A comparison of the inhibitory effects of melatonin and indomethacin on platelet aggregation and thromboxane release

Collagen-induced platelet aggregation and thromboxane release is inhibited, in a concentration response relationship, by preincubation of gel-filtered platelets with melatonin in the concentration range 430 nM – 4.3 mM. Inhibition of platelet aggregation and thromboxane release also occurs in the presence of indomethacin (4.3 nM – 4.3 mM), a known potent inhibitor of prostaglandin synthesis. Arachidonic acid-induced platelet aggregation and thromboxane release was inhibited in the presence of 4.0 mM melatonin. We therefore propose that inhibition of prostaglandin synthesis maybe the mechanism by which melatonin expresses its activity. Its antigonadotropic activity may result from inhibition of PGE₂ synthesis in the hypothalamus and median eminence.     

Induction of rat liver malic enzyme messenger RNA activity by insulin and by fructose

Several human normal and neoplastic cell lines were screened for production of PDGF receptor competing activity. Conditioned medium from two sarcomas and one glioma blocked 125I-PDGF binding to human foreskin fibroblasts in a dose-dependent manner. In each case this effect was abolished when the conditioned medium was pretreated with PDGF-antiserum, indicating that the receptor competing activity was immunologically related to PDGF. Direct evidence for de novo synthesis of a PDGF-like component in the cultures was afforded by 35S-cysteine labeling of the three cell lines, followed by immunoprecipitation with PDGF antiserum. This resulted in the specific precipitation of a 31,000 molecular weight labeled protein, which upon reduction was split into two polypeptides of molecular weights 17,000 and 16,500. The significance of these findings in view of the recently discovered structure homology between PDGF and the transforming gene product of simian sarcoma virus, p28sis, is discussed.     

Stimulation of DNA synthesis in human fibroblasts by a human nonsuppressible insulin-like protein (NSILP).

When nonsuppressible insulin-like protein (NSILP) isolated and purified from human serum was added at concentrations of 5 and 50 ug/ml to cultures of human dermal fibroblasts, both cell proliferation and DNA synthesis were enhanced. However, NSILP, 50 ug/ml, had no effect on glucose uptake. In contrast, insulin, 40 ng/ml (1.0 mU/ml), had no effect on cell proliferation or DNA synthesis, but stimulated glucose uptake. These observations suggest that human NSILP may play an important role in tissue repair or growth by enhancing fibroblast proliferation, but not a significant glucoregulatory role.     

Differential cytotoxic activity of potassium dichromate on nucleoside uptake in BHK fibroblasts.

In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K2Cr2O7) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that K2Cr2O7 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K2Cr2O7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.     

DNA damage and DNA repair in cultured human cells exposed to chromate

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.     

Mutant cell lines resistant to azetidine carboxylic acid: Quantitative and qualitative differences in pyrroline-5-carboxylate synthase activity

Mutant Chinese hamster lung fibroblasts were selected that are resistant to the proline analog L-azetidine-2-carboxylic acid. Resistance in the two mutant cell lines is associated with two distinct alterations in pyrroline-5-carboxylate synthase, the enzyme that catalyzes the proline biosynthetic step leading from glutamic acid to pyrroline-5-carboxylate. In one mutant cell line, pyrroline-5-carboxylate synthase specific activity is increased 30-fold over the level in control cells. In the other mutant line, pyrroline-5-carboxylate synthase activity is not increased, but the enzyme has become insensitive to inhibition by ornithine and proline.     

Transient inability of neonatal rat motoneurons to reinnervate muscle

We studied the reinnervation of internal intercostal muscles of newborn rats. The distal halves were denervated by nerve section at various ages between birth and 6 weeks. Regardless of the age at denervation, neither evoked nor spontaneous nerve-muscle transmission reappeared until the animals were at least 3 weeks old. Older rats recovered a substantial degree of function within 7 days of nerve section. Normally the motor units in this muscle are narrowly distributed, so most axotomized motoneurons lost their entire synaptic periphery. Reinnervation was by axons which had been sectioned, and regenerated motor units were of normal size and number. There was no collateral sprouting from end plates left intact. Motoneurons axotomized at birth did regenerate axons the full length of the muscle within 7 days of operation. Their failure to reinnervate the muscle was due to delay in forming functional end plates. Nerve section in animals aged 1 month or older resulted in an abnormal pattern of reinnervation; reinnervated motor units were diffusely spread through large portions of the muscle, although they still did not overlap with the region left intact. This indicates that thoracic motoneurons respond to axotomy differently in neonatal rats than they do in adults.     

