In vitro effect of aflatoxin B1 on rat liver macrophages (Kuffer cells)

The effect of aflatoxin B1 on the uptake and incorporation of [3H]leucine and [3H]uridine and on phagocytosis of latex particles was studied using cultures of rat liver macrophages (Kuffer cells). Aflatoxin B1 inhibited the incorporation of both isotopes, but inhibition of uridine incorporation was greater than that of leucine, suggesting that RNA synthesis was a major site of inhibition. Aflatoxin B1 also inhibited phagocytosis of latex particles in a time- and dose-dependent manner.     

Effects of ethanol on glycoprotein synthesis and secretion during inflammation-induced stimulation of hepatic glycoprotein secretion

The effects of ethanol on glycoprotein metabolism in liver slices obtained from rats whose synthesis and secretion of glycoproteins had been stimulated by turpentine-induced inflammation were investigated and compared to colchicine, a potent inhibitor of secretion. Inflammation resulted in a large stimulation in liver slices of [¹⁴C]glucosamine and [¹⁴C]leucine incorporation into medium proteins and a lesser stimulation into hepatocellular proteins. Both ethanol (10 mm) and colchicine (50 μm), when present in the incubation medium, markedly decreased the appearance of glucosamine- and leucinelabeled proteins in the medium of both turpentine-stimulated and control livers. However, when microsomes were isolated and fractionated in a fraction rich in secretory contents and another rich in membrane components, the effects of these agents on the labeling of glycoproteins in these microsomal fractions differed. Colchicine did not affect the incorporation of either glucosamine or leucine into the membrane fraction but increased the labeling of the secretory fraction, whereas, ethanol decreased the labeling of both fractions. These effects were observed for both the stimulated and control slices. When protein synthesis was inhibited by cycloheximide in both types of slices, ethanol and colchicine decreased the secretion of glucosamine-labeled protein into the medium while increasing the specific activity of the glycoproteins of the secretory fraction without altering the labeling of the membrane fraction. When fucose, a terminal sugar, was used as a label, both agents decreased the secretion of fucose-labeled proteins into the medium while also causing a retention of labeled glycoproteins in the microsomal secretory fraction. These results indicate that the ethanol-induced inhibition of glycoprotein synthesis and secretion in the normal liver persists in the liver where synthesis and secretion have been greatly stimulated by inflammation.     

Effect of ethanol on cholecystokinin-induced enzyme secretion from isolated rat pancreatic acini

Cholecystokinin octapeptide (CCK8)-stimulated amylase release in isolated rat pancreatic acini was inhibited over 30% by 600 mM ethanol. The configuration of the dose-response curve for CCK8, however, in the presence of ethanol was similar to that of the control. Amylase release elicited by maximal concentrations of CCK8 (300 pM) was inhibited by increasing concentrations of ethanol (0.3 to 1.3 M), and this inhibition was concentration dependent. In addition, the binding of [125I]CCK33 to specific membrane receptors on acini was inhibited by ethanol in a dose-dependent manner. A positive correlation between the inhibitory effects of ethanol on CCK binding and CCK-induced amylase release was observed. Furthermore, these inhibitory effects of ethanol were reversible. Basal amylase release, however, was increased 20-50% by ethanol between the concentrations of 0.3 and 1.3 M; higher concentrations caused a leakage of amylase from the acini both in the absence and presence of 300 pM CCK8. This is confirmed by 51Cr release from prelabeled acini which revealed no significant damage to acinar cell membrane between 0.3 and 1.6 M ethanol, but significant damage to acini at higher concentrations. These data suggest that the 600 mM ethanol-induced inhibition of CCK action in acini is due to reversible perturbation of the acinar cell membrane.     

Ethanol, its effect on the synthesis of proteins by guinea-pig gastric mucosa

Incorporation of L-[U-14C]leucine into proteins is taken to indicate the synthesis of proteins by guinea pig gastric mucosa. Ethanol reduced the synthesis of proteins in vitro by homogenized mucosa, by isolated gastric epithelial cell preparations and by intact tissue. Intact stomach wall incubated without ethanol in phosphate buffered saline showed progressively increasing incorporation of the precursor into tissue proteins and into proteins which were secreted into the mucosal incubation media. On isopycnic CsCl gradient fractionation radioactive tissue proteins were found at the top of the gradient (fraction L1, sp.gr.1.11-1.20) while radioactive secreted proteins sedimented to the bottom of the gradient as the carbohydrate rich high density gastric mucosal glycoprotein fraction L3 (sp.gr.1.29-1.33). Ethanol significantly but reversibly reduced the incorporation of radioactive leucine by intact mucosa into both the tissue proteins and the secreted proteins. Uptake of the precursor into the intracellular acid soluble pool was not impaired by ethanol and no significant differences were detected in the specific activities of free intracellular leucine between the ethanol treated samples and the corresponding controls. It is suggested that the ulcerogenic nature of ethanol may be associated with inhibition of the synthesis of proteins within mucosal epithelium leading to reduction in the output of mucosal secretory glycoproteins with subsequent impairment of the cytoprotective properties of the dynamic mucous barrier.     

