the bacterial cells, salmine sulfate fractionation, ammonium sulfate fractionation, DEAE cellulose column chromatography, calcium phosphate column chromatography, and DEAE-Sephadex column chromatography. The purified enzyme may be utilized for the enzymatic synthesis of tritiated or deuterated 5-aminolevulinic acid (at positions 2 and 3) from appropriately labeled succinic acid precursors. The enzymatic synthesis of 5-aminolevulinic acid stereospecifically labeled at the more sensitive C-5 position using tritiated glycine as the labeled precursor required 5-aminolevulinate synthase of specific activity at least 80 units/mg. The chapter also discusses the molecular properties of 5-aminolevulinate synthase, enzyme activation, and enzyme mechanism.

Amino acid and protein metabolism in dorsal root ganglia of rabbits with experimental allergic neuritis.

Amino acid and protein metabolism has been studied in the dorsal root ganglia of rabbits with experimental allergic neuritis (EAN). The concentrations of a number of nonessential amino acids (glutamine, serine, aspartate, and glutamate) were reduced in the spinal ganglia of EAN animals without any comparable change in the blood plasma. The short-term influx of glycine and GABA was decreased in EAN animals, whereas that of histidine and valine was not altered. The prolonged accumulation of all the four amino acids was unchanged. These results suggest alterations in the cell metabolism of the dorsal root ganglia, rather than unspecific changes in cellular permeability. Furthermore, incorporation of tritiated valine, histidine, and glycine into proteins of EAN-ganglia in vitro was significantly increased. Autoradiography of the protein-bound [3H]-valine indicated alterations in the protein synthesis of the ganglion neurons: A decreased grain density was found in ganglion neurons of EAN animals. The increased grain densities in the affected ganglia were observed in macrophages, and possible in activated Schwann cells, over the demyelinated spots. The results suggest intraneuronal changes in the dorsal root ganglia of amino acid and protein metabolism, possibly in response to peripheral axonal injury and/or to nonspecific cytotoxic effect of active lymphocytes and macrophages.

Effect of estradiol and relaxin on collagen and non-collagen protein synthesis by mammary fibroblasts

In order to determine the effect of relaxin and estradiol on collagen and noncollagen synthesis by mammary gland fibroblasts, fibroblasts isolated from guinea pig mammary glands were grown on plastic or Cytodex-3 collagen coated microcarriers. On plastic, estradiol (600 pg/ml) increased the incorporation of tritiated glycine into collagen. Non-collagenous protein synthesis was increased at all concentrations of estradiol, but it was greatest with 200 pg/ml estradiol. On Cytodex-3, 400 pg/ml estradiol increased the synthesis of collagen and non-collagenous protein. Relaxin (1 μg/ml) did not affect collagen synthesis but decreased the synthesis of non-collagenous protein.

Synthesis of deuterium and tritium labelled phenyl glycine

A new method of synthesis of phenyl glycine labelled with deuterium and tritium was elaborated. Labelled phenyl glycine was obtained by isotope exchange method between phenyl glycine and deuterated or tritiated water at elevated temperature in hydrochloric acid medium using K2PtCl4 as a catalyst. 37 GBq /1 Ci/ HTO was used for the synthesis of tritiated phenyl glycine and labelled product with specific activity of 185 MBq/mole /5 mCi/mole/ was obtained.

Hepatic proline after bile duct ligation in rats.

Since biliary hyperplasia of fascioliasis correlated with hepatic proline level, we examined the occurrence of a similar chemical stimulus during bile obstruction. Uptake of tritiated proline and glycine rose in both hepatocytes and a bile duct enriched cell fraction, following duct ligation in rats. The increased hepatic content of proline but not glycine suggests that proline has a role in post-obstructive biliary proliferation.

Selective uptake of tritiated glycine, GABA and d-aspartate by retinal cells in culture: a study using autoradiography and simultaneous immunofluorescence

Autoradiographic studies on the uptake of 3 tritiated amino acids by cell cultures of rat retina are reported. At 0.2 μM [ 3H]-GABA is taken up with great selectivity by a minority of medium to large multipolar cells. At 2 μM [ 3H]glycine is taken up with slightly less selectivity by a similar proportion of multipolar cells; the proportions of cells labeled by either amino acid are additive which indicates that they are separate populations. In simultaneous autoradiographical and indirect immunofluorescence experiments, both of these cell classes were found to bind tetanus toxin and are therefore presumably neurons. At 0.4 μM [ 3H] d-aspartate is preferentially taken up by virtually all small spheroid cells which lack long processes (putative immature photoreceptors), as well as by a minority of larger multipolar cells. All 3 tritiated amino acids also label flattened cells which include retinal glia. The results are discussed in relation to similar experiments performed on undissociated retina and we conclude that these experiments allow us to identify and quantify immature photoreceptors and certain sub-types of amacrine cells in these cultures.

Energetics and possible mechanisms of ion-beam protein tritiation based on AB initio potential surfaces.

