14C-leucine Uptake Research Articles

MP83-11 Y+LAT2 (SLC7A6) EXPRESSION IN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-11 Y+LAT2 (SLC7A6) EXPRESSION IN CASTRATION RESISTANT PROSTATE CANCER Hideo Otsuki, Toru Kimura, Takeo Kosaka, Takashi Yamaga, Jun-ichi Suehiro, and Hiroyuki Sakurai Hideo OtsukiHideo Otsuki More articles by this author , Toru KimuraToru Kimura More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Takashi YamagaTakashi Yamaga More articles by this author , Jun-ichi SuehiroJun-ichi Suehiro More articles by this author , and Hiroyuki SakuraiHiroyuki Sakurai More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2579AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters can be a novel therapeutic strategy. One of leucine transporters, L-type amino acid transporter (LAT) 1 (SLC7A5), a Na+-independent amino acid transporter, has been reported to be selectively expressed in many cancer cells. Recently it has been shown that primary prostate cancer expresses LAT3 (SLC43A1), while castration resistant or highly aggressive prostate cancer expresses LAT1 as a main leucine transporter. In this study, we examine leucine transporters during acquisition of hormone independence. METHODS A new ″LN-abl″ cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is a model of castration resistant prostate cancer (CRPC) with androgen receptor expression. Uptake of 14C leucine was examined in the presence or absence of LAT inhibitors or Na+. Expression of a major leucine transporter was inhibited by siRNA in LN-abl cells. In silico analysis of leucine transporter expression was examined using Oncomine for different progression status of prostate cancers in clinical data sets. RESULTS Cell viability was decreased to 10% in the absence of leucine. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+-independent. In LN-abl cells, Na+-dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+-independent uptake was only 0.52, therefore, leucine uptake of LN-abl was largely (~85%) Na+-dependent. y+LAT2 (SLC7A6) expression was confirmed in LN-abl, however, expression of LAT1, LAT3, y+LAT1 (SLC7A7), ATB0+ (SLC6A14), B0AT1 (SLC6A19), B0AT2 (SLC6A15) or b0+AT (SLC7A9) were not observed. Knockdown of y+LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%) in LN-abl cells. In silico analysis revealed that more frequent up-regulation of SLC7A6 (y+LAT2) was observed in hormone resistant or metastatic samples in 2 data sets. In addition, some advanced prostate cancer appeared to express high levels of LAT3, suggesting heterogeneity in leucine transporter expression among different prostate cancers. CONCLUSIONS New CRPC cell line with increased expression of y+LAT2 was established in vitro. This is consistent with the fact that at least some advanced stage prostate cancers express y+LAT2 in Oncomine data. Considering the diversity of leucine transporter expression, target of anti-leucine transporter therapy should be individualized. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1110 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Hideo Otsuki More articles by this author Toru Kimura More articles by this author Takeo Kosaka More articles by this author Takashi Yamaga More articles by this author Jun-ichi Suehiro More articles by this author Hiroyuki Sakurai More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Simultaneous radioassays of bacterial production and mercury methylation in the periphyton of a tropical and a temperate wetland

Laboratory radioassays were made to study mercury (Hg) methylation together with bacterial production in the periphyton of two aquatic macrophytes, the submerged Myriophyllum spicatum, from a constructed wetland in Sweden and the floating Eichhornia crassipes, from a eutrophied tropical lake in Brazil. Time course incubations were made by addition of 203HgCl 2 and the methylmercury formed was extracted at pre-defined time intervals. Bacterial production ( 14C-leucine incorporation) was measured at the same time intervals, with plants removed from parallel incubations made with and without addition of cold HgCl 2. For E. crassipes, higher methylmercury production was observed at elevated bacterial production, whereas for M. spicatum, the bacterial production was significantly lower, and Hg methylation was below the detection limit. The combined results confirm the importance of microbial processes for Hg methylation, although other factors are known to influence this process in complex ways. The addition of Hg did not significantly influence bacterial production, while the incubation temperatures used (25 and 35 °C) resulted in different methylation rates. Radiotracer techniques for measurements of bacterial production such as 14C-leucine uptake can provide useful insights into the Hg cycle in aquatic environments, and our data suggest that they may be used as a proxy of mercury methylation potentials.

