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PubMed2000

Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes.

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100-6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol-activated cytolysin family.

Actinomycetaceae↗
PubMed1997

High affinity saturable uptake of oxidized low density lipoprotein by macrophages from mice lacking the scavenger receptor class A type I/II.

Oxidation of low density lipoproteins (LDL) has been implicated as a causal factor in the pathogenesis of atherosclerosis. Oxidized LDL has been found to exhibit numerous potentially atherogenic properties in vitro, including receptor-mediated uptake by macrophages. Oxidized LDL is a ligand for the class A scavenger receptor type I/II (SR-AI/II), but cross-competition studies with cultured macrophages suggested that there is an additional receptor(s) that is specific for oxidized LDL and that does not interact with acetyl LDL or other chemically modified LDL. A number of macrophage membrane proteins, including CD36, FcgammaRII-B2, scavenger receptor BI, and macrosialin/CD68, have been found to bind to oxidized LDL in vitro and have been proposed as candidate oxidized LDL receptors. However, because of overlapping ligand specificity with the SR-AI/II, it has been difficult to evaluate the relative importance of these proteins in the uptake of oxidized LDL by macrophages. In the present report, we have studied the uptake and degradation of oxidized LDL by macrophages from mice in which the SR-AI/II gene had been disrupted. The uptake of acetyl LDL was reduced by more than 80% in macrophages from scavenger receptor knockout mice, confirming that most of the uptake of acetyl LDL by macrophages can be attributed to this receptor. In contrast, the uptake of extensively oxidized LDL was reduced by only 30% and showed high affinity, saturable uptake with apparent Km of about 5 microg/ml, similar to that of the SR-AI/II. This indicates that about 70% of the uptake of oxidized LDL in macrophages is attributable to an alternate oxidized LDL receptor(s). In contrast to findings reported with CD36, mildly oxidized LDL was internalized much more slowly than extensively oxidized LDL. Unlabeled oxidized LDL, polyinosinic acid, phosphatidylserine-rich liposomes, and LDL or bovine albumin modified by fatty acid oxidation products were effective competitors for the uptake of radioiodinated oxidized LDL by macrophages from knockout mice, whereas acetyl LDL and malondialdehyde-modified LDL were relatively poor competitors. This ligand specificity differs from that of CD36-related (class B) scavenger receptors but is similar to the reported specificity of macrosialin/CD68 in ligand blots. However, the rate of uptake of oxidized LDL by knockout macrophages was not increased by phorbol ester or in thioglycollate-elicited macrophages, both of which are expected to increase the amount of macrosialin on the cell surface. In macrophages from SR-AI/II knockout mice, ligand blots of membrane proteins with iodinated, oxidized, or acetylated LDL revealed several bands, with apparent molecular size on SDS-polyacrylamide gel electrophoresis of 60, 94, 124, and 210 kDa, but none of the bands were specific for oxidized LDL. These results provide direct evidence that a receptor other than SR-AI/II is responsible for most of the uptake of oxidized LDL in murine macrophages, but further studies are needed to identify the receptor(s) involved.

Animals↗
PubMed1992

Cyclic AMP inhibition of lipopolysaccharide-induced restriction of Legionella pneumophila growth in macrophage cultures.

The mechanism of the effects of lipopolysaccharide (LPS) on macrophages in terms of replication of intracellular facultative bacteria is unclear. It was found in the present study that the anti-Legionella pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by dibutyryl cyclic AMP (DcAMP). A 24-h pretreatment of murine thioglycolate-elicited macrophages with LPS resulted in an enhanced ability of these cells to inhibit the intracellular growth of L. pneumophila. This anti-L. pneumophila activity of macrophages induced by LPS was inhibited when DcAMP (10(-3) to 10(-5) M) was present during preincubation with LPS. The addition of DcAMP to the cultures was more effective before LPS treatment than after treatment. The effect of DcAMP was dose dependent. The secretion and production of acid phosphatase by LPS-activated macrophages were also inhibited by the addition of DcAMP before LPS treatment. Furthermore, the anti-L. pneumophila activity of macrophages induced by LPS could also be reversed in vitro by treatment with prostaglandin E2, colchicine, isoproterenol, theophylline, or hydrocortisone, all of which are known to increase the intracellular levels of cyclic AMP in various tissues. These observations indicate that the anti-L. pneumophila activity induced by LPS treatment can be modified by mechanisms involving cyclic nucleotide metabolism.

Animals↗
PubMed1987

Studies of the in vitro effect of methylmercury chloride on rat brain neurotransmitter enzymes.

