DNA-protective effects of two components of essential plant oils carvacrol and thymol on mammalian cells cultured in vitro

Many components of essential volatile oils show antioxidant activity and may serve e.g. as a natural replacement of synthetic antioxidant food additives. However, it is important to evaluate such compounds also for their pro-oxidant and toxic properties as their plant origin doesn't secure their safety for living beings, including humans. The aim of this study was therefore to investigate cytotoxic, genotoxic and DNA-protective effects of the long-term (24 h) incubation of mammalian cells with two components of essential plant oils (carvacrol and thymol) in in vitro conditions. Cytotoxicity testing was in all cell lines (human hepatoma cells HepG2, human colonic cells Caco-2 and hamster lung cells V79) performed on the basis of trypan blue exclusion. Plating efficiency was evaluated only in V79 cells which manifest a high colony forming ability. The amount of DNA lesions induced in cells treated with hydrogen peroxide, carvacrol, thymol or combinations of carvacrol or thymol with hydrogen peroxide was measured by standard alkaline single cell gel electrophoresis in human cells HepG2 and Caco-2. Trypan blue exclusion test showed that carvacrol was mildly more cytotoxic than thymol and that Caco-2 cells were mildly more resistant to both carvacrol and thymol than HepG2 and V79 cells. At concentrations = IC20-40, the compounds studied did not induce DNA strand breaks either in human cells HepG2 or in cells Caco-2. Incubation of HepG2 and Caco- 2 cells in the presence of the whole scale of concentrations of carvacrol or thymol led in both cases to a significant protection of the cells studied toward DNA strand breaks induced by a potent oxidant hydrogen peroxide.     

Mucopolysaccharides associated with nuclei of cultured mammalian cells.

Mucopolysaccharides have been isolated, fractionated, and characterized from the nuclei of cultured B16 mouse melanoma cells grown in the presence of (3-H)-glucosamine and (35-S)sulfate. Digestion of the nuclei with DNase followed by Pronase gave a mixture of complex carbohydrates from which the mucopolysaccharides were isolated by precipitation with cetylpyridinium chloride. After fractionation by differential salt extraction and chromatography on controlled pore glass bead columns, the components were identified by chemical and enzymatic methods. The major polysaccharide components were a family of high-molecular-weight chondroitin sulfates with different degrees of sulfation; a minor component has been characterized as heparan sulfate.     

Baculovirus-mediated gene transfer into mammalian cells.

This paper describes the use of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) as a vector for gene delivery into mammalian cells. A modified AcMNPV virus was prepared that carried the Escherichia coli lacZ reporter gene under control of the Rous sarcoma virus promoter and mammalian RNA processing signals. This modified baculovirus was then used to infect a variety of mammalian cell lines. After infection of the human liver cell lines HepG2, >25% of the cells showed high-level expression of the transduced gene. Over 70% of the cells in primary cultures of rat hepatocytes showed expression of beta-galactosidase after exposure to the virus. Cell lines from other tissues showed less or no expression of lacZ after exposure to the virus. The block to expression in less susceptible cells does not appear to result from the ability to be internalized by the target cell but rather by events subsequent to viral entry. The onset of lacZ expression occurred within 6 hr of infection in HepG2 cells and peaked 12-24 hr postinfection. Because AcMNPV is able to replicate only in insect hosts, is able to carry large (>15 kb) inserts, and is a highly effective gene delivery vehicle for primary cultures of hepatocytes, AcMNPV may be a useful vector for genetic manipulation of liver cells.     

Effect of heat-stable enterotoxin of Escherichia coli on cultured mammalian cells

The binding of biologically active 125I-labeled heat-stable enterotoxin (ST) of Escherichia coli with cultured mammalian cells was dose dependent and could be inhibited with low concentrations of unlabeled toxin or by neutralization with specific antiserum. There was positive cooperativity among cell binding sites. A single cultured cell bound approximately 4 X 10(4) molecules of ST; the dissociation constant was 1.33 X 10(-10) M. The specific binding of ST was partially inhibited by Pronase (Sigma Chemical Company, St. Louis, Missouri) and trypsin, but not by lipid- or carbohydrate-specific enzymes, simple sugars, or saccharides. Addition of ST to cultures of rat basophilic leukemia cells resulted in a dose-dependent secretion of histamine. Pharmacologic agents that inhibited calcium uptake or prostaglandin synthesis decreased the amount of histamine released. These data demonstrate the specific binding of ST by cultured cells and support the contention that calcium and prostaglandins may be important in the molecular mechanism(s) whereby ST activates guanylate cyclase.     