Competitive inhibition by procaine of carbachol-induced stimulus-secretion coupling in rat pancreatic acini.

1. Procaine (0.03-10 mM) inhibited carbachol (CCh)-induced amylase release from rat isolated pancreatic acini in a competitive manner. Kinetic analysis of the relation between CCh concentrations and the amount of amylase released in the presence of various procaine concentrations indicated that procaine caused competitive inhibition with the affinity constant (pA2) value of 5.00 +/- 0.08. 2. Receptor binding assay confirmed that procaine (0.01-10 mM) competitively inhibited [N-methyl-3H]-scopolamine chloride ([3H]-NMS) binding to its receptor with binding affinity (pKi) of 4.63 +/- 0.10. 3. Procaine transformed CCh-evoked [Ca2+]i dynamics: the initial rise in [Ca2+]i followed by a gradual decay during continuous stimulation with 3 microM CCh was transformed by 0.3 mM procaine to the oscillatory [Ca2+]i dynamics, which resembled the response to 0.3 microM CCh in the absence of procaine. The initial phase of [Ca2+]i oscillation corresponded to the initial phase of CCh-induced amylase release in isolated perfused acini. 4. Procaine (0.3-3 mM) did not inhibit the secretory response to cholecystokinin octapeptide (CCK-8) in isolated incubated acini. A higher concentration of procaine (10 mM) caused weak but significant inhibition of the response to only limited concentrations of CCK-8, 30 and 100 pM. Procaine lower than 10 mM was ineffective on [125I]-BH-CCK-8 binding, although procaine (10 mM) caused weak but significant inhibition of the binding.     

Effects of complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium dicitrate on the syntheses of DNA, RNA, protein, nucleoprotein and ATP of leukemia L 7712 cells in mice

The incorporations of [3H] thymidine, [3H]uridine and [3H]leucine into DNA, RNA and protein synthesis in leukemia 7712 cells were inhibited by the complex of 3,6-di-(dimethylamino)-dibenzopyriodonium with praseodymium (Pr, rare earth element) dicitrite 34 micrograms/ml for 3-24 h. The degree of inhibition increased in proportion to the incubation time. After being treated with [C17H20N2I]3[Pr(C6H5O7)2] 34 micrograms/ml for 3, 6, 12 and 24 h, the incorporation of [32P]Na2HPO4 into the nucleoprotein of leukemia 7712 cells was inhibited by 49, 57, 65 and 85%, while those into ATP were inhibited by 43, 59, 65 and 83%, respectively. The ID50 of [C17H20N2I]3[Pr(C6H5O7)2] on DNA synthesis in leukemia 7712 cells at 24 h was 22 micrograms/ml. After the complex was removed from the medium entirely, the rate of DNA synthesis decreased with time over 3-12 h. This result indicated that the inhibition mechanism was likely due to damage to the DNA template.     

Inhibition of rat vascular smooth muscle cell proliferation by taurine and taurine analogues

The growth of rat aorta vascular smooth muscle cells (VSMCs) was measured in the presence and absence of taurine. Concentrations of taurine as low as 0.3 mM in the culture medium inhibited the proliferation of the cells, as monitored by measuring cell count, and also inhibited the rate of DNA synthesis, as examined by measuring [3H]thymidine incorporation into DNA. However, even at the highest concentration of taurine (30 mM), the doubling time of the VSMCs was only increased by 38%. Protein content of the VSMCs was decreased by 30 mM taurine. [3H]Leucine incorporation into newly synthesized protein was not affected by the highest concentration of taurine tested (30 mM), indicating that taurine did not inhibit protein synthesis but rather decreased total protein content by inhibiting cellular proliferation. The effects of other amino acids such as alanine, glycine, and serine and of various taurine analogues such as beta-alanine, guanidinoethanesulfonic acid (GES), and isethionic acid also were tested at a concentration of 20 mM for their effects on the growth of the VSMCs. Alanine, glycine, and serine had only a minimal effect or no effect on cell count, quantity of protein, and incorporation of [3H]thymidine into DNA. GES, beta-alanine, and isethionic acid had a significant effect on cell count, protein content, and incorporation of [3H]thymidine into DNA. Beta-alanine was the only analogue tested that significantly depressed [3H]leucine incorporation into newly synthesized protein. It is concluded that taurine, GES, and isethionic acid inhibited proliferation of VSMCs but did not alter normal protein synthesis or survivability of VSMCs. In contrast, other amino acids, alanine, glycine and serine, had minimal effects on VSMC proliferation and protein synthesis, whereas beta-alanine appeared to be toxic, inhibiting both VSMC synthesis and de novo protein synthesis.     

