Studies on the earthy-musty odours in natural water (IV). Mechanism of earthy-musty odour production of actinomycetes

Many reservoirs for water supply have been troubled with earthy-musty odour compounds—2-methylisoborneol (2-MIB) and geosmin. Both of these compounds are terpenoid and related to the metabolite of l-methionine. An experiment using [CD₃]methionine and [¹⁴CH₃]methionine showed that the C-2 methyl group in 2-MIB originate from l-methionine. In the incubation experiment with 10–1000 mg/l of l-methionine, 2-MIB and geosmin appeared in the earlier stages, in greater amounts than in the control. The maximum production of 2-MIB and geosmin increased considerably in the experiments with 10 and 100 mg/l of l-methionine. The effective time for l-methionine addition was after 1 d. Additions after 3 and 5 d were similar to the control. In the incubation experiment with 10–1000 mg/l of folic acid, 2-MIB and geosmin increased only during the 1000 mg/l addition. There seems to be little doubt that l-methionine takes part in the metabolism of 2-MIB and geosmin.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Formation of 2-Aminobutanoic Acid from Threonine and Methionine by Mixed Rumen Ciliate Protozoa

Precursors of 2-aminobutanoic acid (2-ABA), found in the incubation medium of mixed rumen ciliate protozoa, were examined with washed or starved bacteria-free ciliates. Threonine and methionine strongly stimulated the formation of 2-ABA. Formation of 2-ABA by direct conversion of threonine and dethiomethylation of methionine was confirmed by radiotracer experiments with [U-¹⁴C]L-threonine and [carboxyl-¹⁴C] and [methyl-¹⁴C]L-methionine.     

Incorporation of Label from 13C-, 2H-, and 15N-Labeled Methionine Molecules during the Biosynthesis of 2[prime]-Deoxymugineic Acid in Roots of Wheat

The biosynthetic pathway of 2[prime]-deoxymugineic acid, a key phytosiderophore, was investigated by feeding 13C-, 2H-, and 15N-labeled methionine, the first precursor, to the roots of hydroponically cultured wheat (Triticum aestivum L. cv Minori). The incorporation of label from each methionine species was observed during their conversion to 2[prime]-deoxymugineic acid, using 2H-, 15N-, and 13C-nuclear magnetic resonance (NMR). L-[1-13C]Methionine (99% 13C) was efficiently incorporated, resulting in 13C enrichment of the three carboxyl groups of 2[prime]-deoxymugineic acid. Use of D,L-[15N]methionine (95% 15N) resulted in 15N enrichment of 2[prime]-deoxymugineic acid at the azetidine ring nitrogen and the secondary amino nitrogen. When D,L-[2,3,3,-2H3-S-methyl-2H3]methionine (98.2% 2H) was fed to the roots, 2H-NMR results indicated that only six deuterium atoms were incorporated, and that the deuterium atom from the C-2 position of each methionine was almost completely lost. [2,2,3,3-2H4]1-Aminocyclopropane-1-carboxylic acid (98% 2H) was not incorporated into 2[prime]-deoxymugineic acid. These data and our previous findings demonstrated that only the deuterium atom from the C-2 position of L-methionine was lost, and that other atoms were completely incorporated when three molecules of methionine were converted to 2[prime]-deoxymugineic acid. These observations are consistent with the conversion of L-methionine to azetidine-2-carboxylic acid, suggesting that L-methionine is first converted to azetidine-2-carboxylic acid during biosynthesis leading to 2[prime]-deoxymugineic acid. Based on these results, a hypothetical pathway from L-methionine to 2[prime]-deoxymugineic acid was postulated.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

Investigations on the effects of rape oil quality, choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs, on trimethylamine metabolism and on laying performance

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 -75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (P(linear) = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [microg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E(-10) x x(2.71), r2 = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 - 1.5 microg/g, from 2.2 - 34 microg/g and from 18.4 - 75 microg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.     