Primary production and microbial activity in the euphotic zone of Lake Baikal (Southern Basin) during late winter

Three years of regular weekly/biweekly monitoring of seasonal changes in temperature, transparency, chlorophyll a (CHL) and bacteria [erythrosine-stained microscopic counts and cultivable colony forming units (CFUs)] at the vertical profile in the South basin of Lake Baikal (51°54′195″N, 105°04′235″E, depth 800 m) were evaluated. In more detail, the structure and function of phytoplankton and the microbial loop in the euphotic layer at the same site were investigated during the late-winter–early-spring period under the ice. The depth of euphotic zone (up to 1% of surface irradiation) was 35 to 40 m. Primary production was measured three times a week with the 14C method in 2, 10, 20, 30 and 40 m. Maximum production was found in 10 m, with lower values towards the surface (light inhibition) and towards the lower layers. The total production in cells larger than 1 μm in the column (0–40 m) was 204–240 mg C d −1 m −2, 30–40% of it being in cells 1–3 μm (mostly picocyanobacteria), which represented roughly 9% of the total chlorophyll a (estimated from pigment analyses). A major part of phytoplankton biomass was formed by diatoms ( Synedra acus Hust., Asterionella formosa Hass. and Stephanodiscus meyerii Genkal & Popovskaya). Total production (including extracellular, dissolved organic matter) was 235–387 mg C day −1 m −2, and the exudates were readily used by bacteria (particles 0.2–1 μm). This part amounted to 1–5% of cellular production in 2 to 20 m and 11–77% of cellular production in 20–40 m, i.e., in light-limited layers. From 0 to 30 m, chlorophyll a concentration was 0.8 to 1.3 μg l −1, wherefrom it decreased rapidly to 0.1 μg l −1 towards the depth of 40 m. Bacteria (DAPI-stained microscopic counts) reached 0.5–1.4×10 6 ml −1; their cell volumes measured via image analysis were small (average 0.05 μm −3), often not well countable when erythrosine stain was used. Bacterial biomasses were in the range of 6–21 μg C l −1. Numbers of colony forming units (CFUs) on nutrient fish-agar were c. 3–4 orders lower than DAPI counts. The amounts of heterotrophic protists were low, whereby flagellates reached 6 to 87 ml −1 and ciliates, 0.2–1.2 ml −1 (mostly Oligotrichida). Bacterial production was measured in the same depths as primary production using 3H-thymidine (Thy) and 14C-leucine (Leu) uptake. Consistently, bacterial abundances, biomasses, thymidine and leucine production were higher by 30–50% in layers 2, 10 and 20 m compared with that in the deeper 30 and 40 m, where cellular primary production was negligible. Leucine uptake in the deeper layers was even three times lower than in the upper ones. From the comparison of primary and bacterial production, bacteria roughly use 20–40% of primary production during 24 h in the layers 2 to 20 m.

Phytotoxicity of the Fungicide Metalaxyl and Its Optical Isomers.