Direct cell culture assay of thin-layer-chromatographed material without prior substance elution

A method enabling the direct assay in a cell culture system of material separated by thinlayer chromatography (TLC) without prior elution of active material from the TLC sheet is described. After chromatographic development on a plastic cellulose sheet, small disks were punched out from the sheet and transferred to microwells used for cell culture. Lymphoid cells in culture medium were incubated with the disks and the DNA synthesis was measured by [³H]thymidine incorporation. The reliability of the method was tested with l-alanine and thymidine, which are known to enhance the DNA synthesis of lymphoid cells and to interfere with the uptake of labeled thymidine, respectively. The incorporation of [³H]thymidine was stimulated with disks containing l-alanine and inhibited with disks containing unlabeled thymidine, completely in agreement with the expected results. Elution experiments showed that more than 75% of l-alanine and thymidine on the TLC disks was eluted into the culture medium within 1 h. Elution of material of higher molecular weight was also demonstrated. The method was used for the separation and assay of a thymocyte-specific growth factor isolated from calf thymus and greatly facilitated the detection of the active material after TLC.     

Effects of chlorambucil on human chromosomes

No significant amount of chromosomal damage was found in the 48-h cultures of lymphocytes of 18 patients who had been treated with the bifunctional alkylating agent chlorambucil (CBC). However, there was suggestive evidence of chromatid damage (i.e. of types attributable to damage during or after DNA synthesis in the cell cycle). In marrow cells of 3 patients given a single large dose of chlorambucil (equivalent to 2 days' normal treatment) there was also suggestive evidence of induced chromatide-type damage.Extensive series of in vitro experiments yielded evidence that (a) exposure of human lymphocytes over the whole period of culture showed chromatid-type damage; (b) this damage increased sharply from concentrations of 0.5 μg/ml to3.0 μg/ml; (c) although chromatide-type damage always predominated, there was suggestive evidence also of chromosome-type aberrations attributable to damage occuring in the G₀/G₁ period, although some or all of this could be attributed to “derived” chromatid damage; (d) even if lymphocytes were only exposed during the G₀ or G₁ periods of the cycle, damage was found in the subsequent metaphases and it was almost entirely of the chromatid type; (e) much more damage occurred in lymphocytes exposed for varying periods to the drugs after stimulation by phytohaemagglutinins than in those exposed in whole blood, or in medium before stimulation; (f) damaged occurred in lymphocytes exposed to the drug while in S but not exposed only when in G₂; (g) no evidence was found that unschaduled DNA synthesis during G₀ or G₁ was induced by the drug; (h) there appeared to be no delay caused by the drug in the time at which cells reached the first “S” phase in culture but there was some evidence consistent with prolongation of “S” in cells exposed in culture; (i) there was evidence that CBC alone could stimulate lymphocyte tto DNA synthesis, and that a few cells proceeded in the cycle to prophase, or even metaphase. However, there was a considerable amount of cell-killing during CBC-stimulated DNA synthesis.     

Factors influencing the frequency of mitomycin C-induced sister-chromatid exchanges in 5-bromodeoxyuridine-substituted human lymphocytes in culture.

Newly developed techniques for the detection of sister-chromatid exchanges (SCE) require the substitution of 5-bromodeoxyuridine (BrdU) for thymidine in DNA. We investigated the possibility of interactions between BrdU and one mutagen--carcinogen, mitomycin C (MMC) for the induction of both chromosomal aberrations and SCE in human peripheral lymphocytes in culture. No effect on aberration yield was found. Neither comparisons between the yields of SCE by BrdU substitution and differential staining and those detected by tritiated thymidine incorporation and autoradiography nor between the yields of SCE for different levels of BrdU incorporation provided any evidence of synergism. It was found, however, that MMC persists in cultures and continues to increase SCE frequencies for about 30 h. It was also observed that some MMC-induced DNA lesions persist long enough so that some of those present prior to S phase of the first cell cycle cause additional SCE in the third cycle.     

Persistence of phage lambda DNA in genomes of mouse cells transformed by lambda-carrying SV40 vectors.

To test the suitability of simian virus 40 (SV40) DNA as a vector for inserting DNA segments into the chromosomes of mammalian cells, an EcoRI-A fragment of bacteriophage lambda DNA was covalently joined to a fragment of SV40 DNA and used to transform mouse cells in culture. Three independent, morphologically transformed clones were obtained that were positive for SV40 T-antigen by immunofluorescence staining. DNA from each transformant was examined by restriction enzyme analysis and found to contain both lambda and SV40 sequences. Co-migration of some fragments containing lambda and SV40 sequences following digestion of transformed cell DNA by each of four different restriction enzymes indicated that part of the retained lambda and SV40 DNA was linked in two of the three lines. In the third line, however, none of the restriction fragments had both lambda and SV40 sequences. Although the presence of non-integrated lambda DNA was not excluded, at least some of the lambda DNA appeared to be linked to host cell DNA. Results of digestion by EcoRI suggested that in some cases the transforming linear molecule had probably circularized prior to integration.     

Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: Evidence for a second mechanism of spontaneous secretion?

β-Hexosaminidase secreted by peritoneal macrophages in response to stimulation by zymosan or NH₄Cl, or spontaneously by a macrophage-like cell line (P388D₁), is susceptible to receptor-mediated endocytosis by human fibroblasts. This endocytosis is almost completely blocked by exogenous mannose-6-phosphate and therefore seems to depend on a mannose-6-phosphate ligand on the enzyme. It is suggested that macrophage lysosomal enzyme packaging may involve mannose-6-phosphate recognition markers, and that a continuous hypersecretion mechanism may exist which does not depend on a defect in this ligand.     

Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.     

Studies of DNA-strand induced in human fibroblasts by chemical mutagens/carcinogens.

A method for the study of DNA-strand breaks using alkaline denaturation followed by hydroxylapatite chromatography has been modified and used for the detection of chemically induced DNA-strand breaks. A new procedure for the incubation of human fibroblasts with a metabolizing system and the detection of DNA-strans breaks is presented. With this method the induction and repair of DNA-strand breaks have been studied in human fibroblasts exposed to methyl methanesulphonate, melphalan, benzo[a]pyrene and cyclophosphamide. These agents all give rise to DNA-strand breaks. In cells exposed to methyl methanesulphonate, melphalan or benzo[a]pyrene these breaks disappeared within 21 h after re moval of the drug. In cells exposed to the bifunctional alkylating agent cyclophosphamide, studies of DNA-strand breaks suggest the presence of inter-strand cross links.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.     

L-proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.     

Serum-reduced media impacts on cell viability and protein expression in human lung epithelial cells

Serum starvation is a widely used condition in molecular biology experiments. Opti-MEM is a serum-reduced media used during transfection of genetic molecules into mammalian cells. However, the impact of such media on cell viability and protein synthesis is unknown. A549 human lung epithelial cell viability and morphology were adversely affected by growing in Opti-MEM. The cellular protein levels of chloride intracellular channel protein 1, proteasome subunit alpha Type 2, and heat shock 70 kDa protein 5 were dysregulated in A549 cells after growing in serum-reduced media. Small interfering RNA transfection was done in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, and knockdown efficacy was determined compared with Opti-MEM. Similar amounts of knockdown of the target proteins were achieved in DMEM, and cell viability was higher compared with Opti-MEM after transfection. Careful consideration of the impact of Opti-MEM media during the culture or transfection is important for experimental design and results interpretation.     

The effect of trans-stilbene oxide and other structurally related inducers of drug-metabolizing enzymes on glucuronidation

Administration of trans-stilbene oxide, and new type of inducer of drug-metabolizing enzymes, to rats was found to increase hepatic microsomal UDP-glucuronyl transferase activity with both p-nitrophenol and chloramphenicol as substrate. In Triton X-100 activated microsomes the increase with p-nitrophenol as substrate was to approx. 250% of the control value, while the corresponding value for chloramphenicol was about 600%. These observations indicate that trans-stilbene oxide causes a mixed type 'induction' of UDP-glucuronyl transferase(s), i.e., changes in activity which resemble both those seen after induction with phenobarbital and after treatment with 3-methylcholanthrene. We have also shown that the activity of UDP-glucose dehydrogenase, the enzyme which produces UDP-glucuronic acid, is increased to about 300% of the control after administration of trans-stilbene oxide. The time course of this increase and of the return to control activity after cessation of treatment, the dose-response of this increase and the structural features of the trans-stilbene oxide molecule which are essential for the increase have all been examined. The other two enzymes involved in the conversion of glucose 6-phosphate to UDP-glucuronic acid, namely, phosphoglucomutase and UDP-glucose pyrophosphorylase, were found to be only slightly affected (a 30-60% increase) by treatment with trans-stilbene oxide. After induction with trans-stilbene oxide the hepatic level of UDP-glucuronic acid was unchanged.     

Mutagenesis studies on cultured mammalian cells. The sensitivity of the asparagine-requiring phenotype to several chemical agents.

The effect of several chemical agents on the mutation frequency from asparagine dependence to asparagine independence has been studied in Jensen sarcoma cells. It was found that ethylmethanesulfonate brought about a dramatic exponential increase, while nitrosoguanidine was not lighly effective as a mutagen, causing only a modest increase in mutation frequency, and quinacrine HCl was ineffective. The results presented here are compared with those obtained in other systems and with our previous work on the effects of UV on mutation induction in the asparagine system. They suggest that the basis of the asparagine requirement of mammalian cell lines resides in a specific genetic alteration in nuclear DNA which is corrected by the mutagenic action of the agents tested here.