14C-leucine Uptake Research Articles

Role of glycoproteins in the uptake of L-14C-leucine by a synaptosomal fraction.

The present study deals with the effect of modification of membrane glycoproteins on the accumulation of amino acids by isolated synaptosomal fractions, measured by the uptake of 14C-leucine. Superficial sugar receptors were modified by concanavalin A, neuraminidase, and neuraminidase followed by concanavalin A binding. It is demonstrated that neuraminidase treatment followed by concanavalin A binding results in significant diminution of 14C-leucine uptake. A similar effect was observed in experimental hypox followed by concanavalin A binding to isolated synaptosomal fractions. In this case, however, (Na+-K+) -ATPase activity remained unchanged. These results seem to indicate that 14C-leucine uptake was dependent on integrity of glycoprotein structure of synaptosomal membranes.

Effect of whole body irradiation on different tissues. Experiments with 14C leucine in the rat.

The uptake and elimination of 14C leucine were analysed in controls and in rats irradiated 2 h before injection with 8 Gy whole-body irradiation. Plasma, small intestine, kidney and skin were assayed after homogenization for TCA soluble activity curves. In highly differentiated tissues with poor proliferative activity and low protein turnover, the uptake and elimination of the tracer did not appear to be affected by irradiation. In the small intestine differences between control and irradiated animals seemed significant.

Patterns of 14C-Leucine uptake in red spruce and larch seedlings

Summary Incorporation of 14 C-L-leucine into young spruce seedlings ( Picea abies ), a process innibited by NaF, FCCP and azide, is less sensitive to low cyanide concentrations than for larch ( Laris decidua ) seedlings, in keeping with previous observations on biosynthetic capacity.

Aflatoxin B1 influence on excised soya-bean root growth, 14C-leucine uptake and incorporation.

The present work reports a portion of our continuing effort to determine the mechanism(s) whereby aflatoxins cause toxic responses in in vitro cultured plant tissues. Few investigations have dealt with the mode of action of aflatoxin B1 (AFB1) in excised plant tissues. Here is detailed AFB1 influence on growth, uptake and incorporation of 14C-leucine by excised, incubated soya-bean roots. Pure AFB1 was added to culture medium prior to autoclaving. One gram fresh weight portions of roots from three-day old soya-bean seedlings were excised and incubated for 4, 8, 12 and 24 hours. Growth was assayed by following changes in root dry weight. Aflatoxin B1 inhibited root dry weight at both 20 and 30 micrograms/ml. Uptake of 14C-leucine was checked by following its depletion from the medium. Reduced 14C-leucine uptake by roots exposed to 20 micrograms/ml AFB1 suggests that the toxin may alter the plasmalemma. A possible role for AFB1 in modification of membrane-associated amino acid transport mechanisms is discussed. Incorporation of 14C-leucine into trichloroacetic acid-precipitable cytoplasm was assayed. Inhibition of this incorporation at 20 micrograms/ml AFB1 was most apparent at 12 hours. Thus, AFB1 may also impair the ability of excised soya-bean roots to carry out protein synthesis.

Vitellogenesis by the American cockroach: Haemolymph and follicle protein patterns during vitellogenin synthesis

Radioactive 14C-leucine is removed from the blood within 4 hr of injection during the first 2 days of the vitellogenic cycle. Injections during the 3rd to 6th day result in leucine retention and a rise in labelled protein. Label appears in the follicle by day 3 with most of the protein being incorporated during day 5. Comparison of haemolymph and follicle proteins suggests that fat body synthesis, subsequent haemolymph transport and follicle uptake all occur primarily on days 4 and 5 of the cycle. In vivo follicle incubations reveal 14C-leucine uptake during the last 4 days of the cycle. During days 4, 5, and 6, leucine is incorporated into protein by the follicle. Injections of 14C-haemolymph proteins into 6 day females result in the incorporation of label into the terminal oöcytes.

Protein synthesis with special reference to S-100 protein in brain slices from rats receiving a restricted protein supply.

