B and T lymphocyte colony formation in agar: 2-mercaptoethanol-activated albumin can substitute for 2-mercaptoethanol.
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Animals↗
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Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.
The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.
Animals↗
Specific inhibition of sclerotium formation by 2-mercaptoethanol and related sulfhydryl compounds in Sclerotium rolfsii.
Sclerotium formation in Sclerotium rolfsii was completely inhibited by 2-mercaptoethanol at a concentration of 2-4 mM without any adverse effect on mycelial growth. Concentrations lower than 2 mM had no effect on mycelial growth and sclerotium formation, whereas both were inhibited at concentrations higher than 4 mM. Complete inhibition of sclerotium formation with no effect on mycelial growth was also obtained by propyl mercaptan, 1-butyl mercaptan and 2-butyl mercaptan at a concentration of 0.10 mM. Sclerotium formation was also inhibited by benzyl mercaptan and thioglycolic acid at 0.15 mM and 2-4 mM concentration respectively, whereas it was only partially inhibited by L-cysteine and glutathione at 20 mM. Mycelium grown for 21 days in nutrient medium supplemented with mercaptoethanol at a concentration of 3 mM, when transferred into fresh medium without the chemical, grew normally and produced abundant mature sclerotia. Mercaptoethanol inhibited the initiation as well as the further development of young, unpigmented sclerotia. The mechanism of sclerotium formation was arrested completely when mercaptoethanol was added to the growth medium at any time between inoculation and the appearance of sclerotia of the "development" stage. It is suggested that the specific inhibitory action of mercaptoethanol could be used to study the mechanism of sclerotium formation
Cysteine↗
Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuolar membranes and activate their H(+)-ATPases.
Mercaptoethanol and dithiothreitol (DTT) inhibited the acidification of external medium by Saccharomyces carlsbergensis cells and protoplasts during glucose oxidation. The inhibition was also observed when cells were incubated with mercaptoethanol or when mercaptoethanol and DTT were used to prepare protoplasts. Experiments with S. carlsbergensis plasma membrane vesicles and vacuoles showed these thiol reagents to inhibit ATP-dependent generation of delta pH and Em across plasma membrane vesicles and vacuoles but to activate their H(+)-ATPases. Mercaptoethanol and DTT are suggested to de-energize plasmalemma as well as tonoplast by increasing their H(+)-permeability and to disturb the cell ion homeostasis.
Cell Membrane↗
Effects of 2-mercaptoethanol and aging in vitro on 17beta-hydroxysteroid oxidoreductase of guinea pig liver microsomes.
When microsomes were prepared in 2-mercaptoethanol Vmax for 17beta-hydroxysteroid oxidoreductase (17beta-HSD) was greater, the Km for NAD+ was greater and the Km for testosterone lower than in its absence. During storage at 4 degrees Vmax increased in the presence of 2-mercaptoethanol and decreased in its absence; Km values for testosterone and NAD+ increased during storage in both cases. The presence or absence of 2-mercaptoethanol did not affect the extent or time-course of inactivation of 17beta-HSD by trypsin or phospholipase A. Furthermore, no differences were detected in sedimentation properties on sucrose density gradients suggesting that the differences and changes in the kinetic behavior of 17beta-HSD reflect a conformational flexibility at the active site and are not due to extensive changes in the structure of the microsomes. 17beta-HSD exposed to 2-mercaptoethanol was subject to substrate inhibition by testosterone, a type of inhibition not previously reported for this enzyme.
17-Hydroxysteroid Dehydrogenases↗
Thiolysis of Nim-2,4-dinitrophenyl-histidine peptides with 2-mercaptoethanol. An explannation for the pH optimum.
The thiolysis of Nim-2,4-dinitrophenyl-histidine peptides with 2-mercaptoethanol demonstrates an optimal rate between pH 8.5 and 9.0. Base lability of both reactants and product was investigated as a possible cause for the pH optimum. N-2,4-Dinitrophenylimidazole, a model for Nim-2,4-dinitrophenylhistidine peptides, 2-mercaptoethanol and the product of thiolysis, 2,4-dinitrophenyl-S-mercaptoethan-2-ol, were subjected to aqueous basic conditions (pH 8.5 to 12.0). The reactions were followed spectrophotometrically, and products were identified by comparison of their ultra-violet spectra with commercially available or synthetic compounds. Mechanisms and rate constants for base hydrolysis of 2,4-dinitrophenyl-thio ethers are presented. Although N-2,4-dinitrophenylimidazole and 2,4-dinitrophenyl-S-mercaptoethan-2-oldohydrolyze, it is the oxidation of 2-mercaptoethanol to 2-mercaptoethanol disulfide which results in the decreasing rate of thiolysis above pH 9.0.