Replication and packaging of Norwalk virus RNA in cultured mammalian cells

Human noroviruses, the most common cause of nonbacterial gastroenteritis, are characterized by high infectivity rate, low infectious dose, and unusually high stability outside the host. However, human norovirus research is hindered by the lack of a cell culture system and a small animal model of infection. Norwalk virus (NV) is the prototype strain of human noroviruses. We report here replication of NV viral RNA and its packaging into virus particles in mammalian cells by intracellular expression of native forms of NV viral RNA devoid of extraneous nucleotide sequences derived from the expression vector by the use of replication-deficient vaccinia virus MVA encoding the bacteriophage T7 RNA polymerase (MVA/T7). Expressed genomic RNA was found to replicate; NV subgenomic RNA was transcribed from genomic RNA by use of NV nonstructural proteins expressed from genomic RNA and was subsequently translated into NV capsid protein VP1. Viral genomic RNA was packaged into virus particles generated in mammalian cells. The cesium chloride (CsCl) density gradient profile of virus particles containing genomic RNA was similar to that of NV purified from stool. These observations indicate that the NV cDNA constructed here is a biologically infectious clone, and that mammalian cells have the ability to replicate NV genomic RNA. This work establishes a mammalian cell-based system for analysis of human norovirus replication and, thus, makes it feasible to investigate antiviral agents in mammalian cells.     

Transduction of a bacterial gene into mammalian cells.

The transduction of an Escherichia coli gene into mammalian cells is described. A supressor tRNA gene was linked to a simian virus 40 (SV40) vector in vitro and the recombinant was used to transfect rat embryo cells and monkey kidney cells. The hybrid SV40 genome, SV40-su+ III, retained genetic information required for autonomous replication and cellular transformation and had a 1300-base-pair DNA segment in the late gene region (between the restriction endonuclease sits Hpa II at 0.735 and EcoRI at 0/1.0 on the SV40 genetic map) replaced by an 870-base-pair bacterial DNA segment containing the suppressor tRNA gene, su+ III (tRNATyrsu+III). The structure and fate of the SV40-su+III chimera were determined by DNA reassociation kinetic analysis and restriction enzyme cleavage of the total cellular DNA from transformed rat embryo cells and persistently infected monkey cells. Hybridization with radiolabeled probes specific for vector (SV40) or su+III DNA sequences revealed primarily nonintegrated or free hybrid genomes. In cloned lines of both cell types, the bacterial DNA segment was recovered intact, as judged by the length of the segment excised by restriction endonucleases and its ability to hybridize to the radiolabeled bacterial DNA probe and not to the SV40 probe.     

Functional reintroduction of human telomeres into mammalian cells.

Telomeric sequences of eukaryotes consist of short tandem repeats organized in arrays of variable length in which the guanine-rich strand runs 5'----3' toward the chromosomal end. The terminal repeats in yeast are the only elements necessary for telomere function in this organism. To test whether mammalian terminal repeats can function after reintroduction into a mammalian cell, a repeat-containing terminal fragment from a human chromosome was electroporated into a hamster-human hybrid cell line. In 6 of 27 independent transformants analyzed, the introduced sequences were found at the ends of chromosomes, based on all available criteria. Terminal restriction-fragment heterogeneity and the survival of these chromosomes demonstrate that these telomeres are functional. Cytogenetic evidence from one of these cell lines suggests that chromosome breakage with healing at the integration site is the mechanism responsible for the terminal location.     

