The action of reducing agents on the dissociation of iron transferrin with special reference to thioglycollate.
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Ascorbic Acid↗
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Results for “THIOGLYCOLLIC ACID”
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Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages.
The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.
Animals↗
Uptake and incorporation of saturated and unsaturated fatty acids into macrophage lipids and their effect upon macrophage adhesion and phagocytosis.
Murine thioglycollate-elicited peritoneal macrophages were cultured in the presence of a variety of fatty acids added as complexes with bovine serum albumin. All fatty acids tested were taken up readily by the cells and both neutral and phospholipid fractions were enriched with the fatty acid provided in the medium. This generated a range of cells enriched in saturated, monounsaturated or polyunsaturated fatty acids, including n-3 acids of fish oil origin. Saturated fatty acid enrichment enhanced macrophage adhesion to both tissue culture plastic and bacterial plastic compared with enrichment with polyunsaturated fatty acids. Macrophages enriched with the saturated fatty acids myristate or palmitate showed decreases of 28% and 21% respectively in their ability to phagocytose unopsonized zymosan particles. Those enriched with polyunsaturated fatty acids showed 25-55% enhancement of phagocytic capacity. The greatest rate of uptake was with arachidonate-enriched cells. Phagocytic rate was highly correlated with the saturated/unsaturated fatty acid ratio, percentage of polyunsaturated fatty acid and index of unsaturation, except for macrophages enriched with fish-oil-derived fatty acids; they showed lower phagocytic activity than expected on the basis of their degree of unsaturation. These results suggest that membrane fluidity is important in determining macrophage adhesion and phagocytic activity. However, in the case of phagocytosis, this effect may be partially overcome if the cells are enriched with fish-oil-derived fatty acids. Thus it may be possible to modulate the activity of cells of the immune system, and so an immune response, by dietary lipid manipulation.
Animals↗
Dietary n-3 fatty acids alter murine peritoneal macrophage cytotoxicity.
Male CD-1 mice were fed diets containing 10 wt% fat composed of a constant amount of n-6 polyunsaturated fatty acids (PUFA; safflower oil, 1.5 wt%) and increasing levels of n-3 PUFA (up to 1.5 wt%); the remaining fat was a 50:50 mixture of tripalmitin and triolein. Four days prior to sacrifice, the animals were injected intraperitoneally with sterile thioglycollate. Peritoneal macrophages were analyzed for phospholipid fatty acids, prostaglandin E2 (PGE2) synthesis and cytotoxicity towards L929 target cells. Increasing dietary n-3 PUFA increased macrophage phospholipid n-3 PUFA from 4.4 to 11.0 mol% with a concomitant decline in n-6 PUFA from 15.5 to 10.9 mol%, and PGE2 synthesis declined from 2.6 to 1.5 pmol/million cells in thioglycollate-elicited macrophages. Macrophage cytotoxicity toward target L929 cells declined by approximately 50% with increasing n-3 PUFA concentration.
Animals↗
The utilization of casein and amino acids by Streptococcus sanguis P4A7 in continuous culture.
Streptococcus sanguis P4A7 was grown in glucose limited conditions in continuous culture at pH 7.0 in a chemically defined medium containing either free amino acids or casein as the organic nitrogen source. Apart from aspartate and threonine, which were poorly utilized at the higher dilution rates, all amino acids in the free-amino-acid medium were utilized to various extents. At the higher dilution rates, aspartate actually increased in concentration, probably due to deamidation of asparagine. The amino acid most utilized at all dilution rates was arginine, with up to 99% of the amino acid being consumed. Both casein and its alpha s1-casein fraction supported growth at a level only slightly lower than that obtained with the free-amino-acid medium, provided that either cysteine or thioglycollate was present. With the exception of tyrosine, nearly all of the amino acyl residues of alpha s1-casein were utilized to some degree. In general, the higher the concentration of each amino acid in the medium (whether free or as part of alpha s1-casein) the higher the extent of utilization by S. sanguis P4A7. Only 50% of the arginyl residues (0.16 mM) of alpha s1-casein were utilized compared with 99% of free arginine (1.5 mM) under similar conditions, suggesting that only 50% of the alpha s1-casein arginine was accessible to the organism.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Effects of noninsulin-dependent diabetes mellitus on the uptake of very low density lipoproteins by thioglycolate-elicited mouse peritoneal macrophages.