Metalaxyl is a systemic fungicide commonly used in citrus production to control Phytophthora root rot and foot rot. When applied as a drench, injury was observed on newly planted, young citrus trees. The visual injury symptoms ranged from light- to bright-yellow leaves. It was learned that the commercial formulation of metalaxyl contained various isomers and that these isomers may vary in phytotoxic effects on citrus leaves. The objective of this study was to determine the difference in herbicidal activity between the two optical isomers of metalaxyl on pepper plants and citrus leaves. The phytotoxicity of the fungicide metalaxyl and its optical isomers (CGA76538, S+; CGA76539, R-) was determined using a pepper seedling growth bioassay and by measuring protein synthesis as estimated by the incorporation of 14C-leucine by citrus mesophyll cells. The two isomers and metalaxyl differed in their herbicidal activity to pepper plants and citrus cells. Pepper seedlings treated with R- had significantly higher mean fresh weight than plants treated with metalaxyl or S+ at 0.1, 1.0, 10.0, and 100 ppm. Protein synthesis, as measured by the inhibition of 14C-leucine incorporation by citrus mesophyll cells, also was inhibited more by metalaxyl and the S+ isomer than by the R- isomer. After 30 min of incubation at 100 μM, the R- isomer inhibited 14C-leucine incorporation by 29%, whereas incorporation of 14C-leucine in the metalaxyl and the S+ isomer treatments was higher (46 and 81%, respectively). Similarly, the highest 14C-leucine uptake at 60 min was obtained by R- at all concentrations. The assays showed that the R- and S+ isomers differ in their biological activity as expressed by weight loss of pepper plants and inhibition of protein synthesis and that the S+ isomer is responsible for the phytotoxicity of metalaxyl. The findings in this study show that the phytotoxicity of metalaxyl was due to the presence of the S+ optical isomer. Removal of this isomer from metalaxyl has enabled the continued use of this fungicide for control of foot rot and root rot in citrus.

Altered tissue amino acid metabolism in acute T-2 toxicosis.

T-2 toxin is a Fusarium trichothecene mycotoxin that has been shown to alter brain neurochemistry and eating behavior in animals eating contaminated diets. Experiments were conducted to determine the role of altered tissue amino acid metabolism in the etiology of acute T-2 toxicosis. Fasted weanling rats were orally dosed with 0 or 2.0 mg T-2 toxin/kg body weight. Blood, brain, liver, and muscle tissue were excised 4 and 8 hr after dosing, and amino acid concentrations were determined. Hepatic enlargement coupled with reduced liver concentrations of free small neutral, large neutral, and basic amino acids were seen 4 hr after dosing. Brain and muscle amino acid concentrations were largely refractory to treatment, while the plasma concentrations of tyrosine and lysine, and the sum of the basic amino acids fell. Hepatic amino acid concentrations returned to control levels 8 hr after dosing at which time aminoacidemia was seen. This was due partially to an increase in plasma concentrations of large neutral amino acids including particularly the branched-chain amino acids. A subsequent experiment was conducted to determine the effect of T-2 toxin on 14C-leucine uptake and incorporation into protein in liver slices 4 hr after dosing. Exposure to T-2 toxin reduced total (free + protein-bound) uptake of leucine due primarily to reduced incorporation of leucine into newly-synthesized hepatic protein. It was concluded that reduced amino acid uptake by liver preceded aminoacidemia in acute T-2 toxicosis, although it is not clear how this might influence subsequent changes in brain neurochemistry and behavior.

Serum free medium increases expression of markers of differentiation in human colonic crypt cells.

In colitis, colonic epithelial cells have a shortened life span but show normal or increased expression of phenotypic markers of differentiation. This study examined the effect of differing culture conditions on the expression of such markers in colonic crypt cells. Crypt cells were enzymatically isolated from macroscopically normal large bowel mucosa resected because of neoplasia, inflammatory bowel disease or non-neoplastic non-inflammatory conditions. Cells cultured in the presence of serum exhibited a doubling of the rate of protein synthesis (measured by 14C-leucine uptake; p < 0.001) compared with autologous cells cultured in the absence of serum without evidence of loss of cell viability (assessed by 51Cr release from prelabelled cells) or of change in the rate of cell proliferation (assessed by total DNA content and 3H-thymidine uptake). Irrespective of the underlying colonic disease, crypt cells cultured in the absence of serum exhibited increased expression of phenotypic markers of differentiation compared with those cultured with serum: the rate of glycoprotein synthesis relative to that of protein synthesis increased by a mean of 59% and the cellular expression of brush border glycoproteins, alkaline phosphatase, and carcinoembryonic antigen significantly increased. The effects seen could not be mimicked by addition of dexamethasone or insulin to serum free medium. Thus, under less optimal (serum free) culture conditions, colonic crypt cells express phenotypic markers of differentiation at an accelerated rate suggesting that unfavourable microenvironmental conditions themselves are probably in part responsible for the normal or increased expression of such markers in colitis.

