Trichloroacetic Acid-insoluble Material Research Articles

12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

Pentagastrin-induced protein synthesis in the parotid gland of the anaesthetized rat, and its dependence on CCK-A and -B receptors and nitric oxide generation

In parotid glands of pentobarbitone-anaesthetized rats, the incorporation of [3H]leucine into trichloroacetic acid-insoluble materials, reflecting protein synthesis, increased by 17% (compared to saline-treated rats) in response to infusion of pentagastrin (20 microg kg(-1), i.v. for 1 h) under muscarinic and alpha- and beta-adrenoceptor blockade. Both the CCK-A receptor antagonist lorglumide (48 mg kg(-1), i.v.) and the CCK-B receptor antagonist itriglumide (5.5 mg kg(-1), i.v.), given separately, prevented the expected increase in pentagastrin and, in addition, reduced the glandular protein synthesis by 16 and 12%, respectively, below the level of saline-treated rats. In rats treated with saline only, the glandular protein synthesis was reduced by 22% by the CCK-A receptor antagonist and by 17% by the CCK-B receptor antagonist; combined, the two antagonists caused no further reduction (20%). There was no increase in the glandular protein synthesis of pentagastrin-treated rats compared to that of the saline-treated rats when both groups of rats were exposed to a combination of the two types of CCK receptor antagonists. In pentagastrin-treated rats, the protein synthesis in the parotid glands was 23% less in the presence of the non-selective nitric oxide (NO) synthase inhibitor L-NAME (30 mg kg(-1), i.v.) than in its absence; the result was the same (23%) when the neuronal NO synthase inhibitor Nomega-propyl-L-arginine (N-PLA; 30 mg kg(-1), i.v.) replaced L-NAME. The protein synthesis in rats treated with saline only was not reduced by L-NAME; nor was the protein synthesis of saline-treated rats different from that of pentagastrin- and L-NAME-treated rats. Thus, under 'basal' conditions, a portion of the glandular protein synthesis, as well as the whole increase in synthesis in response to administration of pentagastrin, engaged both types of CCK receptors. Furthermore, NO generation, owing to neuronal NO synthase activity, was required for the increase to occur in response to pentagastrin, whereas a non-NO-dependent pathway was responsible for the protein synthesis under 'basal' conditions.

Parasympathetic nerve-evoked protein synthesis, mitotic activity and salivary secretion in the rat parotid gland and the dependence on NO-generation

Incorporation of radiolabelled leucine and thymidine into trichloroacetic acid-insoluble material of the parotid gland was used as indices of protein synthesis and mitotic activity, respectively, following electrical stimulation of the parasympathetic auriculo-temporal nerve for 30 min in pentobarbitone-anaesthetized rats under adrenoceptor blockade (phentolamine and propranolol, 2mg/kg intravenous of each) in the absence or presence of atropine (2mg/kg intravenous) and without or with nitric oxide synthase inhibitors. In atropinized rats, the parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked mean increases in protein synthesis at a frequency of 10 Hz (142%) and 40 Hz (200%) were not affected in a statistically significant way (124 and 275%, respectively) by the neuronal type NO-synthase inhibitor N(w)propyl-l-arginine (N-PLA) (30 mg/kg intravenous). Neither were the increase (175%) in protein synthesis at 10 Hz in non-atropinized animals affected by N-PLA (180%). The increase (65%) in mitotic activity, 19 h after the end of stimulation at 40 Hz, in the presence of atropine, was not affected by N-PLA (55%). Neither were the increase (95%) in gland content of amylase at this point of observation statistically significant affected by N-PLA (144%). The secretion of fluid and output of amylase from the parotid gland upon nerve stimulation was not affected by N-PLA. When examining the non-selective NO-synthase inhibitor l-NAME (30 mg/kg intravenous) in atropinized rats subjected to stimulation at 10 Hz, neither the increase in protein synthesis nor the evoked fluid response or amylase outputs were affected. Hence, in contrast to an NO-dependent sympathetic-induced protein synthesis and mitosis in the parotid gland, involving the activity of the neuronal type NO-synthase, no support for a parasympathetic-induced protein synthesis (and gain in gland amylase) and mitosis, depending on NO-generation, was found. Likewise, the present findings provide no evidence for a role of NO in the parasym pathetic nerve-evoked fluid secretion and amylase output.