The synthesis of S-100 protein and that of soluble and total proteins was investigated using cerebral slices from rats fed a 20% or 3% protein containing diet for 6 days. Incorporation of radioactive amino acids into S-100 protein was significantly higher when rats were fed a diet containing 20% protein. No significant differences were obtained in the radioactivity incorporated into total or soluble proteins between the 2 dietary groups. 14C-leucine of aspecific radioactivity of 55 mCi/mmol or 3.2 mCi/mmol incorporated with time into total protein was similar for the 2 dietary groups. The time-dependent uptake of 14C-leucine by the slices and theinulin space remained unaffected by the dietary conditions used; and amino acid analyser estimates of the free amino acid pool showed no significant differences. Brain wet weight was 1.54+/-0.02 g and1.39+/-0.02 g for protein-fed and protein-restricted rats respectively. The corresponding body weight increased by 7.8 g/day or fell by 0.5 g/day. Although the differences observed in total protein synthesis were small the synthesis of a nervous tissue specific protein S-100 was markedly affected by short-term protein restriction.

Effects of protein malnutrition during development on protein synthesis in brain and peripheral tissues

Rats born of mothers fed a low protein diet (8% casein versus a normal 25% casein diet) starting 5 weeks prior to mating showed a 50–100% increase in protein synthesis in the brain and kidney on the day of birth. This effect was due to a 50–100% increase in the uptake of IP injected 14C-leucine in the malnourished rats. The proportion of total tissue radioactivity in the trichloroacetic acid-protein precipitates was the same in the 8% and 25% casein groups. For the most part, there were no significant diet related changes in uptake or incorporation of 14C-leucine in the brain, liver or kidney in the 8% and 25% casein groups on Days 5, 10–11 and 21. While the physiological basis of the diet related changes seen on the day of birth is unknown, the present data represent a previously undescribed effect of prenatal protein malnutrition.

RNA and Protein Synthesis during Progesterone-Induced Germinal Vesicle Breakdown in R. pipiens Ovarian Tissue

Rana pipiens oocytes with intact follicle cells were induced to undergo meiotic maturation in vitro under the influence of progesterone or related steroids or with pituitary extract. Uptake and incorporation of 3H-uridine and 14C-leucine were measured using pulse-labelling techniques. A transient increase in uridine incorporation (expressed as percent intracellular acid soluble pool) occurred 1–4 h after exposure to inducing levels of progesterone with both amino acid-containing and amino acid-free incubation media; but total incorporation was greatest in amino acid-containing medium. Nanomolar concentrations of a single amino acid such as leucine, tryptophan, or arginine (but not glycine) stimulated uridine incorporation into TCA precipitable material 8–10-fold in non-induced follicles, but had little effect on total uridine uptake. Hydrocortisone or testosterone also induced germinal vesicle breakdown, but did not stimulate uridine incorporation above the control level. A comparison of pulse-labelled RNA recovered in ovulated eggs with that in follicular tissue indicated that progesterone-induced RNA synthesis occurred primarily in the oocyte. Induction in Cal+-free Ringer's solution inhibited uridine incorporation more than 95%, with no effect on total uridine uptake and only 30% inhibition of germinal vesicle breakdown. Twenty μg/ml ethidium bromide inhibited uridine incorporation about 70% in non-induced follicles and only about 30% in progesterone-induced follicles, with no effect on germinal vesicle breakdown. 10–40 μg/ml actinomycin D did not significantly effect either uridine incorporation or germinal vesicle breakdown in non-induced or progesterone-induced follicles. These findings suggest that progesterone (or its metabolites) may act at a transcriptional as well as a post transcriptional level and confirm that the induction of germinal vesicle breakdown is post-transcriptional.

In vitro uptake of labeled amino acids by red blood cells of children with protein-calorie malnutrition

The in vitro uptake of 14C-glycine, 14C-leucine and 35S-methionine by red blood cells as well as the red blood cells and plasma amino acid patterns were studied in 20 children with protein-calorie malnutrition. Blood samples were obtained from each patient on admission, following clinical recovery and, when possible, several months later. Amino acid uptake as well as levels of red blood cells and plasma amino acids are quantitatively changed in protein-calorie malnutrition. Significantly increased uptake of 14C-glycine but decreased uptake of 14C-leucine and 35S-methionine were observed in patients on admission. Following clinical recovery the uptake values of all three amino acids were markedly reduced to levels significantly below normal. Several months later values for glycine and leucine uptake were found to be normal. No significant correlation, however, was found between glycine, leucine, or methionine uptake values and concentrations of these amino groups in RBCs or plasma of patients. It is concluded that the elevations observed in red blood cell amino acids of protein-calorie malnutrition patients are not purely a result of increased amino acid uptake of RBCs and that normalization of both amino acid uptake values and their concentrations in RBCs require longer than the treatment period.