The lipid-laden foam cells from atherosclerotic lesions appear to be derived from macrophages which have accumulated lipids from plasma lipoproteins. When examined in vitro, thioglycolate-elicited mouse peritoneal macrophages do not accumulate lipids when exposed to normal low density lipoproteins (LDL), but take up very low density lipoproteins (VLDL) or chemically modified LDL with resultant lipid accumulation. Patients with noninsulin-dependent diabetes mellitus (NIDDM) have an increased incidence of atherosclerosis, due in part to disturbances in lipoprotein metabolism. We investigated the possibility that VLDL isolated from patients with NIDDM are taken up by mouse peritoneal macrophages more avidly than normal. Two groups of patients with NIDDM were studied; one group was normotriglyceridemic and the other group was hypertriglyceridemic. The VLDL from both normotriglyceridemic and hypertriglyceridemic patients were enriched in cholesterol and triglyceride compared to that from normal subjects. Thioglycolate-elicited mouse peritoneal macrophages bound and degraded greater amounts of VLDL isolated from patients with NIDDM (both normo- and hypertriglyceridemic) than of VLDL from normal subjects. While normal VLDL caused a marked increase in cellular triglyceride and cholesteryl ester contents in macrophages, VLDL isolated from patients with NIDDM resulted in an even greater cellular accumulation of lipids. These results suggest that the VLDL of patients with NIDDM have alterations in their composition and metabolic behavior. The increased uptake of VLDL by macrophages may contribute to the enhanced atherosclerosis present in NIDDM.
Adult↗
Mercaptans decrease selenium-dependent glutathione peroxidase activity in the chick.
A variety of mercaptans, especially the beta-mercaptocarboxylic acids, inhibited the selenium (Se)-dependent glutathione peroxidase (SeGSHpx) activity of chick liver postmitochondrial supernatant or cytosol and of purified bovine erythrocyte SeGSHpx. The effects of mercaptans and a glutathione analogue on Se utilization were determined by subcutaneous injection of test compounds into vitamin E-deficient chicks fed diets containing 0.1 ppm Se (as Na2SeO3) at times when greater than 50% of vitamin E- and Se-deficient chicks showed the vitamin E-, Se-deficiency disease exudative diathesis (ED). D-(-)-Penicillamine hydrochloride (the positive control model compound), sodium beta-mercaptopyruvate, t-butyl mercaptan and S-methylglutathione (nonmercaptan glutathione analogue) decreased SeGSHpx activity in chick liver postmitchondrial supernatants within 24 h of injection and increased the incidence of ED within 4 d. Other mercaptans tested did not increase ED incidence or affect liver, kidney or plasma SeGSHpx activities. Although mercaptosuccinic acid, N-(2-mercaptopropionyl)glycine and sodium thioglycolate each strongly inhibited SeGSHpx in vitro, each also significantly increased chick mortality in the dosage range tested; therefore, their effects on SeGSHpx in vivo could not be evaluated. It appears that the beta-mercaptocarboxylic acids, mercaptans with a high degree of steric hinderance in close proximity to the thiol group and a close structural analogue of glutathione are capable of altering selenium status in chicks.
Animals↗
The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum.
