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PubMed1980

Biochemical properties of CO2-dependent streptococci.

A total of 153 clinical isolates and 10 reference strains were employed in an investigation of CO2-dependent streptococci. Their selection was based on a lactic acid homofermentative end product. This group of organisms grew best in 5 to 10% CO2, and several species, including Streptococcus mutans, S. intermedius, Streptococcus MG, S. anginosus, and S. constellatus, required increased CO2 for primary recovery. A basal medium of thioglycolate with 0.1% Tween 80 and phenol red was prepared and used with selected carbohydrates. This media provided luxuriant growth. Serological testing failed to give any definitive correlation with species identification. A shortened differentiation scheme, combined from previous studies, was proposed.

Carbohydrate Metabolism↗
PubMed1985

Release of galactosyltransferase from peritoneal macrophages during acute inflammation.

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2-4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophage-enriched, Fc receptor-bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4 degrees C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.

Animals↗
PubMed1998

Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis.

The protozoan parasite, Giardia duodenalis, shares many metabolic and genetic attributes of the bacteria, including fermentative energy metabolism which relies heavily on pyrophosphate rather than adenosine triphosphate and as a result contains two typically bacterial glycolytic enzymes which are pyrophosphate dependent. Pyruvate decarboxylation and subsequent electron transport to as yet unidentified anaerobic electron acceptors relies on a eubacterial-like pyruvate:ferredoxin oxidoreductase and an archaebacterial/eubacterial-like ferredoxin. The presence of another 2-ketoacid oxidoreductase (with a preference for alpha-ketobutyrate) and multiple ferredoxins in Giardia is also a trait shared with the anaerobic bacteria. Giardia pyruvate:ferredoxin oxidoreductase is distinct from the pyruvate dehydrogenase multienzyme complex invariably found in mitochondria. This is consistent with a lack of mitochondria, citric acid cycle, oxidative phosphorylation and glutathione in Giardia. Giardia duodenalis actively consumes oxygen and yet lacks the conventional mechanisms of oxidative stress management, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. In their place Giardia contains a prokaryotic H2O-producing NADH oxidase, a membrane-associated NADH peroxidase, a broad-range prokaryotic thioredoxin reductase-like disulphide reductase and the low molecular weight thiols, cysteine, thioglycolate, sulphite and coenzyme A. NADH oxidase is a major component of the electron transport pathway of Giardia which, in conjunction with disulphide reductase, protects oxygen-labile proteins such as ferredoxin and pyruvate:ferredoxin oxidoreductase against oxidative stress by maintaining a reduced intracellular environment. As the terminal oxidase, NADH oxidase provides a means of removing excess H+, thereby enabling continued pyruvate decarboxylation and the resultant production of acetate and adenosine triphosphate. A further example of the bacterial-like metabolism of Giardia is the utilisation of the amino acid arginine as an energy source. Giardia contain the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, but not in other eukaryotes apart from trichomonads and Chlamydomonas reinhardtii. The pathway includes substrate level phosphorylation and is sufficiently active to make a major contribution to adenosine triphosphate production. Two enzymes of the pathway, arginine deiminase and carbamate kinase, are rare in eukaryotes and do not occur in higher animals. Arginine is transported into the trophozoite via a bacterial-like arginine:ornithine antiport. Together these metabolic pathways in Giardia provide a wide range of potential drug targets for future consideration.

Amino Acids↗
PubMedDate not supplied

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine.

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of 75Se from 75Se-labeled selenite (conc. 10 mumol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique. L-Cysteine (L-Cys) stimulated 75Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum. D-Cys was less effective in the jejunum and similarly effective in the cecum. L-Leucine (L-Leu) and L-glutamic acid significantly reduced the stimulatory effect of L-Cys on Se uptake in the distal jejunum, whereas the respective effect of D-Cys was not diminished by L-Leu. Cysteamine stimulated mucosal 75Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced 75Se uptake in the distal jejunum. The stimulatory effects of L-Cys, mercaptopyruvate, and thioglycolate were Na(+)-dependent, whereas the effect of cysteamine also occurred in the absence of Na+. Mercaptosuccinate, D-penicillamine, ergothioneine, and thiosulfate did not enhance mucosal 75Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na(+)-dependent and Na(+)-independent mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.

Analysis of Variance↗
PubMed2000

Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages.

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.

Adjuvants, Immunologic↗
PubMed1974

In vitro synthesis and secretion of lysozyme by mononuclear phagocytes.