Effect of Pyributicarb on Lipid Metabolism in Gramineous Plants.

Herbicidal selectivity of the new thiocarbamate compound pyributicarb [O-3-tert-butylphenyl 6-methoxy-2-pyridyl (methyl)-thiocarbamate] and its effect on plant metabolism were examined under root-applied condition.Gramineous plants such as barnyardgrass, crabgrass and corn were more susceptible to pyributicarb than broadleaved plants such as soybean and cucumber. Among the examined gramineous plants, rice was the most tolerant to the herbicide.Pyributicarb reduced the incorporation of 14C-acetate into lipid fraction in young corn root tips at 10-7 and 10-6M concentrations. Reduction of uptake and incorporation of other 14C-labeled precursors, 14C-thymine, 14C-uracil, 14C-leucine and 14C-glucose were not observed.Lipid fractions metabolized from 14C-acetate in corn roots under pyributicarb treatment (10-7 and 10-6M) were investigated by thin layer chromatography. Clear spots of squalene were detected in these lipid fractions; in the absence of pyributicarb, however, the spots of squalene decreased in size with the lapse of time. Thus, it was recognized that the herbicide induced the accumulation of squalene in corn roots.Pyributicarb appears to inhibit the squalene metabolism.

Effect of prostaglandin E2 on proline uptake and protein synthesis by cultured human mesangial cells

PGE2 production by glomeruli is increased in a variety of glomerular diseases. Potentially, this process may affect mesangial cell protein synthesis and mesangial cell growth. Thus studies have been undertaken, using cultured human mesangial cells, to assess the effects of PGE2 on proline uptake, protein synthesis and cell proliferation. In the presence of 140 mM NaCl, incubation of mesangial cells with 0.01 to 1 microM PGE2 for 72 hours resulted in a marked decrease of 14C proline uptake, but did not modify 14C leucine uptake. Substitution of choline to sodium inhibited 14C proline uptake by 85% which became independent of PGE2, indicating that this PG specifically altered sodium-dependent proline uptake. Inhibition of this component reached 35 to 50% with 1 microM PGE2. The inhibitory effect of PGE2 on sodium-dependent proline uptake required a lag time of 48 hours, and was suppressed by ouabain, an inhibitor of Na+, K+ ATPase activity. PGE2 did not modify the Vmax of the transport system (1.007 vs. 1.023 nmol/mg/min) but increased (P less than 0.01) its Km (1.179 vs. 0.823 mM). 8-bromo-cyclic AMP also inhibited sodium-independent proline uptake, and PGE2 markedly increased cyclic AMP production. Taken together, these results suggested that PGE2 acted via cyclic AMP stimulation. PGE2 under identical conditions (1 microM, 72 hr incubation) produced a decrease in collagen synthesis estimated by the relative rate of collagen production after incubation of mesangial cells with 14C proline (percentage of 14C radioactivity in collagenase-sensitive proteins over total proteins). PGE2 also diminished the intracellular free proline pool. More generally, PGE2 inhibited cell proliferation and cell total proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein synthesis in trifluralin-treated corn root tips

The effect of trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) on protein synthesis in corn (Zea mays) root tips was determined. 14C-leucine uptake and subsequent incorporation into protein in intact and excised root tips was measured. Root tips were treated with and without 15 µM trifluralin for 2 hr; incorporation of 14C-leucine was observed during a 20-min interval. Total amino acid content in the soluble pool and protein hydrolysate was reduced in the excised tissue by the herbicide. Kinetic analysis showed trifluralin had no effect on endogenous leucine pool size nor on the rate of protein synthesis in intact tissue. Uptake was unaffected; however, in excised tissue uptake increased 100% over the control. While 14C-leucine content was greater in both the soluble pool and protein in treated, excised root tips, analysis showed the apparent increase in protein synthesis was in response to increased pool size.

Study of cardiac hypertrophy. Humoral factors that stimulate protein metabolism o cultured rat heart cells.