The influence of dietary vitamin E, fat, and methionine on blood cholesterol profile, homocysteine levels, and oxidizability of low density lipoprotein in the gerbil

A 90-day feeding study with gerbils was conducted to evaluate the influence of dietary vitamin E levels (25 mg/kg diet, 75 mg/kg, 300 mg/kg, and 900 mg/kg), two levels of dietary methionione (casein or casein+L-methionine (1% w/w)) and two sources of lipid (soybean oil [20%] or soybean oil [4%]+coconut oil [16%, 1:4 w/w]) upon serum lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol). In addition, this study examined the effects of diet-induced hyperhomocysteinemia and supplemental dietary vitamin E on the oxidation of low density lipoproteins. Tissue vitamin E (heart, liver, and plasma) demonstrated a dose response (P≤0.001) following the supplementation with increasing dietary vitamin E (25, 75, 300, and 900 mg/kg). In addition, tissue vitamin E levels were found to be higher (P≤0.001) in those animals receiving a combination of coconut oil+soybean oil as compared to the group receiving soybean oil solely. Blood cholesterol profiles indicated an increase (P≤0.001) in total cholesterol and LDL cholesterol by the influence of saturated fat and supplemental methionine. Low-density lipoprotein cholesterol profile demonstrated a reduction (P≤0.001) at the higher dietary vitamin E levels (300 and 900 mg/kg) as compared to the 25 mg/kg and 75 mg/kg dietary vitamin E. Plasma protein carbonyls were not influenced by dietary vitamin E nor by supplemental methionine intake. In vitro oxidation of LDL showed that vitamin E delayed the lag time of the oxidation phase (P≤0.001) and reduced total diene production (P≤0.001). On the contrary, supplemental methionine decreased (P≤0.001) the delay time of the lag phase, whereas total diene production was increased (P≤0.001). Plasma lipid hydroperoxides were significantly reduced (P≤0.05) with supplemental dietary vitamin E, whereas supplemental L-methionine (1%) resulted in a significant (P≤0.05) increase in lipid plasma hydroperoxide formation. Plasma homocysteine was elevated (P≤0.001) with supplemental dietary L-methionine (1%) as well as the inclusion of dietary saturated fat. The present data showed that 1) a combination of dietary lipids (saturated and unsaturated fatty acids) as well as vitamin E and methionine supplementation altered blood cholesterol lipoprotein profiles; 2) in vitro oxidation parameters including LDL (lag time and diene production) and plasma hydroperoxide formations were affected by vitamin E and methionine supplementation; and 3) plasma homocysteine concentrations were influenced by supplemental methionine and the inclusion of dietary saturated fat.     

氯喹对伯氏疟原虫摄取次黄嘌呤、甲硫氨酸、精氨酸及其氨基酸组成的影响

伯氏疟原虫氯喹敏感株和抗氯喹株感染的RBC,与0.4mmol/L氯喹一起培养2小时后,敏感和抗氯喹株感染的RBC,对[~3H]次黄嘌呤、[~(14)C]精氨酸和[~3H]甲硫氨酸的摄入量分别被抑制67.3％、41.8％和35.7％以及65.4％、45.6和46.9％。 感染疟原虫的小鼠,经氯喹10mg/kg肌注20小时后,各氨基酸组成,在敏感株疟原虫中普遍的较不服药的对照组上升,而在抗氯喹疟原虫中,升高的氨基酸主要是与多胺、谷胱甘肽有关,如精氨酸、鸟氨酸、甲硫氨酸、脯鼠酸、甘氨酸和半胱氨酸。     

Generation of one-carbon units in methionine-supplemented Euglena cells

The possible effect of L-methionine supplements on the folate metabolism of division-synchronized Euglena gracilis (strain Z) cells has been examined. Cells receiving 1 mM L-methionine for four cell cycles were examined for folate derivatives, prior to and during cell division. Before cell division, methionine-supplemented cells contained less formylfolate but more methylfolate than unsupplemented cells. During division, both types of folates were present in lower concentrations in the supplemented cells. Growth in methionine for 10 and 34 hr also increased the levels of free aspartate, threonine, serine, cysteine and methionine relative to the controls. Methionine-supplemented cells contained ca 50% of the 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) activity per cell of unsupplemented control cultures and specific enzyme activity was reduced ca 90%. Supplemented cells contained almost twice as much serine hydroxymethyltransferase (EC 2.1.2.1) activity per cell but comparable levels of glycollate dehydrogenase. Growth in methionine also reduced the incorporation of formate-¹⁴C] into serine, RNA, DNA, adenine and protein methionine. In contrast, incorporation of glycine-[2-¹⁴C] and serine-[3-¹⁴C] into folate-related products was not greatly altered by this treatment. Levels of radioactivity in these products suggested that formate was a more important C₁ unit source than glycine or serine when growth occurred in unsupplemented medium. It is concluded that methionine reduces formylfolate production by an effect on the cellular levels of formyltetrahydrofolate synthetase.     