大鼠胰腺腺泡中胆囊收缩素类似物JMV—180诱导的胞内钙振荡是由三磷酸肌醇介导的

目的:研究三磷酸肌醇在胆囊收缩素类似物JMV-180引起的胰腺腺泡分泌反应中的作用。 方法:胰腺腺泡胞浆中钙离子浓度变化用Fura-2双波长比例测量法记录。结果:在灌流中的分离大鼠胰腺腺泡细胞,一个新的可通透细胞膜的三磷酸肌醇受体抑制性调控剂2-氨基乙氧基苯硼酸(2APB)抑制了JMV-180诱导的胞浆中钙离子波峰的出现,2APB 100μmol·L~(-1)引起了钙波峰的立即、完全的抑制。结论:JMV-180持续性刺激所诱导的重复性胞内钙离子波峰的出现是由三磷酸肌醇介导的胞内钙库中钙离子释放引发的。     

Changes in protein and RNA synthesis in rat brain neurons after a single dose of methylmercury

Methylmercury was given to 30-day-old Wistar rats, At 1-7 days later the protein and RNA synthesis was studied in vivo by injecting a mixture [14C]leucine and [3H]uridine. Protein and RNA were precipitated from isolated cerebral and cerebellar neurons. The reduction of RNA synthesis coincides or precedes the reduced protein synthesis.     

Inhibition of human lymphocyte transformation by two aryloxyalkylamidines

The incorporation of [3H]thymidine into human lymphocytes stimulated by phytohaemagglutinin (PHA) was inhibited by anilino-N-2-m-chlorophenoxypropylacetamidine (501C) and xylamidine. These amidines antagonize 5-HT, but 5-HT did not alter [3H]thymidine incorporation. 501C inhibited PHA-induced lymphocyte transformation as observed by [3H]thymidine incorporation, [3H]uridine incorporation, [3H]leucine incorporation, DNA content, potassium content, and histological examination. 501C also inhibited increased [3H]thymidine incorporation in human mixed lymphocyte cultures. The IC50 of 501C for inhibition of these processes lay between 4 and 8 microM. When added late in culture (after 6-8 h) 501C was less effective. Possible mechanisms by which 501C inhibits transformation are discussed.     

Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1)

Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4'-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC50 values of 9.5 +/- 3.2, 59.1 +/- 19.6 and 1423.3 +/- 212.2 ng/ml respectively. No inhibition of [3H]uridine incorporation was observed using actinomycin 4-4'-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 values for inhibition of [3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 "translocation" from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45-55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75-85% inhibition, only a uniform nucleoplasmic fluorescence was observed.     

Central nervous system myelination in rats exposed to ethanol in utero

Pregnant rats were pair-fed using isocaloric control or 6.6% (v/v) ethanol liquid diets. At 18, 25, and 53 days, the in vivo incorporation of [3H] leucine and [14C] glucose into proteins and lipids of three central nervous system (CNS) myelin subfractions was examined in the offspring (control and ethanol pups) of control and ethanol-treated females. With few exceptions, the pattern of CNS myelination appeared near-normal in the ethanol pups. The ethanol pups had brain weights and total myelin protein content within the normal range. In addition, the amount of protein in light, medium, and heavy myelin was normal at 18 and 25 days. However, the ethanol pups had an excess of the chemically and morphologically immature heavy fraction at 53 days. The incorporation of [3H] leucine and [14C] glucose into myelin subfractions and separated proteins and lipids was similar in control and ethanol pups. The findings of near-normal CNS myelination in the offspring of female rats fed an ethanol liquid diet during gestation differ from our previous findings of a premature onset and slowdown of active CNS myelination in the offspring of female rats that consumed an ethanol liquid diet for one month prior to conception as well as during gestation.     