Recent extensive studies suggested that some humoral factors may contribute to the development and/or reversal of cardiac hypertrophy in hypertension. The purpose of this study is to examine whether there exists humoral factor(s) for cardiac hypertrophy in experimental perinephritic hypertension in dogs. Hypertension was induced by the method of Page with some modifications. Humoral factors were studied using a microassay system that was independent of pressure overload, hemodynamic effects, and other factors. L(-)-isoproterenol (ISO) and angiotensin II (AngII) increased the uptake of 3H-uridine into 10-day-old cultured heart cells. The maximal response to ISO or AngII was obtained by 10(-5) M ISO or 10(-10) M AngII, and the percent increments in the uptake of 3H-uridine was 38.4 +/- 20.4%, and 45.1 +/- 22.5%, respectively. Moreover, ISO, DL-noradrenaline, and AngII stimulated protein synthesis of 7-day-old or 14-day-old cultured heart cells in this order (9.5 +/- 1.5, 7.3 +/- 1.2, 2.0 +/- 0.5 micrograms/6 x 10(5) by 7-day-old heart cells; 23.8 +/- 8.3, 19.0 +/- 9.4, 4.4 +/- 1.1 micrograms/6 x 10(5) by 14-day-old heart cells). Heart and kidney extracts were obtained from experimental perinephritic hypertensive dogs and sham-operated dogs. The heart extract obtained from hypertrophied left ventricle (LV) of the experimental hypertensive dogs, but not of the sham-operated dogs, increased the uptake of 3H-uridine into 10-day-old heart cells. The mean percent increment in uptake of 3H-uridine induced by the hypertrophied LV heart extract at a final concentration of 5 x 10(-3)% V/V (1-3 micrograms) in different experiments was about 15%. High performance liquid chromatography (HPLC) demonstrated that the LV heart extract obtained from the perinephritic hypertensive dogs contained at least 16 molecules. Among them, one with a molecular weight of approximately 11,200 daltons stimulated uptake of both 3H-uridine and 14C-leucine into cultured heart cells. These results demonstrated that heart extract obtained from hypertrophied LV of perinephritic hypertensive dogs contained at least one factor which may induce and/or modulate myocardial hypertrophy in this model for hypertension.

Soluble factor from the hypertrophied left ventricle of dogs in experimental hypertension: ability to stimulate protein metabolism of cultured heart cells.

Our study concerned the existence of humoral factor(s) in cardiac hypertrophy arising from experimental perinephritic hypertension in dogs. Hypertension was induced by the method of Page with some modifications. A microassay technique was used on cultured neonatal rat heart cells. Heart extract from the hypertrophied left ventricle of dogs with experimentally induced renal hypertension, but not sham-operated dogs, increased the uptake of 3H-uridine by cultured rat heart cells. At a final heart extract concentration of 5 X 10(-3)% v/v (1-3 micrograms/ml), 3H-uridine was increased by a mean of about 15%, from four experiments. High performance liquid chromatography showed that the heart extract contained at least 16 molecules. Among them, a molecule of approximately 11,200 molecular weight stimulated the uptake of both 3H-uridine and 14C-leucine by cultured rat heart cells. These results indicate that the heart extract from the hypertrophied left ventricle of dogs with experimentally induced renal hypertension contained a factor that might induce and/or modulate myocardial hypertrophy in the model of hypertension.

Cartilage sulfation inhibitor from rat liver: Partial characterization of properties and biologic action