Pure cultures of three types of mononuclear phagocytes-mouse peritoneal macrophages, unstimulated or after thioglycollate stimulation, and human monocytes-synthesize and secrete large amounts of lysozyme in vitro. The macrophage lysozyme is indistinguishable from authentic lysozyme in its ability to lyse M. lysodeikticus, inhibition by specific antisera, a similar size of 14,000 and cationic charge. Lysozyme secretion in culture is characterized by a large net increase in total lysozyme, 4-20-fold in 3 h, 75-95% of which is in the medium, and its continued extracellular accumulation over at least 2 wk in culture. Lysozyme is the major (14)C-labeled protein secreted into the medium by both unstimulated and thioglycollate-stimulated macrophages and the 0.75-1 microg produced per 1 x 10(6) cells/day represents 0.5-2.5% of the total cell protein. Lysozyme is a cell-specific marker for mononuclear phagocytes and the PMN, which contains preformed enzyme, since it is absent in lymphoid cells and a variety of fibroblast and epithelioid cell lines. Lysozyme production is also a useful measure of mononuclear phagocyte cell number. The rate of lysozyme production and secretion is remarkably constant for all cell types under a variety of culture conditions. Production by the mouse macrophage increases threefold on the 2nd day in culture and then remains linear with time. Production is optimal at a relatively low serum concentration, but can be maintained, in the absence of serum, in lactalbumin hydrolysate or, at a reduced level in basal media. The production and secretion of lysozyme are independent of the production of macrophage acid hydrolases. Net increase and secretion of lysozyme occur under conditions where acid hydrolases like N-acetyl beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, and cathepsin D are neither accumulated nor secreted. Massive phagocytosis of latex particles has no effect on lysozyme production and secretion. Lysozyme production can be rapidly inhibited by treatment with cycloheximide (0.4 microg/ml) whereas inhibition of its production by colchicine (10(-6) M) occurs only after a lag period of more than 8 h, and is probably due to a secondary effect. These results show that mouse macrophages provide a simple in vitro system to measure lysozyme secretion and its control. These studies also indicate the possible importance of mononuclear phagocytes in the secretion of a variety of biologically active products and in the modification of their environment.

Amino Acids↗
PubMed1988

The effects of malnutrition on murine peritoneal macrophages.

Differences were detected between peritoneal macrophages (both resident and elicited) from mice on a low protein diet and from normal animals. The concentration of resident peritoneal macrophages was lower in animals on low protein diets than in normal controls. Although total protein (and therefore cell mass) of resident macrophages from malnourished mice was increased, their contents of thiamine pyrophosphatase, succinate dehydrogenase, and non-specific esterase were disproportionately reduced. In addition they did not ingest as many glutaraldehyde-fixed sheep erythrocytes or attach to as many adherent C3b sensitized sheep red blood cells as those from normal animals, although reduction of nitroblue tetrazolium was unaffected. Initially (24 hr after thioglycollate), elicited macrophages from malnourished mice did not divide as frequently as those from normal mice but by 48 hr the differences were insignificant. The elicited macrophage possessed lower levels of total protein (indicating a reduced cell mass); the levels of acid phosphatase, thiamine pyrophosphatase, succinate dehydrogenase, and nonspecific esterase and nitroblue reducing activity were also proportionately reduced. They ingested fewer glutaraldehyde-fixed erythrocytes and reacted with fewer C3b sensitised sheep red blood cells than those from normal mice; ingestion of IgG-coated sheep erythrocytes, on the other hand, was somewhat increased. These abnormalities may influence adversely the efficiency of early phlogistic responses and favor the establishment of infection in malnourished animals.

Animals↗
PubMed1983

Measurement of phagocytosis utilizing 51Cr-labeled tannic acid treated erythrocytes.

Macrophages recognize and ingest tannic acid treated erythrocytes (TAE) by a mechanism which does not involve the well characterized receptors for immunoglobulin and complement. Since the TAE are susceptible to osmotic lysis, a clear distinction can be made between erythrocytes which are ingested from those attached to the phagocytes' surface. We present a quantitative assay for macrophage phagocytosis using 51Cr-labeled TAE. Uptake of TAE was linearly related to the incubation time during the first 40 min, and increased with the TAE concentration in a log fashion. Furthermore, both attachment and ingestion were positively related to the concentration of tannic acid used to pretreat the erythrocytes. As an example of the application of the method, we compared the ingestion of TAE with that of opsonized erythrocytes (EA) by resident and elicited mouse peritoneal macrophages. While the uptake of EA by thioglycollate-induced macrophages was increased by a factor of approximately 7 over the resident population, the uptake of TAE was only stimulated 2.3-fold. The method will be useful in comparative physiological and pharmacological studies of immunological and non-immunological phagocytosis by macrophages.