To assess the effects of inflammation on the generation of circulating DNA from dead and dying cells, plasma DNA levels were determined in BALB/c mice, administered apoptotic or necrotic Jurkat cells following induction of peritonitis by treatment with thioglycollate (TG), peptone (PT), or sodium periodate (NaIO(4)). In mice receiving TG or NaIO(4), plasma DNA levels following intraperitoneal administration of Jurkat cells were significantly reduced compared with controls, whereas they were not affected in mice receiving PT. To determine the basis of these differences, the cellular composition of peritoneal fluids prior to the administration of the dead cells was analyzed. Among agents tested, TG administration led to the largest increase in cells, both neutrophils and monocytes. As shown by flow cytometry, the exudates contained apoptotic neutrophils and macrophages, with the highest levels in the TG-induced exudates. Analysis of DNA and caspase 3 in the fluids also showed differences. TG exudates showed increases in DNA and caspase 3, while NaIO(4)-induced exudates had an increase only in DNA. Fluid from PT-treated mice did not have increases in DNA or caspase 3. Together, these results indicate that prior inflammation can affect the generation of blood DNA from apoptotic or necrotic cells, although this effect may vary depending on the composition of the exudates with respect to cells as well as DNA.
Animals↗
Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy.
Macrophage-derived foam cells are a prominent component of developing atherosclerotic lesions. We describe an in vitro model of foam cell formation which mimics some aspects of the evolution of foam cells in mature atherosclerotic lesions. Thioglycollate-elicited mouse peritoneal macrophages were incubated with copper-oxidized LDL (ox-LDL) for periods up to 168 hr. Identifiable foam cells were present after incubation with ox-LDL at 24, 72, and 168 hr. Control cells incubated without ox-LDL did not form foam cells. Fluorescence microscopy after staining with Nile red exhibited progressive accumulation of lipids, and transmission electron microscopy (TEM) showed distinct ultrastructural changes over time. Macrophages at 24 hr had a few non-membrane-bound lipid droplets but were otherwise identical to control cells. These lipid droplets fluoresced yellow-gold after Nile red staining. After 72 hr of incubation with ox-LDL, in addition to increased numbers of non-membrane-bound lipid inclusions, macrophages contained membrane-bound multilamellar lipoid structures. These multilamellar structures corresponded to areas of reddish-orange fluorescence after Nile red staining. In macrophages incubated with ox-LDL for 168 hr, the amount of cellular lipid was further increased and cholesterol crystal profiles were apparent within some multilamellar lipoid structures. Biochemical analysis showed that the total cholesterol content steadily increased over 168 hr. The increase in total cholesterol was accompanied by a dramatic increase in free cholesterol between 72 and 168 hr. These results demonstrate that long-term incubation of macrophages with ox-LDL increased lipid deposition in cultured cells and that, under the conditions studied, cholesterol crystals formed in macrophage foam cells. Moreover, this system allows investigation of the evolution of foam cells showing some characteristics of those found in atherosclerotic lesions.
Animals↗
DECLINE OF SPECIFIC INFECTIVITY OF COWPEA CHLOROTIC MOTTLE VIRUS IN VIVO.
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Antioxidants↗
Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages.
J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.
Ammonium Chloride↗
Effects of erdosteine treatment against doxorubicin-induced toxicity through erythrocyte and plasma oxidant/antioxidant status in rats.
The clinical use of doxorubicin (Dxr), an antineoplastic agent, is limited by its extensive toxicity which is mostly mediated by oxidant injury. We have studied the effect of erdosteine, a mucolytic drug showing antioxidant properties, in preventing Dxr-toxicity to improve future Dxr therapy. Male Sprague-Dawley rats were divided into four groups. The first group that underwent no medication was accepted as control group; the second group was treated with a single i.p. injection of Dxr (20 mg kg(-1) b.wt.); the third group was treated with oral erdosteine alone (10 mg kg(-1) b.wt. day(-1) for 12 days); and in the last group erdosteine was administered starting before Dxr injection for 12 days. The thiobarbituric acid reactive substances (TBARS) level of Dxr group was higher in both plasma and erythrocyte than the other groups. In plasma and erythrocyte, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased in Dxr plus erdosteine group in comparison with control group, and the activities of GSH-Px were increased in Dxr plus erdosteine group in comparison with Dxr group. The erythrocyte catalase (CAT) activity of Dxr plus erdosteine group was higher than control and Dxr groups. Plasma xanthine oxidase activities and nitric oxide (NO) levels were not significantly different between groups, however erythrocyte NO level of Dxr group was higher than control. In conclusion, Dxr administration resulted in increased lipid peroxidation in plasma as well as erythrocyte and erdosteine treatment helped to prevent oxidative injury by increasing antioxidant enzymes, especially SOD, GSH-Px and CAT, in rats.