Pokeweed Antiviral Protein Inhibits Brome Mosaic Virus Replication in Plant Cells

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

ADP-ribosylation in permeable HeLa S3 cells.

ADP-ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4-5-times greater in metaphase cells. 17-22% was in the soluble fraction which contained material released from the cells, 16-22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP-ribosylated proteins corresponding to 78 000, 54 000 and 36 000 Da. In addition metaphase cells contained several other ADP-ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-Da region indicating ADP-ribosylation of core histones. The pattern of ADP-ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP-ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP-ribose) and oligo(ADP-ribose). The results do not lend support to a special function of ADP-ribosylated histones in the mitotic event while certain ADP-ribosylated non-histone proteins may be specific for metaphase cells.

Native Replication Intermediates of the Yeast 20 S RNA Virus Have a Single-stranded RNA Backbone

20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo. A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome. When in vitro products, after phenol extraction, were analyzed in a time course, radioactive nucleotides were first incorporated into double-stranded RNA (dsRNA) intermediates and then chased out to the final single-stranded RNA products. The positive and negative strands in these dsRNA intermediates were non-covalently associated, and the release of the positive strand products from the intermediates required a net RNA synthesis. We found, however, that these dsRNA intermediates were an artifact caused by phenol extraction. Native replication intermediates had a single-stranded RNA backbone as judged by RNase sensitivity experiments, and they migrated distinctly from a dsRNA form in non-denaturing gels. Upon completion of RNA synthesis, positive strand RNA products as well as negative strand templates were released from replication intermediates. These results indicate that the native replication intermediates consist of a positive strand of less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91. Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 degrees C, is not an intermediate in the 20 S RNA replication cycle, but a by-product.

Influence of parasympathetic non-adrenergic, non-cholinergic mechanisms on [ 3H]thymidine uptake in rat parotid salivary glands in response to mastication following upon a liquid regimen

A role for parasympathetic non-adrenergic, non-cholinergic (NANC) mechanisms in parotid gland cell hyperplasia in response to mastication was investigated. Following a liquid regimen for 1 week, aiming at glandular inactivity, rats were offered hard chow for 2 h to throw their parotid glands into secretory activity. In response to the increasing demands, [ 3H]thymidine incorporation into the trichloroacetic acid-insoluble material increased by 76% in normally innervated glands, while there was no increase in glands parasympathetically denervated in advance. In innervated glands of rats pretreated with atropine and α- and β-adrenoceptor blockers the increase was 46%. The results suggest a potential contribution of parasympathetic NANC mechanisms to the mitotic response of the parotid gland under natural feeding conditions.

Reflex-elicited increases in female rat parotid protein synthesis involving parasympathetic non-adrenergic, non-cholinergic mechanisms.

Normal rats or rats parasympathetically denervated on one side by cutting the auriculo-temporal nerve were maintained on a liquid diet for 1 week. After fasting overnight, experimental rats were then offered hard, pelleted food over a period of 2 h, while unfed animals served as controls. The reflex-induced increase in the [3H]leucine incorporation into trichloroacetic acid-insoluble material of the parotid glands following intake of the food in the presence of the muscarinic blocker atropine and alpha- and beta-adrenoceptor antagonists was greater (104%) than in the absence of the blockers (75%) in normal rats. The picture was the same when the innervated glands of rats subjected to unilateral parasympathetic denervation were examined. In this case, the protein synthesis increased by 108% in the presence of the blockers and by 63% in their absence. Analysis of the parasympathetically denervated and contralateral innervated glands revealed no support for a cholinergic or adrenergic contribution to this response. The increase in protein synthesis in the innervated glands was 86% in the rats treated with atropine, whilst it was 76% in those treated with alpha- and beta-adrenoceptor antagonists. The protein synthesis in the denervated glands increased by 57% in the absence of receptor blockade. Neither atropine (51%), nor a- and b-adrenoceptor antagonists (52%), nor a combination of the three blockers (55%) affected the response of the denervated glands. A large part of the response in the presence of the traditional autonomic receptor blockers was thus dependent on the parasympathetic auriculo-temporal nerve. Under natural feeding conditions, the parasympathetic non-adrenergic, non-cholinergic (NANC) mechanisms are likely to contribute to the synthesis of secretory proteins.