The in vitro effect of methylmercury (MM) on the enzymatic activities of brain cell specific marker enzymes, choline acetyltransferase (CAT), glutamic acid decarboxylase (GAD), 2',3'-cyclic nucleotide phosphohydrolase (CNP), glutamine synthetase (GS) and enolase was examined. The results demonstrate that at 100 microM MM, GS activity was not affected whereas a small decrease in the activity of both GAD (20%) and enolase (10%) was observed. CNP and CAT activity appeared to be more sensitive toward MM with 100 microM MM producing inhibition of 50% and 30%, respectively. The addition of sulfhydryl protecting reagents such as DTT or sodium thioglycolate can restore the enzyme activities to normal control levels despite prior exposure of the enzymes to MM.

Animals↗
PubMed1989

Molecular cloning and characterization of the complementary DNA coding for the B-chain of murine Clq.

cDNA clones coding for the B-chain of murine Clq were isolated from a mouse macrophage library. The characterized clones include the total coding region plus a leader sequence. High homology was found with human Clq B-chain in the coding region (81%). Northern blot analysis of total RNA from different tissues of Balb/c mice showed one band of approximately 1.2 kb. The highest signal was found in RNA preparations of thioglycolate-activated peritoneal macrophages. The probe also hybridized with mRNA from spleen, thymus and heart. Extremely weak signals were found in liver, kidney, lung and intestine tissues.

Animals↗
PubMed1994

A flow cytometric assay reveals an enhancement of phagocytosis by platelet activating factor in murine peritoneal macrophages.

Phagocytosis of fluorescein isothiocyanate-labeled latex beads by peritoneal macrophages from thioglycollate-stimulated mice was examined in vitro by means of flow cytometry. This assay revealed that platelet activating factor (PAF), a known inflammatory mediator, enhanced the phagocytosis in a dose-dependent manner. The enhancement was completely suppressed by a PAF antagonist, Y-24180, and partly suppressed by another antagonist, CV-3988. The phagocytosis of both nonstimulated and PAF-stimulated macrophages was suppressed in Ca(2+)-free ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid-containing solution, but the phagocytosis was enhanced by PAF even in this solution. These results suggest that phagocytosis by macrophages is enhanced by PAF in allergic and inflammatory reactions.

Animals↗
PubMed1981

Endocytosis of native and cationized horseradish peroxidase by cultured mouse peritoneal macrophages. Variations in cell surface binding and intracellular traffic and effects of colchicine.

Thioglycollate-elicited mouse peritoneal macrophages were cultivated in vitro and endocytosis of native and cationized horseradish peroxidase was studied electron microscopically and biochemically. Native peroxidase (HRP) was ingested by fluid-phase endocytosis and accumulated in lysosomes. Cationized peroxidase (CHRP) bound diffusely to the macrophage surface in a saturable manner. It was then internalized via membrane folds and transferred not only to lysosomes but also to the Golgi complex, mainly those parts referred to as GERL and positive for acid phosphatase activity. Following initial uptake, surface staining for CHRP was lost, although the tracer remained present in the medium. This indicates that anionic binding sites were internalized together with the ligand and not immediately replaced. Accordingly, continued uptake of CHRP occurred at a rate similar to that for HRP. Exposure of the macrophages to cationized ferritin (CF) decreased their ability to bind CHRP. However, after 2 to 4 h in CF-free medium, the CHRP-binding ability returned and raised to 2 to 3 times higher values than in cells not exposed to CF. Treatment with cycloheximide at a concentration that effectively inhibits protein synthesis did not clearly affect this regeneration. These findings support the concept of recirculation of plasma membrane constituents. They further suggest that there exists an intracellular membrane pool which rapidly exchanges with the cell surface. Colchicine removed cytoplasmic microtubules, caused a characteristic disorganization of the Golgi complex, and inhibited uptake of both HRP and CHRP. Additionally, no transport of CHRP to the Golgi complex or GERL was observed in colchicine-treated cells. The regeneration of surface anions after exposure to CF was also delayed. Contrarily, lumicolchicine was without effect on cell morphology and uptake as well as intracellular transport of the tracers. Nevertheless, the effects of colchicine on endocytosis were not necessarily due to a direct role of microtubules. They could be secondary to a disturbed function of the Golgi complex or other organelles after collapse of the microtubular cytoskeleton.

Animals↗
PubMed1980

Killing of the S and Re forms of Salmonella minnesota via the classical pathway of complement activation in guinea-pig and human sera.