Dinitrobenzenes↗
[Use of 2-mercaptoethanol in cell culture].
Survival and growth in in vitro cultivation of lymphocytes, lymphoma cells and some other cells including human carcinomas are profoundly improved by 2-mercaptoethanol. These cells hardly take up cystine, an essential nutrient in the culture medium, but in the presence of 2-mercaptoethanol they can utilize cystine. Recently it has been found that 2-mercaptoethanol is effective in the in vitro cultivation of pathogenic trypanosomes and in the in vitro development of bovine embryos. The mechanisms by which 2-mercaptoethanol improves the survival and growth of these cells are described.
Animals↗
Role and mechanism of peripheral fatty acid mobilization in 2-mercaptoethanol-induced fatty liver.
2-Mercaptoethanol-induced fatty liver involves an increased free fatty acid mobilization which is primarily due to an inhibition of free fatty acid reesterification in adipose tissue. Furthermore, increased free fatty acid mobilization as well as fatty liver induction are not induced by 2-mercaptoethanol per se but result most probably from 2-mercaptoacetate through oxidation of 2-mercaptoethanol.
Adenosine Triphosphate↗
Effect of mercaptoethanol in the radioactive thin layer chromatography assay of NAD+-15-hydroxyprostaglandin in dehydrogenase.
The use of mercaptoethanol in the assay of rat kidney 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to have minimal effect on activity assayed with the spectrophotometric and substrate loss assays. However, mercaptoethanol appeared to inhibit PGDH when assayed by thin-layer chromatography, based upon conversion of 3H-PGE1 to 15-keto-3H-PGE1. Mercaptoethanol reacted with 15-keto-PGE1 to alter its chromatographic mobility and to suppress the U.V. absorption spectrum of 15-keto-PGE1. The implication of the use of ME in radiometric assays is discussed.
Animals↗
Role of mercaptoethanol and endotoxin in stimulating B lymphocyte colony formation in vitro.
Mercaptoethanol is necessary to permit B lymphocyte colony formation in semi-solid agar cultures of cells from normal mouse lymphoid organs. Transfer studies on developing colonies showed that, in part, this was a direct action on B lymphocyte colony cells but evidence was produced that in the presence of mercaptoethanol lymphoid organ cells release a factor promoting colony growth. Endotoxin strongly potentiated B lymphocyte colony formation in vitro by a direct action on colony cells but in the absence of mercaptoethanol did not allow cell survival or proliferation.
Animals↗
An absolute requirement for 2-mercaptoethanol in the in vitro primary immune response in the absence of serum.
Using a serum-free medium, high recoveries of plaque-forming cells (PFCs) were obtained up to 5 days of incubation in the in vitro primary immune response. Under these conditions, the presence in the medium of 2-mercaptoethanol was an absolute requirement. In order to obtain maximal recoveries of PFCs, the antigen and 2-mercaptoethanol had to be present from the start of the culture, while the addition of fetuin could be delayed for 24 h without any loss of PFCs. The 2-mercaptoethanol could be replaced by (dialysed) human serum.
Animals↗
Relationship between a long-term treatment of 2-mercaptoethanol and protein metabolism in the ageing rat.
Female RLEF1/Lati rats were chronically treated with 2-mercaptoethanol in a dose of 13 micrograms.100 g bw-1.day-1 dissolved in drinking water. During a 48-h experiment 15N-labelled glycine was given orally in a dose of 5 mg 15N.kg bw-1 and urine samples were collected and analysed by an emission spectrometric isotope method. Protein synthesis and nitrogen excretion rate constants were calculated according to the three-pool model, and 3-methylhistidine excretion rates were also determined. 2-Mercaptoethanol appears to influence protein metabolism; however, the slower rates of protein synthesis proved to be apparent in almost all groups of treated rats. Protein synthesis and nitrogen excretion rate constants have exceptionally high values in 2-year-old rats, possibly explained by the occurrence of hypercompensation mechanisms in old age. These were reflected by the excretion rates of 3-methylhistidine which were reduced as a result of sulphhydryl group interactions in age-dependent cellular metabolic changes.
Aging↗
Loss of thyroid hormone receptor activity in primary cultured rat hepatocytes is reversed by 2-mercaptoethanol.