鹿茸有效成分对小鼠肝脏RNA和蛋白质合成的影响

多次给小鼠po鹿茸多胺30mg/kg,对[³H]leucine和[³H]uridine掺入肝组织蛋白和RNA有明显的促进作用,而庸茸非多胺则无此作用;当腐胺剂量为21mg/kg时,不仅促进[³H]leucine和[³H]uridiae掺入肝组织蛋白和RNA,也促进[³H]uridine掺入肝细胞核的RNA中,并增强RNA聚合酶的活性;精脒在剂量为8mg/kg时,仅对[³H]leucine掺入肝组织蛋白有促进;而精胺在1mg/kg时,没有观察到上述各种现象。此结果提示,鹿茸多胺类物质是鹿茸中刺激小鼠肝组织蛋白和RNA合成的主要活性物质,这 种刺激小鼠肝组织蛋白和RNA合成效应是由于鹿茸多胺能够显著地增强RNA聚合酶的活性。     

Zinc stimulation of bone protein synthesis in tissue culture. Activation of aminoacyl-tRNA synthetase

The present investigation was undertaken to clarify the effect of zinc on bone protein synthesis in tissue culture. Calvaria were removed from 3-week-old male rats and cultured for periods up to 96 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The calvaria were incubated at 37 degrees in 5% CO2/95% air in the medium containing 10(-6)-10(-4) M zinc. Zinc content in bone cells was increased when the culture was treated with 10(-5) and 10(-4) M zinc for 48 hr. When calvaria cultured in the presence of 10(-4) M zinc were pulsed with [14C]uridine, the incorporation of [14C]uridine into the bone RNA was not increased significantly. In the pulse with [3H]leucine, the presence of 10(-5) to 10(-4) M zinc in the medium caused a significant increase in the incorporation of [3H]leucine into the acid-insoluble residues of bone tissue. This increase was blocked completely by treatment with 10(-7) M cycloheximide, an inhibitor of protein synthesis. When [3H]leucine was added into the reaction mixture containing the 5500 g supernatant fraction of the homogenate prepared from calvaria cultured in the presence of 10(-4) M zinc, the in vitro protein synthesis was increased about 2-fold. The activity of [3H]leucyl-tRNA synthetase in the 105,000 g supernatant fraction (cytosol) of the bone homogenate was increased about 2-fold by the culture with 10(-4) M zinc. The presence of 10(-4) M dipicolinate, a specific chelator of zinc, in the culture medium negated the effect of zinc on [3H]leucyl-tRNA synthetase activity. The addition of 10(-7) to 10(-6) M zinc into the reaction mixture containing enzyme extracts obtained from uncultured rat calvaria caused a 2-fold increase of [3H]leucyl-tRNA synthetase activity. These results clearly indicate that zinc induces the stimulation of protein synthesis at the translational level in bone cells. The present study further supports the view that zinc increases protein synthesis in bone cells and that the metal induces bone formation.     

Cortisol effect on protein synthesis and ribonucleic acid polymerase activity in rat thymus

The time course for cortisol to effect a decrease in the amount of soluble (extractable) thymic nuclear RNA polymerase activity was determined. Decreases in soluble polymerase activity were compared with the cortisol-mediated inhibition of [³H]leucine ([³H]leu) incorporation into cells and cell fractions. [³H]leu incorporation into total cell protein was inhibited by cortisol before measurable effects on extractable nuclear RNA polymerase were detected. Cortisol inhibition of [³H]leu incorporation into protein of nuclear extract (soluble at pH = 8.0) and protein associated with sucrose gradient purified RNA polymerase lagged behind [³H]leu incorporation into total cell protein but preceded the decrease in extractable RNA polymerase activity. These data suggest that the decrease in extractable RNA polymerase seen 12 hr after cortisol treatment is due, at least in part, to a decreased synthesis of the enzyme. The decrease in RNA polymerase activity, however, does not occur early enough to explain the marked inhibition of protein and RNA synthesis seen early (3 hr) after cortisol injection.     