Animals↗
Inhibitory effects of metabolite I of erdosteine on the generation of nitric oxide and peroxynitrite chemiluminescence by human neutrophils.
Polymorphonuclear neutrophils (PMNs) can generate superoxide anions and nitric oxide (NO), which is not only an important mediator of various cellular activities, but can also react with superoxide anions to produce peroxynitrite anions (ONOO-). Peroxynitrite is a potent and potentially toxic oxidant that damages various types of biomolecules. It preferentially mediates the oxidation of thiolic groups in protein and non-protein molecules, thus altering their functions. The aim of this study was to examine whether, in addition to its ability to reduce the respiratory bursts of human PMNs, the SH metabolite I (Met I) of erdosteine, can interfere with NO and NO-derived peroxynitrite production, thus extending its antioxidant activity. This was done by means of the luminol amplified chemiluminescence (LACL), which has been widely used to detect the production of reactive oxidant species (ROS) by PMNs under various conditions. At 5 and 10 microg/ml, Met I significantly reduced LACL after fMLP and PMA stimulation. When L-Arg was added to the reaction medium, as a NO donor, the chemiluminescence of fMLP increased by up to 67% and that of PMA by up to 132%, but was once again significantly reduced by 5 and 10 microg/ml of Met I. In a cell-free system, the use of linsidomine (SIN-1) makes it possible to investigate the behavior of LACL induced by peroxynitrite release, which was significantly reduced by Met I concentrations ranging from 1.25 to 10 microg/ml. Our findings indicate that Met I, a molecule with a SH group, reacts with ROS, NO and NO-derived peroxynitrite, and has both antioxidant and scavenging activity. This is of interest for the strategy of protecting against damage induced by radical species in the pulmonary cell environment, in which they can induce a phlogogenic loop, and suggests that adding exogenous thiols may be useful in antagonizing the toxic effects of reactive molecules on endogenous thiols.
Adult↗
Effects of erdosteine on smoking-induced lipid peroxidation in healthy smokers.
AIM: Oxidative stress caused by smoking has been implicated in many pulmonary diseases. Smoking causes reductions in plasma nitrate plus nitrite (NOx) concentrations and increases in plasma malondialdehyde (MDA) concentrations, which indicate oxidative stress and lipid peroxidation, respectively. In this study, we investigated the acute effects of smoking a single cigarette on the plasma concentrations of NOx and thiobarbituric acid reactive substances (TBARS) including MDA, and whether administration of erdosteine, a mucolytic and antioxidant agent, affects these parameters. METHODS: Thirty healthy smokers were included in the study. Subjects smoked a single cigarette in 10 minutes on the study day. For analysis of NOx, TBARS and cotinine, blood was drawn from each subject before and 5 and 30 minutes after smoking. The subjects were then randomly divided into two groups, one receiving placebo and the other erdosteine suspension 175mg/5mL twice daily for 1 month. After this treatment period, the same study protocol was carried out. Two subjects in the placebo and five subjects in the study group were excluded because of noncompliance. RESULTS: Twenty-three (14 female, 9 male) subjects completed the study. Their mean age was 32 +/- 8 years and their smoking history was 14 +/- 9 pack-years. Baseline NOx, TBARS and cotinine concentrations were similar between the groups. NOx concentrations decreased significantly after smoke exposure. At the end of the treatment period there were no significant differences in NOx, TBARS or cotinine concentrations between the groups. The concentration of TBARS after smoking decreased significantly in the erdosteine-treated group (at 5 minutes: 2.8 +/- 0.5 micromol/L before treatment and 2.3 +/- 0.3 micromol/L after treatment, p < 0.05; at 30 minutes: 2.8 +/- 0.5 micromol/L before treatment and 1.8 +/- 0.7 micromol/L after treatment, p < 0.05). Smoking history was significantly correlated with cotinine concentrations. CONCLUSION: Acute smoke exposure decreased plasma NOx concentrations in healthy smokers, and this was not changed with erdosteine treatment. However, significant decreases were noted in TBARS concentrations after smoke exposure in the group that received erdosteine, suggesting that short-term erdosteine administration might help prevent smoking-induced lipid peroxidation.