Ab initio self-consistent field potential energy surfaces for the approach of T, T2, T+, T3+ and HeT+ to glycine in the gas phase have been determined and this data used to obtain insight into mechanisms of experimental ion-beam protein tritiation processes. Results of these calculations show that the ionic species T+, T3+ and HeT+ can form stable adducts with glycine (Gly) and that each functions as a tritiation agent forming the complex GlyT+. Neutral T and T2 experience a purely repulsive interaction with Gly and do not form an intermediate complex. These neutral species are expected to be less effective tritiation agents than the respective ions, in agreement with experimental observations. The fate of the stable GlyT+ complex is discussed and it is proposed that this species is neutralized by electron capture to give GlyT which spontaneously dissociates to either Gly+T or tritiated glycine (Gly*)+H, with the latter reaction product channel favored statistically. The most likely site of exchange is predicted to be at the amine nitrogen although significance exchange is expected to occur at the alpha-carbon site by a somewhat more complex reaction mechanism.

The nature of formate metabolism in greening barley leaves

The metabolism of [ 3H]formate has been examined in etiolated and greening leaves of barley ( Hordeum vulgare), dwarf bean ( Phaseolus vulgarls), broad bean ( Vicia faba) and corn ( Zea mays). Tritium was extensively incorporated by primary leaves incubated for 20-min periods in light or dark. The organic acids and free amino acids were the principal products of formate metabolism but these and other products were more heavily labelled in green tissues. Time course experiments with barley leaves revealed a rapid labelling of serine, accompanied by increasing amounts of 3H in glycine and aspartate as the feeding period was extended. These amino acid products were formed throughout a 4-day greening period with an approximate doubling in total incorporation being due to large accumulations of tritiated glycine and aspartate. The involvement of tetrahydrofolate-dependent reactions in formate metabolism was indicated by inhibition of [ 14C] and [ 3H]formate incorporation by the folate antagonist, aminopterin. Labelling of glycine and serine was also strongly inhibited (up to 90%) when the leaves were incubated with increasing concentrations of isonicotinylhydrazide.

Die Entwicklung der Sch�delknochen des wachsenden Hamsters

Hamsters were labelled by tritiated glycine 1,3, 5,7,9, and 17 days after birth. The radioactivity in bone matrix and osteogenic cells was studied. The desmal ossification showed no differences in matrix production patterns in comparison to ossicles and alveole of the mandible. Activity indicating an increased glycine turnover at the bone production site appeared at the same time as osteoid could be recognized in the HE staining.

Effect of microwave radiation on cells treated with membrane-injuring substances

WISH cells grown in vitro were pretreated with subcytotoxic concentrations of digitonin, cortisol and purified bacterial toxins -- staphylococcal beta-haemolysin or Clostridium perfringens alpha-toxin and irradiated with 3 GHz electromagnetic wave (microwaves) at the field power densities 5 or 40 mW/cm2. At 40 mW/cm2 increase in temperature of the culture medium of about 2-3 degrees C was noted, while at 5 mW/cm2 no detectable increase in temperature was found. Control and pretreated WISH cells after irradiation in the microwave field were used for evaluation of their viability, incorporation of tritiated thymidine, glycine and uridine and level of intracellular cyclic AMP. Irradiation with microwaves resulted in lowering of thymidine and glycine incorporation along with changes in the intracellular amount of cAMP (decrease in cells exposed to 5 mW/cm2 and increase in those exposed to 40 mW/cm2). Under both conditions viability of the cultures was normal. Pretreatment of cells with digitonin or purified bacterial toxins followed by irradiation with microwaves resulted in enhancement of the cytotoxic effect with lowering of cell viability, especially after exposition to power density of 40 mW/cm2. Cortisol led to decrease in 3H-glycine and 3H-uridine incorporation into WISH cells, but did not influence the reaction of the cells to microwave radiation. In view of the results presented it may be concluded that substances injuring cell membranes sensitize cell cultures to electromagnetic radiation of the microwave range and may enhance the specific (non-thermal) effect of microwaves.

Effect of salicylate on the surgical inducement of joint degeneration in rabbit knees

Degenerative joint changes were produced in one knee of each of fourteen rabbits by surgical induction of instability. The involved knee in rabbits with and without systemic salicylate treatment was compared with the knee not operated on. Salicylate did not significantly change the activities of lysosomal enzymes in cartilage or synovial fluid, the uptake of tritiated thymidine, glycine, or 35S-inorganic sulphate by cartilage, or the histological manifestations of cartilage degeneration.

THE ABSORPTIVE CAPACITIES OF BROMELIAD TRICHOMES

Tritiated glycine and leucine and microautoradiographic techniques were employed to determine whether various bromeliad species produce foliar trichomes capable of absorbing these two solutes from solutions placed on intact leaf surfaces. All trichomes borne by members of subfamily Tillandsioideae, whether on the leaf blade or sheath, accumulated both amino acids in substantial quantities. Trichomes located on the leaf sheaths of tank‐forming members of Bromelioideae absorbed much smaller quantities while trichomes located on the blades of the same tank‐producing bromelioids, as well as those of nontank‐forming members of Pitcairnioideae and Bromelioideae, took up little or none at all. The evolution of the bromeliad trichome is considered in the light of these results.