Mechanism of action of nicotine on amylase release by isolated pancreatic acini

The effects of nicotine on the pH of acinar suspension, amylase release and on amylase response stimulated by carbachol were examined in isolated rat pancreatic acini. Additions of nicotine at concentrations ranging from 10 microM to 30 mM caused dose-dependent increases in pH of acinar suspension with simultaneous amylase release (p less than 0.05). There was no increase in amylase release when acinar cells were incubated with nicotine adjusted to pH 7.40. Carbachol alone released amylase whereas nicotine (pH 7.40) at a concentration of 10 mM caused a significant and nonparallel inhibition of amylase release in response to graded doses of carbachol. At concentrations ranges between 3 microM and 10 mM, nicotine at pH 7.40 inhibited amylase release stimulated by 1 microM carbachol, with a half maximal inhibition at 0.8 +/- 0.2 mM. These results indicate that in isolated rat pancreatic acini nicotine at pH 7.40 has no effect on basal nonstimulated amylase release but it inhibits carbachol-stimulated amylase response in a noncompetitive manner. These observations may have direct implications in underlying mechanism of pancreatic disorders.     

The effect of ethanol on enzyme synthesis and secretion in isolated rat pancreatic lobules

This study investigates the effect of ethanol on enzyme synthesis and secretion in rat pancreatic lobules. Ethanol caused a dose-dependent inhibition of 3H-leucine incorporation into total protein. Examination of the time dependence showed that ethanol inhibited protein synthesis at each time point. Removal of ethanol partially reversed this inhibition. An autoradiograph of the newly synthesized proteins separated on SDS-PAGE showed that ethanol inhibited synthesis of all proteins. 14C-cycloleucine uptake was not altered by ethanol, excluding inhibition of amino acid uptake as the mechanism for the decreased protein synthesis induced by ethanol. Electron microscopy revealed no ultrastructural damage. Ethanol had no effect on the stimulated release of (i) amylase from zymogen granules nor (ii) newly synthesized pulse labelled enzymes. Acetaldehyde had no inhibitory effect on enzyme synthesis or secretion indicating that ethanol per se and not its metabolite is inhibitory. The decreased synthesis after acute exposure to ethanol with preservation of exocytosis would limit the autodigestive potential of pancreatic tissue. This may explain why isolated toxic doses of ethanol are rarely if ever associated with pancreatitis.     

Effects of trifluoroacetic acid, a halothane metabolite, on C6 glioma cells

Effects of trifluoroacetic acid (TFA) on cell growth, DNA, glycoprotein, and dolichol-linked oligosaccharides synthesis and ribonucleotide triphosphate concentrations were examined in exponentially growing C6 murine glioma cells. One day of treatment with TFA caused a slight concentration-dependent enhancement of cell growth and [3H]thymidine incorporation. Exposure for 1 or 5 d to TFA (0.5-7.0 mM) elevated the [3H]leucine incorporation in a dose- and time-dependent manner. The results suggested that TFA stimulated cell growth and enhanced protein synthesis. TFA also affected [3H]mannose incorporation into glycoproteins and dolichol-linked oligosaccharides in a dose-dependent fashion. In addition, it was found that TFA accelerated lectin-induced cell agglutination. These data suggest that TFA, the principle halothane metabolite, alters plasmalemmal glycoprotein synthesis. These findings should form a basis for further understanding on the mechanism underlying halothane-associated neurotoxicity.     

Effects of rifampicin on RNA and protein synthesis in isolated rat liver mitochondria

Isolated rat liver mitochondria incorporated [³h]utp in an essentially linear fashion for at least 60 min in the presence and absence of exogenous ribonucleoside triphosphates. Preincubation of isolated mitochondria with rifampicin at 0° in the absence of ribonucleoside triphosphates produced a consistent inhibition of [³h]utp incorporation at 30° that ranged up to 75 per cent and was independent of preincubation time with rifampicin or the length of the assay period. Incorporation of [³H]UMP into mitochondria prepared in the presence of the proteolytic enzyme Nagarse was similarly inhibited by rifampicin. When mitochondria prepared in the presence of Nagarse were preincubated with rifampicin, incorporation of [³h]utp was inhibited by approximately 75 per cent within 2 min, while [³H] leucine incorporation continued in a linear fashion for 4–5 min before decreasing with first-order kinetics. These data demonstrate that rifampicin primarily affects RNA synthesis in mitochondria, and leads secondarily to an inhibition of protein synthesis. A 75 per cent inhibition of mitochondrial RNA synthesis resulting in a 45 per cent inhibition of mitochondrial protein synthesis permits an estimate of 3.3 mm as the half-life of mitochondrial RNA serving a messenger function. It is hypothesized that this rapidly turning-over mitochondrial RNA which is closely linked to protein synthesis serves a regulatory role in oxidative phosphorylation.