Adult↗
[Hydrolysis of complex levomycetin esters].
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Acrylates↗
Direct and indirect assays of serum iron with a sensitive new color reagent.
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Blood Proteins↗
Antigenic cancer cells that escape immune destruction are stimulated by host cells.
Cancers induced by UV light in murine skin often regress completely when transplanted into normal syngeneic recipients and grow progressively only in T-cell-deficient hosts. Heritable cancer variants that grow progressively and kill normal mice occasionally evolve in vivo. It is surprising that most of these variants appear to retain their antigenicity and immunogenicity. We have compared three such variants (4102-PRO, 6132A-PRO, and 6134-PRO) with the parental tumors to determine why the variants acquired progressive phenotypes without antigen loss. We found that all three variants grew substantially faster than the parental tumors in T-cell-deficient hosts; one variant, 6132-PRO, also grew faster in vitro. Furthermore, the growth of all of the variants was stimulated by soluble factors released by tumor-induced peritoneal exudate cells, and all attracted more leukocytes than the parental cells. Finally, pretreatment of mice with antigranulocyte antibody reduced the growth of variant but not parental 4102 and 6134A tumor cells. The treatment reduced the growth of both the parental and the variant 6132A lineage cells. We found no evidence for acquired resistance of variant tumors to immune destruction by a host defense mechanism. The parental cells did not grow faster in beige nude mice deficient in natural killer and alpha beta T cells or in SCID mice deficient in B and T cells. The variant parental cells had a similar sensitivity to lysis by polyinosinic-polycytidic acid-induced natural killer cells or thioglycolate- and LPS-induced macrophages. Together, our results are consistent with the notion that these variants escape from immune destruction in vivo by attracting leukocytes that stimulate tumor cell growth.
Animals↗
Dietary myristic acid alters acylated proteins in activated murine macrophages.
After stimulation with select activating agents such as lipopolysaccharide (LPS) or recombinant interferon-gamma (rIFNgamma), several macrophage proteins may be induced, acylated with myristic acid, or both. Our goal in this study was to determine whether altering the levels of myristic acid in the diet would modulate the levels of a specific acylated macrophage protein, MacMARCKS (myristoylated, alanine-rich C kinase substrate), because that fatty acid can be found in substantial quantities in some foods. Thioglycollate-elicited peritoneal macrophages from groups of mice fed diets with various levels of myristic acid (from 0.2 to 99 g/100 g fatty acids) were treated with LPS, phorbol myristate acetate (PMA), or rIFNgamma plus LPS, which are well-established macrophage activating agents. Levels of MacMARCKS were measured by enzyme-linked immunosorbent assay using a rabbit anti-mouse polyclonal antibody against the first 10 amino acids of murine MacMARCKS. A 42-kDa protein with the same molecular weight as MacMARCKS was identified in macrophage lysates by Western analysis using the antibody. Lipopolysaccharide- and PMA-activated macrophages from mice fed the trimyristin diet had significantly greater levels of MacMARCKS than LPS- and PMA-activated macrophages of mice fed the safflower oil-containing diet. The levels of MacMARCKS were also greater in lysates of LPS plus rIFNgamma-stimulated macrophages from mice fed the trimyristin diet and mice fed a diet containing a moderate level of myristic acid (12 g/100 g fatty acids) compared with the lysates of macrophages from mice fed the safflower oil diet. These results indicate that altering the level of myristic acid in the diet may alter the production of specific proteins that may be involved in macrophage activation.
Acylation↗