Enhancing polyethylenimine's delivery of plasmid DNA into mammalian cells

The effect of various chemical modifications of nitrogen atoms on the efficiency of polyethylenimines (PEIs) as synthetic vectors for the delivery of plasmid DNA into monkey kidney cells in vitro has been systematically investigated. The resultant structure-activity relationship has both provided mechanistic insights and led to PEI derivatives with markedly enhanced performance. For example, N-acylation of PEI with the molecular mass of 25 kDa (PEI25, one of the most potent polycationic gene delivery vectors) with alanine nearly doubles its transfection efficiency in the presence of serum and also lowers its toxicity. Furthermore, dodecylation of primary amino groups of 2-kDa PEI yields a nontoxic polycation whose transfection efficiency in the presence of serum is 400 times higher than the parent's and which exceeds 5-fold even that of PEI25.     

Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells.

Cultured Schwann cells from sciatic nerves of newborn rabbits and rats have been examined with patch-clamp techniques. In rabbit cells, single sodium and potassium channels have been detected with single channel conductances of 20 pS and 19 pS, respectively. Single sodium channels have a reversal potential within 15 mV of ENa, are blocked by tetrodotoxin, and have rapid and voltage-independent inactivation kinetics. Single potassium channels show current reversal close to EK and are blocked by 4-aminopyridine. From these results, and from comparisons of single-channel and whole-cell data, we show that these Schwann cells contain voltage-dependent sodium and potassium channels that are similar in most respects to the corresponding channels in mammalian axonal membranes. Cultured rat Schwann cells also have sodium channels, but at a density about 1/10th that of rabbit cells, a result in agreement with saxitoxin binding experiments on axon-free sectioned nerves. Saxitoxin binding to cultured cells suggests that there are up to 25,000 sodium channels in a single rabbit Schwann cell. We speculate that in vivo Schwann cells in myelinated axons might act as a local source for sodium channels at the nodal axolemma.     

Inhibition of sonic hedgehog autoprocessing in cultured mammalian cells by sterol deprivation

Sonic hedgehog (Shh) is a signaling molecule that is important for defining patterning in the developing vertebrate central nervous system. After translation, Shh autoproteolyzes and covalently attaches cholesterol to the newly formed carboxyl terminus, a modification crucial for normal Shh signaling. Presented here is evidence that acute severe sterol deprivation in cultured Chinese hamster ovary cells expressing mouse Shh (mShh) inhibits autoprocessing of the protein. These conditions allowed the first detailed kinetic analysis of mShh autoprocessing and turnover rates revealing that cells rapidly degrade both precursor and mature mShh regardless of sterol content and sterol deprivation increases the rate of precursor degradation. Inhibition of mShh autoprocessing also allowed the determination of the subcellular localization of mShh precursor which accumulates in a pre-medial Golgi intracellular compartment. Finally, the precursor form of mShh that results from autoprocessing inhibition appears to accumulate as an amide rather than a stable thioester.     

Distinctive higher-order chromatin structure at mammalian centromeres

The structure of the higher-order chromatin fiber has not been defined in detail. We have used a novel approach based on sucrose gradient centrifugation to compare the conformation of centromeric satellite DNA-containing higher-order chromatin fibers with bulk chromatin fibers obtained from the same mouse fibroblast cells. Our data show that chromatin fibers derived from the centromeric domain of a chromosome exist in a more condensed structure than bulk chromatin whereas pericentromeric chromatin fibers have an intermediate conformation. From the standpoint of current models, our data are interpreted to suggest that satellite chromatin adopts a regular helical conformation compatible with the canonical 30-nm chromatin fiber whereas bulk chromatin fibers appear less regularly folded and are perhaps intermittently interrupted by deformations. This distinctive conformation of the higher-order chromatin fiber in the centromeric domain of the mammalian chromosome could play a role in the formation of heterochromatin and in the determination of centromere identity.     

Glass needle-mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells

A novel glass needle-mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix-coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34(+), CD34(+)/CD38(-), and CD34(+)/CD38(-)/Thy-1(lo) human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% +/- 8.4% of CD34(+) cells and 42% +/- 14% of CD34(+)/CD38(-)cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34(+) and CD34(+)/CD38(-) cells, and retention of the ability of CD34(+)/CD38(-) cells to generate progenitor cells. Delivery of DNA constructs (currently 相似文献   

An antigen in metaphase chromatin and the midbody of mammalian cells binds to scleroderma sera