Studies of the Effect of Tenuazonic Acid on Plant Cells and Seedlings

Abstract The biological and biochemical studies of the effect of tenuazonic acid on plant cells and seedlings were carried out. Tenuazonic acid exhibited a conspicuous stunting effect on the seedling-growth of rice plant, mung bean, radish and turnip, and on the growth of suspension cultured cells of soybean and rice plants. Tenuazonic acid exhibited no effect on the O2-uptake and the activity of SH-enzyme of the plant, but inhibited the incorporation of 14C-Ieucine into the protein fraction and that of 14C-adenine into nucleic acid fraction of suspension cultured soybean cells as well as these uptake into the cells. And then it has been proved that these incorporation-inhibitions were not merely due to the inhibition of 14C-leucine and 14C-adenine uptake into the cells but based on the intrinsic inhibition of protein and nucleic acid syntheses, respectively.

Maternal hyperphenylalaninemia induced experimentally: decreased incorporation of 14C-leucine into protein in the brain of the fetus.

14C-leucine was intraperitoneally injected to pregnant rats with experimental hyperphenylal-aninemia. One hour following the 14C-leucine injection the radioactivities of amino acid fraction and protein fraction of the brain and liver were measured both in maternal and in fetal rats. Little inhibitory effect of an excess of phenylalanine was observed on the uptake of 14C-leucine by the liver or on its incorporation into the liver protein in the maternal rats. The striking inhibition of the uptake and incorporation of 14C-leucine was found in the brain of fetuses from the phenyl-alanine-loaded rats, in contrast with those of controls (p<0.001). In the fetal liver and maternal brain a similar inhibitory effect was also noted (p<0.05). These findings suggest that an excess of phenylalanine in maternal blood transfers across the placenta into the fetus, concentrates in fetal blood, and interferes with protein synthesis in fetal organs, particularly in the brain. These mechanisms may play an important role on the development of mental retardation and microcephaly of fetus in maternal PKU.

Studies of the Effect of Tenuazonic Acid on Plant Cells and Seedlings

The biological and biochemical studies of the effect of tenuazonic acid on plant cells and seedlings were carried out. Tenuazonic acid exhibited a conspicuous stunting effect on the seedling-growth of rice plant, mung bean, radish and turnip, and on the growth of suspension cultured cells of soybean and rice plants. Tenuazonic acid exhibited no effect on the O2-uptake and the activity of SH-enzyme of the plant, but inhibited the incorporation of 14C-Ieucine into the protein fraction and that of 14C-adenine into nucleic acid fraction of suspension cultured soybean cells as well as these uptake into the cells. And then it has been proved that these incorporation-inhibitions were not merely due to the inhibition of 14C-leucine and 14C-adenine uptake into the cells but based on the intrinsic inhibition of protein and nucleic acid syntheses, respectively.

Inhibition of brain protein synthesis by doses of L-DOPA that disaggregate brain polyribosomes.

Rats receiving doses of L-DOPA sufficient to cause disaggregation of brain polyribosome profiles exhibitin vivo inhibition of uptake of14C-leucine or14C-lysine into brain protein coincident with the disaggregation.

Separate biochemical actions of inhibitors of short- and long-term memory

In vitro effects of the antibiotics - cycloheximide, puromycin and chloramphenicol, and the sodium pump blockers - ouabain, lithium and copper, on protein synthesis in the crude mitochondrial, postmitochondrial and synaptosomal fractions of chicken forebrain were studied. In the synaptosomal fraction there was a correlation between the inhibitions produced by the sodium pump blockers, of Na +/K + ATPase activity, 14C-leucine uptake and the incorporation of 14C-leucine into protein; whereas the antibiotics only inhibited leucine incorporation into protein. The effects of these drugs on the in vivo incorporation of 14C-leucine into protein in various fractions of chicken forebrain were also studied. The results suggest that there is a biochemical link involving a sodium pump and the transport of amino acids, between nerve impulse activity and neuronal protein synthesis. This link may also interconvert short-term and long-term storage of memory.

Moniliformis dubius: Uptake of leucine and alanine by adults

Uptake of 14C-leucine by Moniliformis dubius in 2-min incubations was not linear with respect to substrate concentration and appeared to involve a combination of diffusion and mediated transport. During a 90-min incubation in 1 m M 14C-leucine, the total pool of free leucine increased from an initial concentration of 0.46–12.21 m M; less than 2% of the absorbed leucine was metabolized during this time period. The concentrations of leucine in the pseudocoelomic fluid and extracts of body walls, measured before and after incubation, were the same in either case. Uptake of 14C-leucine was insensitive to the external concentration of Na +, Ca 2+, Mg 2+, K +, and Tris(hydroxymethyl) aminomethane. Uptake of 14C-alanine also appeared to be mediated, however, the alanine pool was not altered after a 30-min incubation in 1 m M 14C-alanine. Following a 30-min incubation in 14C-alanine, only 38% of the absorbed radioactivity was present as labeled alanine; the remaining radioactivity was detected in aspartic acid, cysteic acid, taurine, and urea.