原核生物中土霉味化合物二甲萘烷醇和2-甲基异茨醇生物合成研究进展

由原核生物蓝藻和放线菌引起的水体土霉异味问题备受世界的关注。本文以土霉味化合物二甲萘烷醇和2-甲基异茨醇的研究历史为主线,综述了土霉味化合物二甲萘烷醇和2-甲基异茨醇的化学特性、生物合成途径以及生物合成相关基因和酶等方面的研究进展,探讨了土霉味化合物二甲萘烷醇和2-甲基异茨醇生物合成研究的重要意义,并展望了土霉味化合物二甲萘烷醇和2-甲基异茨醇生物合成研究存在的问题及发展的方向。     

Effects of gibberellic acid and L-methionine on C1 metabolism in aerated carrot disk

Aeration of carrot storage tissue disks in water was accompanied by net folate synthesis and by changes in the specific activities of key folate-dependent enzymes. Disks aerated in 0.1 mM gibberellic acid (GA₃) for 48 hr contained higher concentrations of methyltetrahydrofolates but aeration in 5 mM L-methionine reduced net folate synthesis. Gibberellic acid also increased the specific activities of 5,10-methylenetetrahydrofolate reductase (E.C. 1.1.1.68), serine hydroxymethyltransferase (E.C. 2.1.2.1) and 5-methyltetrahydrofolate: homocysteine transmethylase. The levels of these enzymes in disks aerated in L-methionine (5 mM) were comparable or slightly higher than those of disks aerated in water. Activity of the reductase and 10-formyltetrahydrofolate synthetase (E.C. 6.3.4.3) was inhibited by L-methionine in vitro. Aeration increased ability to incorporate formate [¹⁴C] into serine, glycine and methionine. Disks aerated for 36 hr in 0.1 mM GA₃ incorporated greater amounts of ¹⁴C into free methionine but those aerated in L-methionine (5 mM) had less ability to metabolize formate and the specific radioactivities of free glycine, serine and methionine were low.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

Biosynthesis of tritium-labeled S-adenosyl-L-methionine in yeast cells

Biosynthetic preparation of S-adenosyl-L-[methyl-3H]methionine from L-[methyl-3H]methionine by cultivation of diploid yeast Saccharomyces cerevisiae (methionine-auxotrophic) in a cultural medium with the high concentration of L-methionine is described. The radiochemical purity was over 95%. Biological activity of the preparations has been shown in transmethylation reactions in the presence of the yeast homocysteine-methyltransferase.     

Methionine metabolism and ultrastructural changes with D-galactosamine in isolated rat hepatocytes

The biochemical and morphological effects of 2, 10 and 100 mM of D-galactosamine (GalN) were studied in isolated rat hepatocytes during 2 h of incubation. Lactate dehydrogenase (LDH), alanine aminotransferase (ALAT) and cell viability did not change, whatever the concentration used. The variations observed, which were dose dependent, included a large drop in ATP levels and inhibition of RNA and protein synthesis. A very high concentration of GalN was necessary, however, to induce a significant decline in methionine adenosyltransferase activity compared to control cells.The use of L-[methyl-¹⁴C]methionine during cell incubation with GalN demonstrated a decrease of S-adenosyl-L-methionine (SAMe) and an accumulation of L-methionine content related to the GalN concentration. These results suggested that an hepatotoxic agent such as GalN was able to induce disturbances of methionine metabolism.Some of the ultrastructural changes observed were different from those previously found in vivo, in rats given GalN intraperitoneally, underlining the marked difference between in vivo and in vitro intoxication.     

Effects of sodium and temperature on naloxone binding in brain tissues of a urodele amphibian

1. Partially purified brain membranes obtained from male rough-skinned newts (Taricha granulosa) were used to determine the effects of NaCl and temperature on the specific binding of the opioid receptor antagonist [3H]naloxone. 2. The addition of NaCl to the incubation medium at concentrations up to 400 mM produced a dose-related increase of the specific binding of [3H]naloxone. 3. The addition of other salts to the incubation medium had less pronounced effects: KCl and MgCl2 slightly increased and decreased, respectively, the specific binding of naloxone, and CaCl2 had no effect. 4. Results of an equilibrium saturation experiment showed that the addition of 200 mM NaCl resulted in over a 10-fold increase in the number of high affinity (KD = 0.61 nM) binding sites for naloxone, with no changes in the number of low affinity (KD = 21.8 nM) binding sites. 5. Changes in NaCl concentrations did not significantly affect either dissociation constant. 6. The binding of [3H]naloxone was temperature-dependent; it increased when the incubation temperatures were elevated from 0 degree C to 37 degrees C. 7. Results obtained for this urodele amphibian are compared with those available for other vertebrate species.     