Antibodies from 5 patients with systemic sclerosis reacted with an antigen localized to the metaphase chromatin, the cleavage furrow and the midbody of anaphase and telophase HEp-2 cells. The titer of antimidbody antibodies ranged from 1:160 to 1:1280. Four patients had systemic sclerosis and one had idiopathic Raynaud's phenomenon. In situ biochemical characterization of the antigen revealed that it was resistant to DNase I, micrococcal nuclease and RNase A, but was sensitive to trypsin treatment. The antigen remained insoluble in 400 mM acetic acid but was extracted from the cells with 400 mM hydrochloric acid. The antibody was not seen in sera from 2500 normal female blood donors, 120 patients with systemic lupus, 60 patients with rheumatoid arthritis, 15 patients with linear scleroderma or 25 patients with Raynaud's disease.     

Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

An orthogonal tryptophanyl-transfer RNA (tRNA) synthetase (TrpRS)-mutant opal suppressor tRNA(Trp) (mutRNA(UCA)(Trp)) pair was generated for use in mammalian cells. The anticodon loop of the Bacillus subtilis tRNA(Trp) was mutated to UCA, three positions in the D arm were mutated to generate an internal promoter sequence, and the mutRNA(UCA)(Trp) gene was inserted between the 5' and 3' flanking sequences of the tRNA(Trp-1) gene from Arabidopsis to enhance its expression in mammalian cells. In vitro aminoacylation assays and in vivo opal suppression assays showed that B. subtilis TrpRS (BsTrpRS) charges only the cognate mutRNA(UCA)(Trp) and no endogenous mammalian tRNAs. Similarly, the mutRNA(UCA)(Trp) is specifically charged by B. subtilis TrpRS and not by endogenous synthetases in mammalian cells. Site-directed mutagenesis was then used to alter the specificity of BsTrpRS to uniquely charge 5-hydoxy-l-tryptophan. The resulting mutant BsTrpRS-mutRNA(UCA)(Trp) pair allows the efficient and selective incorporation of 5-hydroxy-l-tryptophan into mammalian proteins in response to the codon, TGA. This amino acid can be used as a fluorescence probe and also undergoes electrochemical oxidation in situ to generate an efficient protein crosslinking.     

Mitogenic effects of hydroxyapatite and calcium pyrophosphate dihydrate crystals on cultured mammalian cells

Synthetic hydroxyapatite (HA) crystals in 1% serum stimulated 3H thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures, as did 10% serum. The onset of stimulation and peak uptake of thymidine after crystal addition were delayed by 2-3 hours as compared with the effects produced by 10% serum. Stimulation of 3H thymidine uptake was proportional to the serum concentration used. HA crystals (50 micrograms/ml) stimulated nuclide uptake at each serum concentration used. 3H thymidine uptake was also proportional to the dose of HA or calcium pyrophosphate dihydrate crystals, although larger doses of the latter crystal were required to produce equivalent effects. Not all particulates were effective mitogenic agents. Latex beads and diamond crystals had no effect. Monosodium urate crystals modestly stimulated and calcium urate crystals markedly stimulated nuclide uptake. The more complex crystals found in a naturally occurring condition (calcinosis) were as mitogenic as the pure synthetic HA. The synovial cell hyperplasia sometimes associated with crystals might be explained in part by their mitogenic activity.     

Effects of sodium nitrite and potassium sorbate on in vitro cultured mammalian cells

The food additives sodium nitrite and potassium sorbate had cytostatic and cytotoxic effects on in vitro cultured V79 hamster cells and EUE human fibroblasts if administered in an acid environment (pH 4.95). The strong cytotoxic effect of sodium nitrite and that of the combined action of sodium nitrite and potassium sorbate was observed along the inhibition of macromolecular synthesis. In this respect, potassium sorbate was less effective. The decreased plating efficiency of the cells and the inhibition of de novo DNA synthesis induced by these substances aroused the question whether they also have genotoxic effects on V79 cells. Statistical analyses showed that sodium nitrite induced more 6-TG-resistant (6-TGr) mutants as compared to the untreated control. However, this elevation did not correspond to the level of inhibition of DNA synthesis determined during the followed period of time after the removal of the substance. Potassium sorbate and a combination thereof with sodium nitrite, in our experiments, had no mutagenic effects.