Cartilage sulfation (somatomedin) inhibitors (CSI) from rat liver produce reversible inhibition of cartilage growth. After gel filtration Sephadex G-200, CSI appear to have MW ∼100,000 and they are urea- and trypsin-labile factors. To explore further the mechanism of CSI action, we used the chick pelvic rudiment bioassay and studied the effect of CSI on the incorporation on 35S-sulfate (proteoglycan synthesis), 14C-leucine (protein synthesis), 3H-uridine (RNA synthesis), and 3H-thymidine (DNA synthesis). Normal rat serum (NRS) significantly stimulated the incorporation of all four isotopes, as expected. After a 24-hour incubation, CSI significantly blunted cartilage stimulation by NRS regarding total isotope uptake (1), 35S-sulfate (NRS, 96 ± 8 mcg/100 mg cartilage dry weight; NRS + CSI, 48 ± 4, mean ± SEM, n = 29, P < .05); and (2) 14C-leucine (NRS, 2,089 ± 172 cpm/mg dry weight; NRS + CSI, 1,102 ± 141, n = 18, P < .05); and (3) 3H-uridine (NRS, 6,711 ± 832 cpm/mg; NRS + CSI, 3,227 ± 425 cpm/mg, n = 18, P < .05); but not (4) 3H-thymidine (NRS, 3,540 ± 620 cpm/mg; NRS + CSI 3,249 ± 285, n = 19). The inhibition of 35S-sulfate and 14C-leucine uptake by CSI was dose-dependent and reversible. For 35S-sulfate, uptake by cartilage incubated with CSI alone for 40 hours was 13 ± 3 μg/100 mg; with CSI for 16 hours then fresh medium with NRS for 24 hours uptake was 39 ± 12, P < .05. Similarly, for 14C-leucine, uptake with CSI alone for 40 hours was 22.8 ± 5.4 × 10 3 cpm/mg; with CSI 16 hours, then NRS 24 hours, uptake was 59.9 ± 9.3 × 10 3, P < .05. We conclude that partially purified CSI profoundly inhibit the incorporation into chick cartilage of 35S-sulfate, 14C-leucine, 3H-uridine, but not 3H-thymidine. CSI action is dose-dependent and reversible. The data suggest that CSI can exert broad antianabolic effects on normal cartilage.

Physiological Responses to Fluazifop-Butyl in Tissue of Corn (Zea mays) and Soybean (Glycine max)

Effects of fluazifop-butyl {(±)-butyl 2-[4-[(5-(trifluoromethyl)-2-pyridinyl)oxy] phenoxy] propanoate} on cellular uptake and incorporation of14C-leucine,14C-uridine, and14C-thymidine into protein, RNA, and DNA, respectively, were evaluated in corn (Zea maysL.) coleoptiles and soybean [Glycine max(L.) Merr.] hypocotyls. Uptake of14C-leucine in corn coleoptiles was inhibited by fluazifop-butyl at 10-4to 10-7M, which was related to reductions in incorporation of14C-leucine into protein at 10-4to 10-6M. The effect of fluazifop-butyl on14C-leucine uptake and incorporation was reduced by 2 × 10-6M 2,4-D [(2,4-dichlorophenoxy)acetic acid], indicating antagonism between these herbicides. RNA and DNA synthesis were inhibited at 10-5and 10-4M. Protein synthesis in soybean hypocotyls was stimulated by 10-7and 10-6M fluazifop-butyl 33 and 27%, respectively, and inhibited by 10-4M 50%. RNA and DNA synthesis were also inhibited by 10-4M in soybean.

Effect of metalaxyl on the synthesis of RNA, DNA and protein in Phytophthora nicotianae

Metalaxyl is used to control diseases caused by fungi of the order of the Perenosporales. We investigated the action of this fungicid eon nucleic acid and protein synthesis in liquid cultures of Phytophthora nicotianae. The uptake of 32P, 3H-uridine, 3H-thymidine and 14C-leucine as precursors of nuclei acid and protein synthesis by the mycelium was not inhibited by metalaxyl. RNA synthesis as indicated by 3H-uridine incorporation was strongly inhibited (about 80%) by 0.5 micrograms/ml of metalaxyl. The inhibition was visible already few minutes after addition of the toxicant. Since the inhibition of incorporation of 3H-thymidine into DNA and of 14C-leucine into protein became significant 2-3 hours later, we conclude that metalaxyl primarily interfers with RNA synthesis. Synthesis of ribosomal RNA is more affected (more than 90%) than that of tRNA (about 55%) and poly(A)-containing RNA. Since in the presence of actinomycin, in contrast to metalaxyl, protein synthesis is inhibited immediately as a consequence of complete inhibition of RNA synthesis and of the short life-time of mRNA, it is also evident that mRNA synthesis is less strongly inhibited, at least during the early period of metalaxyl action. The molecular mechanism of metalaxyl inhibition of the transcription process remains open. The fungicide did not inhibit the activity of a partially purified RNA polymerase isolated from the fungus. On the other hand, the RNA synthesis (14C-UTP-incorporation) by a cell homogenate and by isolated nuclear fractions was inhibited significantly. Possibilities of the molecular action of metalaxyl are discussed. The RNA synthesis of some plant systems (cell cultures of Lycopersicon peruvianum, isolated nuclei from the same cell cultures, purified RNA polymerase from Spinacia oleracea chloroplasts) was not inhibited by metalaxyl, not even at high concentrations.