Fluorophores at the N Terminus of Nascent Chloramphenicol Acetyltransferase Peptides Affect Translation and Movement through the Ribosome

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Crithidia luciliae: Effect of Purine Starvation on S-Adenosyl-L-Methionine Uptake and Protein Methylation

The utilization of S-adenosyl-L-[ methyl- 3H]methionine ([ 3H- methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate ∼10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an ∼10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [ 3H- methyl]AdoMet, S-adenosyl-L-[ carboxyl- 14C]methionine, L-[ methyl- 3H] methionine and L-[ 35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [ 3H- methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, ∼15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [ 3H- methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.

Cerium stimulates protein biosynthesis in rat heart in vivo.

The aim of the study was to ascertain whether Ce, a lanthanide that has been implicated in the pathogenesis of tropical endomyocardial fibrosis, interferes with the biosynthetic repertoire of the cardiac muscle in vivo. Female Sprague-Dawley rats received Ce chloride iv at 1.3 mg/kg body wt.; controls received an equal volume of physiological saline. Rates of protein synthesis and transcription in cardiac muscle, measured in terms of incorporation of (3H)-phenylalanine and (3H)-uridine, respectively, into trichloroacetic acid-insoluble material were found to be significantly higher in Ce-treated animals. As low levels of Ce were earlier shown to stimulate collagen as well as noncollagen protein synthesis in cardiac fibroblasts in vitro, the stimulatory effect of the element in vivo reported here supports the speculation that it may influence the expression of proteins like collagen in the heart and contribute to their accumulation as in endomyocardial fibrosis.

Differences in the synthesis of total and poly(A)<sup>+</sup>RNA in the Kennebec potato and its dihaploid

RNA synthesis as measured by the incorporation of tritiated uridine into trichloroacetic acid insoluble material was studied in the leaf protoplasts of cv. Kennebec and its parthenogenically derived dihaploid. Protoplasts of cv. Kennebec incorporated tritiated uridine at a greater rate and accumulated more than twice the amount of radioactive materials than did the dihaploid over a 6-h incubation period. Poly(A)+RNA, isolated from the total RNA of the tetraploid and of the dihaploid, by oligo(dT)-cellulose column chromatography, was present in amounts of 11.3 and 5.2 % respectively. The tetraploid synthesized 4.8 times the amount of poly(A)+RNA that was synthesized by the dihaploid.

Insulin-like growth factor 1, but not growth hormone, has in vitro proliferative effects on neonatal foreskin fibroblasts without affecting 5-alpha-reductase or androgen receptor activity.

Clinical observation of patients with congenital growth hormone (GH) deficiency and Laron-type dwarfism suggests that factors such as GH or insulin-like growth factor 1 (IGF-1) might in addition to androgens, be needed for normal phallic growth. We speculated GH or IGF-1 might have direct actions on genital tissues and performed the present study to evaluate the in vitro effects of GH and IGF-1 on cultured neonatal foreskin fibroblasts. Cells derived from foreskins of normal newborns were studied between cell passages 6 and 15. Serum-free media with and without 100 ng/ml GH, IGF-1, or both were added 24 hours prior to and at the time of study. To determine the activity of 5-alpha-reductase (5-alpha-R), 3H-testosterone (T: 2 nM) was added, and 5-alpha-R activity was calculated as femtomoles 3H-dihydrotestosterone and 3H-androstanediol produced/microgram DNA/hour. Androgen receptor (AR) binding was determined by the addition of 3H-dihydrotestosterone (dHT; 0.03125-0.5 nM) in the presence and absence of a 200-fold excess of unlabeled dHT. Specific binding was used in Scatchard analysis for determination of AR number (Bmax) and binding affinity (Kd). The rate of DNA synthesis was determined by incorporation of 3H-thymidine (3H-Thy) into trichloroacetic acid-insoluble material. DNA and protein content were determined on cell lysates. IGF-1, but not GH, had proliferative effects (significant increases in the rate of 3H-Thy incorporation, DNA, and protein content) but no effect on 5-alpha-R activity, Bmax or Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity of CU, NI and Alkylbenzene Sulfonate (LAS) on the naturally occurring bacteria in the Rhône river plume

The toxicity of Cu++, Ni++, and linear alkylbenzene sulfonate to the bacterioplankton in the Rhone River plume (Mediterranean Sea) was assessed by [3H]thymidine incorporation into trichloroacetic acid-insoluble material. This method proved useful to evaluate the toxic effect of three common pollutants to the natural bacterial population in a system with permanent mixtures of fresh and sea water. An important variability in the toxicity has been detected irrespective of bacterial density or activity. The variations in toxicity were associated with the changes of salinity, particulate matter, and nutrients in the river plume.