The S (wildtype) and Re form (heptose-deficient, core-defective mutant) of Salmonella minnesota were killed by treatment with normal guinea-pig serum (GPS). Using C4-deficient GPS and serum containing 0.02 M ethyleneglycol-bis-(beta-aminoethylether)-tetraacetic acid and 0.02 M MgCl2 (EGTA-Mg2+) a reduced killing rate was observed. In normal GPS diluted 1:10 containing 0.02 M EGTA-Mg2+ or in C4-deficient GPS diluted 1:10 no killing occurred, whereas the same serum dilution without EGTA-Mg2+ showed a strong bactericidal effect indicating a dependency upon C4 and Ca2+ ions. Furthermore, in contrast to normal human serum (NHS) no killing occurred in a selective complete C1q-deficient human serum. The bactericidal effect, however, could be restored by addition of highly purified C1q; this is a further indication for a dependency upon the classical pathway of C activation. The C-dependent bactericidal activity was totally abolished when phosphate buffer was used, partially reduced in the presence of veronal-buffered saline (VBS), and not affected by tris-(hydroxymethyl)-aminomethane(Tris) or thioglycollate-buffered system EGTA-Mg2+ alone slightly reduced the growth rate of the bacteria whereas disodium ethylene diaminetetraacetate (EDTA) had a bacteriostatic effect on the S-form. The inhibition of the growth of the Re-form by EDTA was amplified by the addition of serum. Pre-incubation of bacteria with serum for absorption of antibodies did not increase the killing rate of such pre-treated bacteria excluding an antibody-mediated bactericidal reaction. Furthermore, pre-treatment of the bacteria with GPS at 0 degrees reduced the serum sensitivity of both types of bacteria.

Animals↗
PubMed1995

2-Deoxy-D-glucose but not 2-mercaptoacetate increases Fos-like immunoreactivity in adrenal medulla and sympathetic preganglionic neurons.

2-Deoxy-D-glucose (2DG) and 2-mercaptoacetate (MA) are drugs that competitively inhibit metabolism of glucose and fatty acids, respectively. Both 2DG and MA stimulate food intake. In addition, 2DG-induced glucoprivation is a known stimulus for adrenomedullary secretion. However, very little is known about the effects of MA on the sympathoadrenal system. In the present study, we examined effects of 2DG and MA on the activity of preganglionic neurons and the adrenal medulla, as indicated by expression of Fos-like immunoreactivity (Fos-li). 2DG, MA, or saline was administered using a stress-attenuated paradigm incorporating remote drug infusion. Expression of Fos-like immunoreactivity (Fos-li) was subsequently examined in the adrenal medulla and in preganglionic sympathetic neurons throughout the intermediolateral column (IML) of the thoracic and lumbar spinal cord. We found that 2DG increased Fos-li in the adrenal medulla and in the IML primarily at spinal cord segments T7-T10, where adrenomedullary preganglionic neurons reside. In contrast, MA did not induce Fos-li either in the adrenal medulla or in sympathetic preganglionic neurons at any cord level. Results support the hypothesis that decreased fatty acid oxidation is not a stimulus for adrenal medullary secretion and provide evidence for a highly selective stimulation of adrenal medullary preganglionic neurons by 2DG.

Adrenal Medulla↗
PubMed1988

Characteristics of the beta-glucan receptor of murine macrophages.

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.

Animals↗
PubMed1975

The biological fate in rats of vinyl chloride in relation to its oncogenicity.

The main eliminative route for [14C]vinyl chloride after oral, i.v. or i.p. administration to rats is pulmonary; both unchanged vinyl chloride and vinyl chloride-related CO2 are excreted by that route and the other [14C] metabolites via the kidneys. After intragastric administration, pulmonary output of unchanged vinyl chloride is proportional to the logarithm of reciprocal dose. Excretion patterns after i.v. and i.p. injections are predictable from the characteristics of excretion following oral administration. Pulmonary excretion of unchanged vinyl chloride after oral dosing is complete within 3-4 h, but pulmonary elimination of CO2 and renal excretion of metabolites occupies 3 days. In comparison, 99% of a small i.v. dose is excreted unchanged within 1 h of injection; 80% within 2 min. The rate of elimination of a single oral doses of [14C]vinyl chloride is uninfluenced by up to 60 days' chronic dosing with the unlabelled substance. The distribution volume of vinyl chloride as displayed by whole-animal autoradiography agrees with deductions from excretion data. Small localization of 14C in the para-auricular region of appropriate sections occurs in sectioned tubules, belonging possibly to the Zymbal glands. Biotransformation of vinyl chloride into S-(2-chloroethyl) cysteine and N-acetyl-S-(2-chloroethyl) cysteine occurs through addition of cysteine, and biotransformation into: (i) chloroacetic acid, thiodiglycollic acid and glutamic acid, and (ii) into formaldehyde (methionine, serine), CO2 and urea is explicable in terms of an associative reaction with molecular O2 involving a singlet oxygen bonded transition state in dynamic equilibrium with a cyclic peroxide ground state. There is no evidence for chloroethylene oxide formation. Thiodiglycollic acid is the major metabolite of chloroacetic acid in rats; more than 60% of the dose. The interaction of vinyl chloride and of its primary metabolites with the intermediates of mammalian metabolism is discussed in relation to the oncogenicity of that substance.

Acetates↗
PubMed1991

Disinfection of alginate impression material using disinfectants as mixing and soak solutions.