The Uptake of 14C-Leucine by Neutrophils and the Inhibitory Effects of Prostacyclin and of Methyl-Prednisolone

The Uptake of 14C-Leucine by Neutrophils and the Inhibitory Effects of Prostacyclin and of Methyl-Prednisolone

Primary Metabolic Effects of Cadmium in the Brown Alga, Laminaria saccharina

Summary Laboratory — cultured young sporophytes of the brown alga, Laminaria saccharina exhibit a syndrome of physiological and biochemical effects upon a 4 day treatment with 4.5 and 9.0nmoll-1 cadmium supplied as CdCl2. Activity of enzymes involved in primary metabolism and extracted from untreated control plants is not affected by exogenously added Cd. In vivo uptake and incorporation of 14C-leucine is drastically reduced in Cd treated plants. It is concluded from these observations that Cd in the culture medium inhibits one or several steps in protein biosynthesis and thus leads to enzyme deficiency of the specimens.

DNA and protein synthesis in normal and transformed lymphocytes exposed to abrin

The dose dependent effects of abrin, a toxic D-galactose binding plant lectin, on 3H-TdR and 14C-leucine uptake are studied in normal and Epstein Barr Virus (EBV) transformed lymphocyte cultures. Results show that while abrin is highly toxic to both the DNA and protein synthesis in EBV lymphocytes, some toxicity to the normal cells is also seen. It is postulated that lymphocyte DNA synthesis is affected by ribosomal shutdown induced by the abrin.

Studies on the toxicity and binding kinetics of abrin in normal and Epstein Barr virus-transformed lymphocyte culture-I: experimental results - 1.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Activation, stimulation and relative toxicity factor indices are studied as well as possible relationships between the DNA and protein synthesis rates, as measured by simultaneous 3H-TdR and 14C-leucine uptake.

On the dynamics of abrin binding to receptor sites in normal and Epstein Barr Virus transformed lymphocyte cell cultures.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA an protein synthesis of normal and Epstein Barr Virus transformed lymphocytes has been investigated. Activation, stimulation, and relative toxicity factor indices are studied, as well as possible relationships between DNA and protein synthesis rates, as measured by simultaneous tritiated thymidine (3H-TdR) and 14C-leucine uptake. Studies of the two new indices, the metabolic self and cross coupling indices lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin receptor site concentrations. This prediction is supported by SEM morphological differences. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of receptors bound-EBV-lymphocyte needed to exert a biological influence lies in the interval 59,264 receptors/cell to 370.040 receptors/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 receptors/cell and 360,000 receptors/cell.

14C leucine uptake in rat tissues at different times after irradiation.

The uptake of 14C leucine administered at different intervals after irradiation, but always 4 and 8 h before the animals were killed, has been evaluated in tissues with different proliferative activity and protein synthesis. The results have demonstrated an increased uptake and a more rapid elimination of the tracer after irradiation. In the small intestine a lower amount of TCA insoluble fraction was observed when the morphologic injury was evident, while protein synthesis significantly increased during the initial phase of appearance of the injury and mainly during the recovery phase of epithelial cells. Kidney and plasma had levels higher than controls at all intervals.