Functional foles of insulin and insulinlike growth factors in preimplantation mouse embryo development

Growth factors are known to play important roles in cellular proliferation and differentiation. However, little information is available concerning their roles in the earliest stages of mammalian development. The effect of physiologic levels of insulin, insulinlike growth factor-I, and insulinlike growth factor II (IGF-I and -II) on DNA, RNA, and protein synthesis in preimplantation stages of the mouse are described in this study. Quantitative studies of the incorporation of labeled thymidine, uridine, and methionine into trichloroacetic acid-insoluble material by different developmental stages of preimplantation mouse embryos labeled in vitro, indicate that physiologic levels of insulin stimulated DNA, RNA, and protein synthesis with significant effects observed first at the morula stage of development. In contrast, neither IGF-I nor IGF-II stimulated DNA, RNA, or protein synthesis to a significant degree under the same experimental conditions. These results suggest a functional role for insulin at the earliest stages of mammalian embryogenesis.

Inhibition of N-glycan processing affects iodide organification in porcine thyroid cells

The N-glycan-processing inhibitors swainsonine (Sw) and deoxymannojirimycin (dMM) were used to study the influence of N-glycans on iodide organification in cultured porcine thyroid cells. Incubations with [ 125I]NaI were followed by determination of labeled trichloroacetic acid-insoluble material in culture media, follicular contents and cells. In controls, most of this material was in the follicular contents. With Sw and dMM, total acid-insoluble material was less than 10% of control. Iodide uptake was slightly inhibited and hydrogen peroxide release was not affected by inhibitors. Cell-surface thyroid peroxidase (TPO) activity, assayed by its ability to iodinate bovine serum albumin, was strongly inhibited. Pronase glycopeptide analysis indicated that with drugs the content in complex-type N-glycans was strongly decreased while that in hybrid or oligomannosidic type was increased. In conclusion, inhibition of N-glycan processing prevents iodide organification in cultured porcine thyroid cells by decreasing the recovery of cell-surface TPO activity.

Effect of Norspermidine and Its Related Triamines on the Cell‐Free Polyphenylalanine Synthesizing System from Vibrio parahaemolyticus

The effect of norspermidine and its structurally related triamines on the cell-free polyphenylalanine synthesizing system from Vibrio parahaemolyticus was examined in connection with the requirement of the system for monovalent cation. In the absence of norspermidine, the maximal incorporation of [14C]phenylalanine into hot trichloroacetic acid insoluble material was observed under ionic conditions of 12 mM Mg2+ and 50 mM NH4+. K+ could partially substitute for NH4+, but Na+ could not. The addition of norspermidine to the polyphenylalanine synthetic reaction mixture not only lowered the optimal Mg2+ concentration, but it also stimulated the polyphenylalanine synthesis up to 2-fold with no significant increase in misincorporation of [14C]leucine. Other triamines having one or two methylene chains more than norspermidine were also effective in eliciting these effects. Furthermore, Na+ could not support the polyphenylalanine synthesis even in the presence of norspermidine and, on the contrary, inhibited the polyphenylalanine synthesis induced by NH4+ regardless of whether norspermidine was present or not. These findings are discussed in comparison with the properties of other bacterial cell-free systems.