Prostaglandin synthesis by day-6 rabbit blastocysts in vitro

Day-6 rabbit blastocysts were recovered from superovulated donor animals, washed in ice-cold Krebs-Ringer-bicarbonate (KRB) buffer, pooled and randomly allocated to polypropylene incubation tubes, usually 10 blastocysts in 1 ml KRB. The blastocysts were ruptured with a dissecting needle and incubated at 37 degrees C for periods of 1-3 h with 10 microCi [3H]arachidonic acid/tube. A control tube without blastocysts was run in each experiment. At the end of the incubation, the samples were acidified, extracted with ethyl acetate, dried down and resuspended in h.p.l.c., using a solvent system for prostaglandins (PGs), was subtracted from each experimental run in the same experiment. The remaining radioactivity constituted 0.14% of the original [3H]arachidonic acid added to each incubation tube. This was considered to have been the result of conversion of the radiolabelled arachidonic acid to prostanoids. In the absence of 10 mM-EDTA no conversion occurred, whereas in its presence peaks of radioactivity co-eluting with [3H]PGF-2 alpha and [3H]PGE-2 were seen. A third peak that eluted was either 15-keto metabolites of these PGs or PGD-2. These 3 peaks were always significantly above background, and usually did not differ from each other. No differences in amount of conversion could be related to incubation time. Addition of indomethacin (100 micrograms/ml) or radioinert arachidonic acid (10 micrograms/ml) inhibited production of [3H]PG, even in the presence of EDTA. Removal of calcium from the incubation medium was per se without effect. Addition of atropine (0.15 mM) or carbachol (0.15 mM) in the presence or absence of EDTA did not change the pattern of conversion of [3H]arachidonic acid to [3H]PGs. These experiments demonstrate that rabbit blastocysts have the capacity for de-novo synthesis of PGs from exogenous substrate, when utilization of endogenous substrate is inhibited. The extent of conversion observed may not be a true reflection of the capacity for conversion of endogenous substrate.     

Investigations on the effects of rape oil quality,choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs,on trimethylamine metabolism and on laying performance

Abstract

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000 mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 – 75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (p _linear = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [μg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E^?10 ? x^2.71, r² = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000 mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 – 1.5 μg/g, from 2.2 – 34 μg/g and from 18.4 – 75 μg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.     

Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10^–5M and 10^–8M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with [³H] and [³⁵S] methionine and [³H]ethanolamine as precursors showed an increased methylation of [³H]ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to [³H]choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with [³H]- and [³⁵S]methionine. This was confirmed after electrophoretic separation of a protein extract with increased³H-and³⁵S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when [³H] methionine was the precursor. The treatment with gangliosides increased the incorporation of [methyl-³H] label after incubation of neurons with [³H] methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.     

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2-¹³C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using ¹³C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-¹³C]glycine, cell extracts and incubation media were analyzed for ¹³C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-¹³C]glycine, the amount of de novo synthesized [2-¹³C]creatine was two-fold increased, and in addition, ¹³C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-¹³C]glycine was utilized for the synthesis of glutathione in astroglial cells. ¹³C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-¹³C]serine, [3-¹³C]serine, and [2,3-¹³C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.     

Growth stimulating activity on 3T3 fibroblasts of the molecular weight 6,500-peptide purified from rat pancreatic juice

Growth stimulating activity of the molecular weight 6,500-peptide purified from rat pancreatic juice was measured on 3T3 fibroblasts. This peptide was reported to be a cholecystokinin-releasing peptide and to stimulate pancreatic enzyme secretion in the rat small intestine in response to food intake. Incorporation of [3H]thymidine and [35S]methionine into 3T3 was significantly stimulated and the cell number was also increased after 24-48 hr incubation with 10-100 ng/ml of the peptide. The increase in [3H]thymidine incorporation was dose-related and started 12 hr after the incubation, a peak being reached 24 hr after the incubation. These results show that this peptide exhibits growth stimulating activity to the mammalian cells, and suggest that the peptide might have a physiological effect in vivo.