Growth and development of larval Taenia crassiceps (Cestoda) : III. The relationship between larval biomass and the uptake and incorporation of 14C-leucine

The rate of increase in larval biomass in the abnormal ORF strain of Taenia crassiceps was significantly greater than in the normal KBS strain during 26 weeks of infection. Larvae of both strains (in terms of biomass) increased more rapidly in female mice than in male mice. In vitro uptake and incorporation rates of 14C-leucine were found to be inversely proportional to the increase in larval biomass of both ORF and KBS strains, when measured at intervals of 3 weeks throughout a 26-week infection period. There was no correlation between larval biomass, sex of host, and ratios of uptake rate to incorporation rate of 14C-leucine, in either strain. The mean ratio of uptake to incorporation rate of 14C-leucine was significantly less in the ORF strain, indicating that ORF larvae incorporate leucine at significantly higher rates than KBS larvae.

Inhibition of selected plant systems by stereoisomers of chloramphenicol

The stereospecificity of chloramphenicol isomers on the inhibition of several plant systems was investigated. l- Threo, d- erythro, l- erythro and the antibiotic d- threo-chloramphenicol were effective inhibitors of auxin-induced elongation, 14C-leucine uptake and 14C-leucine incorporation into the protein fraction of coleoptiles from Avena sativa and Triticum vulgare. The isomers also inhibited Avena coleoptile uptake of 14C-α-aminoisobutyric acid and the de novo synthesis of α-amylase by aleurone layers from Hordeum vulgare seeds. All four compounds inhibited these processes to about the same extent and over a similar high concentration range (5 × 10 −4 to 5 × 10 −3 M). Bioassay of extracts from Avena coleoptiles treated with the non-antibiotic isomers revealed no tissue conversion into d- threo-chloramphenicol.

The Uptake of 14C-Leucine and 14C-Histidine by Cell Suspensions of Isolated Strata of Neonatal Rat Epidermis

The epidermis of neonatal rat skin is separated from the dermis with trypsin, and preparations of the four cell strata are obtained by elution with EDTA or trypsin. The four preparations contain a predominance of each of the four cell types and are adequate for the examination of protein synthesis at the different levels of epidermis. The preparations were tested for protein synthesizing activity in two ways: 1) time course of incorporation of 14 C-histidine and 14 C-leucine by whole cells, and 2) their distribution in the buffer (BSS) soluble, NaOH soluble and NaOH insoluble protein fractions obtained from homogenized cell suspensions. The specific activity ranking of the preparations was spinous cells > basal cells > granular cells. The cornified cells were inactive. Early granular cells incorporated 14 C-histidine preferentially into their NaOH insoluble fraction.

Effect of morphine on protein synthesis in synaptosomes and mitochondria of mouse brain in vivo.

The effect of morphine on incorporation of14C-leucine into proteins of synaptosomes and mitochondria of mouse brain was studied in vivo. Morphine significantly decreased the uptake of14C-leucine into the synaptosomes by about 20%. It did not affect the uptake into the mitochondria, however. The effect was dose-dependent and could be prevented by naloxone. Barbital, on the other hand, depressed the incorporation of14C-leucine into mitochondria more than into synaptosomes, thus showing a different pattern of inhibitory effects on protein metabolism. The depressing effect of morphine on uptake of14C-leucine into the synaptosomes in vivo is probably due to inhibition of axoplasmic transport of proteins, and is an effect of higher specificity than that of barbital.

Incorporation of 14C-leucine into brain protein in rats with hyperaminoacidemia.

In in vivo experiments on rats with experimentally induced hyperaminoacidemia, it was observed that when the plasma concentration of a single amino acid, such as phenylalanine, methionine, histidine or valine was elevated, both the uptake of 14C-leucine by the brain and the incorporation of 14C-leucine into the brain protein were inhibited. These findings suggest that the reduction of brain protein synthesis due to an imbalance of amino acid concentrations in serum may play an important role in the development of the brain damage seen in inbor, errors of amino acid metabolism.