Animals↗
PubMed1971

Anaerobic roll tube media for nonselective enumeration and isolation of bacteria in human feces.

Medium 10 (M10), developed for rumen bacteria and containing small amounts of sugars, starch, volatile fatty acids, hemin, Trypticase, yeast extract, cysteine, and sulfide, plus agar, minerals and CO(2)-HCO(3)-buffer, was used with the Hungate anaerobic method as a basal medium to evaluate the efficacy of various ingredients. Three-day-old colony counts from adults on normal diets (17 samples) were 0.55 x 10(11) to 1.7 x 10(11) per g (mean, 1.15 x 10(11)) for M10. Single deletion of volatile fatty acids, Trypticase, yeast extract, or sulfide did not reduce counts. Deletion of hemin or both Trypticase and yeast extract significantly lowered counts. Addition of fecal extract, rumen fluid, 1% dehydrated Brain Heart Infusion (BHI) or 2 to 6% liver infusion did not increase counts; 1% dehydrated bile or 3.7% BHI markedly depressed them. Decreasing the gas-phase CO(2) concentration from 100 to 5% with N(2) and correspondingly lowering the HCO(3) had little effect. Counts in supplemented Brewer Thioglycollate (Difco), BHI, and Trypticase soy agar were similar or lower than in M10; ease in counting was best in M10. Comparison of features of 88 predominant strains of fecal bacteria randomly isolated indicated that M10 supported growth of as many or more species of bacteria as compared to supplemented BHI. The results suggest that predominant bacteria of human feces, in general, are not as nutritionally fastidious as rumen bacteria and indicate that media for counts or isolation containing large amounts of rich organic materials are neither necessary nor desirable when adequate anaerobic techniques are used.

Adult↗
PubMed1993

Structure, expression, and evolution of guinea pig serum amyloid P component and C-reactive protein.

The structure and expression of the pentraxins, serum amyloid P component (SAP), and C-reactive protein (CRP), have been investigated in the guinea pig. Northern blot analysis of hepatic RNA from animals in which acute inflammation had been induced by intraperitoneal injection of thioglycollate established that neither SAP or CRP is a major acute phase reactant in the guinea pig. Genomic clones of SAP and CRP were isolated and sequenced, and the gene and the derived protein sequences were compared with other mammalian homologues. Both genes have organizations typical of the pentraxin genes of other species, but some differences were defined in the regions that potentially determine the capacity of the pentraxin gene to be induced during acute inflammation. Nucleotide substitutions in coding regions have occurred at similar rates in the two pentraxin genes. Nonsynonymous substitution rates indicate that SAP and CRP are subject to similar, relatively low levels of constraint; at the amino acid sequence level the rate of evolution is approximately two replacements per site per 10(9) years. An estimate of the phylogenetic relationship among the pentraxin genes suggests that SAP and CRP arose as the result of a gene duplication event that occurred very early in mammalian evolution, but subsequent to the divergence of the reptilian ancestors of the mammalian and avian lineages. This raises doubts about the identity of proteins from fish, which have been previously characterized as CRP and SAP.

Acute-Phase Reaction↗
PubMed1982

Toxicokinetics of chloroethanol in the rat after single oral administration.

The excretion and tissue distribution of 14C-labelled chloroethanol were studied in rats following single oral administration of 5 and 50 mg/kg body weight. At both dose levels, the radioactivity was rapidly eliminated, mainly in the urine. On the first day after application of 5 mg/kg body weight, 77.2% of the dose were found in the urine, 1.7% in the faeces, and 1.0% as carbon dioxide in the expired air. Only 2.8% were excreted by these routes during the following 3 days. The residual radioactivity remaining in the tissues after 4 days was almost equally distributed and amounted to about 0.4% of the dose in the liver and 3% in the whole organism. At the higher dose level, excretion rates and tissue concentrations were similar. Examination of the urine by anion exchange chromatography on DEAE-Sephadex revealed two metabolites which were identified by GC/MS analysis as thiodiacetic acid and thionyldiacetic acid. These metabolites represented almost the whole urinary radioactivity. They were excreted in approximately equal amounts at the low dose whereas the thiodiacetic acid predominated with about 70% of the urinary radioactivity at the high dose. Unchanged chloroethanol, chloroacetic acid, S-carboxymethylcysteine and sulphonyldiacetic acid were not found in the urine.