In primary cultures of rat hepatocytes, specific thyroid-hormone-binding activity diminished with time and was hardly detectable at 24 h. In accordance with the loss of 3,5,3'-tri-iodothyronine (T3) binding, responses to the hormone disappeared, as indicated by low induction of the thyroid-hormone-responsive gene S14. In contrast, thyroid hormone receptor proteins were present, as determined by immunostaining with a specific antibody against the receptor. Thus the loss of T3 binding was due to receptor inactivation. After various attempts to restore the T3-binding activity, we found that 2-mercaptoethanol, a reducing agent, when added to the culture medium restored the hormone binding activity in a dose- and time-dependent manner. The observed kinetics and experiments using cycloheximide suggested that mercaptoethanol prevented inactivation of the newly synthesized receptors. Oxidoreductive conditions within cells may have a role in determining the level of activity of thyroid hormone receptors.
Animals↗
The stability of 99Tcm directly labelled to an Fab' antibody via stannous ion and mercaptoethanol reduction.
The anti-CEA FO23C5 F(ab')2 antibody was directly radiolabelled with 99mTcm by two methods (stannous ion and mercaptoethanol reduction) and compared in vitro and in vivo for label stability. By both methods, reduction of the F(ab')2 fragment produced primarily Fab' fragments. By both methods, the label was stable to 99Tcm-pertechnetate formation in vitro. Analysis by high performance liquid chromatography (HPLC) of serum, urine, kidney and liver homogenates from mice injected with 99Tcm-antibodies by both methods consistently showed a prominent radiolabelled peak with an estimated molecular weight of about 300 daltons. An identical peak was observed in the analysis of patient samples in a related investigation from this laboratory. Cysteine was radiolabelled with reduced 99Tcm and analysed by HPLC and thin layer chromatography (TLC); one of the 99Tcm-cysteine species so produced showed the same chromatographic behaviour as that of the 300 dalton species. In conclusion, the FO23C5 and other antibodies are stably labelled with 99Tcm via either stannous ion or mercaptoethanol reduction. In mice and in patients, the labelled proteins are either catabolized or, more likely, the 99Tcm label is transchelated such that the label is present on several low molecular weight species, the most prominent of which is postulated to be 99Tcm-cysteine.
Animals↗
Two tris urea mercaptoethanol extractable polypeptides found uniquely in scales of patients with psoriasis.
This study was designed to chemically characterize the principal structural proteins of psoriatic scales. Cornified cells were obtained from 40 patients with psoriasis, 21 patients with other scaly diseases, and 13 normal individuals. Cells were washed with Tris-HCl buffer and incubated in 8 M urea containing 2-mercaptoethanol (pH 9.0) at 30 degrees C for 7 hr. Extracted proteins were subjected to SDS polyacrylamide gel electrophoresis and protein patterns from normal and diseased scales were compared. The 67,000 dalton constituent of normal cornified cells could not be identified in protein from psoriatic scale and instead, a pair of polypeptides of approximately 54,000 and 57,000 daltons appeared. These extra bands were not found in protein extractions from other skin diseases, uninvolved skin of psoriasis patients, or normal skin. In order to analyze further normal and psoriatic scale proteins, the immunoreaction of rabbit antisera to human 67,000 dalton polypeptide with extracted psoriasis protein and with frozen biopsy sections, was studied using immunoprecipitation tests and indirect immunofluorescence microscopy. Both techniques demonstrated the existence of the 67,000 dalton protein in psoriasis, but as a minor component. These results indicate that additional unique urea mercaptoethanol soluble proteins are formed in psoriatic lesions, and this unusual protein synthesis may reflect the morphological changes in this disease.
Humans↗
Inhibition of aflatoxin formation by 2-mercaptoethanol.
2-Mercaptoethanol inhibits growth of Aspergillus parasiticus NRRL 3240 and aflatoxin formation by the fungus. When added to the resuspended medium, 2-mercaptoethanol inhibited [1-14C]acetate incorporation into both aflatoxins and neutral lipids, thereby showing that it acts at an early stage of aflatoxin biosynthesis. The inhibition is probably due to its chelating action on zinc, which is essential for aflatoxin production. It is proposed that any chelating agent that selectively binds to zinc will inhibit aflatoxin formation.
Acetates↗
Selective antibacterial action of 2-mercaptoethanol on propionibacteria in skin cultures.
2-Mercaptoethanol applied to the surface of agar medium had a selective antibacterial effect on Propionibacterium acnes and Propionibacterium granulosum without interfering with the growth of Peptococcus saccharolyticus or staphylococci in anaerobic cultures of skin or in pure cultures. In aerobic broth culture, 2-mercaptoethanol inhibited aerobes and stimulated anaerobes, consistent with its action as a reducing agent.
Aerobiosis↗
Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.
A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.
Bacterial Proteins↗