Disinfectant solutions were used as water substitutes in the preparation of specimens of alginate impression material. Chlorhexidine gluconate and sodium peroxysulphate solutions were tested. The alginate specimens were infected by sucking, subsequently disinfected in the solution used in the alginate preparation, and then tested for infectivity by 24-h incubation in thioglycollate broth. Bacterial growth was measured by changes in turbidity using a turbidimeter. The results indicate that chlorhexidine gluconate is an effective alginate disinfectant, when it is used as the liquid for alginate preparation and post-setting disinfection solution.

Alginates↗
PubMed1970

Effect of bile and desoxycholate on gram-negative anaerobic bacteria.

The bile tests for characterizing gram-negative anaerobic bacilli were reevaluated in prereduced anaerobically sterilized peptone-yeast-glucose broth, in thioglycollate broth, and on blood agar plates. Blood agar plates were unsatisfactory. The combination of 20% bile with 0.1% desoxycholate inhibited Fusobacterium, Bacteroides melaninogenicus, and B. oralis and sometimes Sphaerophorus necrophorus, but not B. fragilis or other Sphaerophorus species studied. Ten per cent bile with 0.05% desoxycholate was less satisfactory. There was no significant difference between fresh and commercial powdered bile. Desoxycholate (0.1% in thioglycollate broth) inhibited B. fragilis, Fusobacterium, B. melaninogenicus, B. oralis, and S. necrophorus, but not S. varius or S. mortiferus/S. ridiculosus. The bile and desoxycholate tests are simple to perform and helpful for characterization and classification of gram-negative anaerobic bacilli.

Animals↗
PubMedDate not supplied

[The bioutilization of thiodiglycol (a breakdown product of mustard gas): isolation of degraders and investigation of degradation conditions].

The Alcaligenes xylosoxydans subsp. denitrificans strain TD1 capable of degrading thiodiglycol (TDG), a breakdown product of mustard gas, was isolated from soil contaminated with breakdown products of this chemical warfare agent. The selected stable variant of TD1 (strain TD2) can grow on TDG with a lag phase of 4-8 h and a specific growth rate of 0.04-0.045 h-1. Optimal conditions for the biodegradation of TDG (pH, the concentration of TDG in the medium, and its relative content with respect to the bacterial biomass) were determined. TDG was found to be degraded with the formation of diglycolsulfoxide and thiodiglycolic acid. The data obtained can be used to develop approaches to the bioremediation of mustard gas-contaminated soils.

Alcaligenes↗
PubMed2004

2-Mercaptoacetate does not stimulate chow intake in periweanling rats.

In adult rats, administration of drugs that suppress oxidation of fatty acids, like mercaptoacetate (MA), produces increases in food intake. During development, the consequences of administration of MA are more varied. For example, in very young pups, intake of milk diets is unaffected by MA, while pups aged 12 to 15 days demonstrate increases in intake. However, in 18- and 21-day-old rats, milk intake is suppressed by administration of MA. Typically, the paradigms used to test rat pups differ significantly from those used to assess intake in adult rats. The present experiments were designed to examine whether 18-day-old pups tested with adultlike paradigms showed adultlike responses to administration of MA. In the first experiment, rat pups aged 18 days were injected with 0 or 68.4 mg/kg MA, then given 60-min tests while consuming either milk or chow diets that were novel, or to which they had previously been exposed. The results demonstrated that chow intake was not affected by administration of MA, but milk intake in experienced animals was suppressed by MA. Experiment 2 demonstrated that in contrast to administration of MA, 18-day-old pups deprived of food overnight showed increases in intake of chow and milk diets. In Experiment 3, when the effects of a range of doses of MA (22.8, 45.6, 68.4 and 91.2) on chow intake over a 4-h period were assessed, all doses of MA produced a significant suppression of chow intake in 18-day-old pups. Taken together, the data suggest that alterations in fatty acid oxidation produced by administration of MA do not stimulate chow intake in periweanling pups tested in an adultlike fashion.

Analysis of Variance↗
PubMed1989

[Determination of mercapturic acid in urine samples for the monitoring of occupational exposure to xenobiotics].

The determination of mercapturic acids in urine samples of employees exposed to carcinogenic substances requires highly sensitive and specific methods such as GC/MS or HPLC/Fluorimetry. We applied GC/MS to the analysis of thiodiglycolic acid, which is an indicator metabolite of vinyl chloride exposure and of S-phenylmercapturic acid which can be used to control benzene exposure. S-cyanoethylmercapturic acid appears as a degradation product during acrylonitrile metabolism. This metabolite can be determined by HPLC combined with a sulfur specific fluorimetric detector. This procedure which is of general use offers the possibility to improve the biological monitoring in case of exposures to carcinogenic substances.

Acetylcysteine↗