Cu,Zn superoxide dismutase and copper deprivation and toxicity in Saccharomyces cerevisiae

A wild-type strain of the yeast Saccharomyces cerevisiae grown at a medium [Cu] of less than or equal to 50 nM contained less Cu,Zn superoxide dismutase (SOD) mRNA (60%), protein (50%), and activity (50%) in comparison with control cultures grown in normal synthetic dextrose medium ([Cu] approximately 150 nM). A compensating increase in the activity of MnSOD was observed, as well as a smaller increase in MnSOD mRNA. These medium [Cu]-dependent differences were observed in cultures under N2 as well. Addition of Cu2+ (100 microM) to Cu-depleted cultures resulted in a rapid (30 min) increase in Cu,ZnSOD mRNA (2.5-fold), protein (3.5-fold), and activity (4-fold). Ethidium bromide (200 micrograms/ml of culture) inhibited by 50% the increase in Cu,ZnSOD mRNA, while cycloheximide (100 micrograms/ml of culture) inhibited completely the increase in protein and activity. Addition of Cu2+ to greater than or equal to 100 microM caused no further increase in these parameters but did result in a loss of total cellular RNA and translatable RNA, a decline in the population of specific mRNAs, a decrease in total soluble protein and the activity of specific enzymes, and an inhibition of incorporation of [3H]uracil and [3H]leucine into trichloroacetic acid-insoluble material. Cu,ZnSOD mRNA, protein, and activity appeared relatively more resistant to these effects of Cu toxicity than did the other cellular constituents examined. When evaluated in cultures under N2, the cellular response to [Cu] of greater than or equal to 100 microM was limited to the inhibition of radiolabel incorporation into trichloroacetic acid-insoluble material. All other effects were absent in the absence of O2. The data indicated that medium (cellular) Cu alters the steady-state level of Cu, ZnSOD. This regulation may be at the level of transcription. In addition, Cu,ZnSOD exhibits the characteristics of Cu-stress protein in that it and its mRNA are enhanced relative to other cellular species under conditions of Cu excess. This observation and the O2-dependence of some of the manifestations of Cu excess suggest that one mechanism of Cu toxicity involves the superoxide radical anion O2-.

Protective effect of S-adenosyl-L-methionine against CCl4-induced hepatotoxicity in cultured hepatocytes.

Effect of S-adenosyl-L-methionine disulfate tosylate salt (SAMe-ST) and L-methionine (L-Met) on primary cultured rat hepatocytes were studied. In cultured hepatocytes treated with CCl4, SAMe-ST and L-Met suppressed the decrease in urea-nitrogen secretion as well as the leakages of GOT and GPT. The membrane-protective action of these two compounds was verified by the histological data. Failure of SAMe-ST to counteract CCl4-induced reduction of radioactive leucine incorporation into the trichloroacetic acid-insoluble materials in hepatocytes indicates that the observed effects of SAMe-ST or L-Met do not involve acceleration of protein synthesis. The present results indicate that SAMe-ST remarkably protects hepatocytes from CCl4-induced hepatotoxicity, probably by either changing the structure or compositions of membrane phospholipids or by modifying the interaction of CCl4 with the intracellular drug-metabolizing enzyme systems.

Effects of Toxic Substances on Natural Bacterial Assemblages Determined by Means of [ 3 H]Thymidine Incorporation

The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were examined by means of [H]thymidine incorporation into trichloroacetic acid-insoluble material. Results from a large number of coastal marine and freshwater samples suggest the following. (i) The effects of the three toxicants included reductions in the bacterial cell number as well as changes in rates of [H]thymidine incorporation and in [H]thymidine incorporation per cell. The concentrations that inhibited [H]thymidine incorporation by 50% ranged from 3 to 11 mg liter for 3,5-dichlorophenol, 6 to 10 mg liter for 2,4-dinitrophenol, and 21 to 123 mg liter for potassium dichromate, with a tendency to higher values in bacterial assemblages from more eutrophic environments. (ii) The effects of 3,5-dichlorophenol and potassium dichromate determined by [H]leucine incorporation into bacterial protein were similar or larger than those obtained from [H]thymidine incorporation. (iii) Two to four hours of exposure to the toxicants was necessary before stable maximum effects were found in [H]thymidine incorporation. (iv) Storage of natural environmental samples should be avoided, since tests with water stored for 1 to 3 days sometimes produced results different from results obtained from in situ tests. (v) The effects of 3,5-dichlorophenol, 2,4-dinitrophenol, and potassium dichromate on natural bacterial assemblages were relatively constant during periods with different growth rates in the assemblages, during various periods of the year, and between samples from freshwater and marine localities. With some precautions, [H]thymidine incorporation can be used as a quick and sensitive method for determining the effects of toxicants on aquatic bacterial assemblages from natural environmental samples.