Animals↗
PubMed2006

Mercaptoacetate fails to block the feeding-inhibitory effect of the beta3-adrenergic receptor agonist CGP 12177A.

Peripherally administered beta3-adrenergic receptor (beta3-AR) agonists stimulate lipolysis and inhibit food intake. To test the hypothesis that this inhibition of feeding is due to a substrate-driven increase in hepatic fatty acid oxidation (FAO), we assessed the ability of the FAO inhibitor mercaptoacetate (MA) to reverse the feeding-inhibitory effect of the beta3-AR agonist CGP 12177A (CGP). Adult male Sprague-Dawley rats received intraperitoneal injections of 1 mg/kg CGP, of 45.6 mg/kg MA, or of both drugs, and the effects on food intake, plasma free fatty acids (FFA), and plasma beta-hydroxybutyrate (BHB), an indicator for hepatic FAO, were assessed. Control rats received saline injections. CGP significantly reduced food intake after 0.5 and 6 h and increased plasma FFA and BHB at 0.5 h, suggesting increased lipolysis and hepatic FAO. MA completely reversed the increase in plasma BHB and thus appeared to effectively abolish CGP's effect on hepatic FAO, but MA failed to affect CGP's feeding-inhibitory action. These findings do not support the hypothesis that the beta3-AR agonist CGP inhibits feeding by enhancing hepatic FAO or ketogenesis. Although the beta3-AR agonist CGP reduced saccharin intake in a one-bottle condition taste aversion test, it seems unlikely that the hypophagic effect of CGP is elicited by malaise.

3-Hydroxybutyric Acid↗
PubMed1976

The respiratory metabolism of Mycobacterium lepraemurium.

The respiratory metabolism of Mycobacterium lepraemurium isolated from Sprague-Dawley rats lepromata using several substrates was investigated. None of the intermediates of the glycolysis cycle as well as of the tricarboxylic acid cycle except succinate was oxidized by purified whole suspensions of M. lepraemurium. Likewise, many sulfur compounds such as cystine, thiourea, thioacetate, thiodiglycol, mercaptoact and some sulfhydryl compounds, e.g., cysteine, dithioerythritol, dithiorthritol, and penicillamine were readily oxidized by murine bacillary suspensions, whereas thioglycolate, thioglucose, and reduced glutathione were oxidized at a slow rate. Succinate was not or was very poorly oxidized by normal cells probably because of impermeability of the cell wall but the addition of succinate to the cell suspensions frozen for 1 min at -40 degrees C considerably enhanced oxygen uptake over the endogenous value. The oxidation of succinate was unaffected by inhibitors rotenone, atabrine, and amytal but was markedly inhibited by thenoyltrifluoroacetone, antimycin A, 2-N-heptyl-4-hydroxyquinoline-N-oxide, and cyanide. The thiol-binding agents, p-hydroxymercuribenzoate and N-ethylmaleimide were also effective inhibitors of succinate oxidation but the process was not affected by uncouplers dinitrophenol, dibromophenol, pentachlorophenol, and carbonyl-cyanide-m-chlorophenylhydrazone. The results indicated that succinate oxidation by M. lepraemurium was mediated by oxidative enzymes involving an electron transport chain with oxygen as the terminal electron acceptor.

Antimycin A↗
PubMed1995

Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages.

This study investigated the effects of feeding mice lipids with different fatty acid compositions upon the ability of stimulated macrophages to produce inflammatory mediators. Weanling mice were fed for 8 weeks on a low-fat (LF; 2.5% by weight) diet or on diets containing 20% by weight of hydrogenated coconut oil (HCO), olive oil (OO), safflower oil (SO), or menhaden (fish) oil (MO). Thioglycollate-elicited peritoneal macrophages were isolated. Macrophages isolated from MO-fed mice produced less PGE2, 6-keto-PGF1 alpha, TXB2, and interleukin-6 in response to lipopolysaccharide (LPS) stimulation than those from mice fed each of the other diets. Macrophages from mice fed the OO, SO, or MO diets produced less tumor necrosis factor alpha in response to LPS stimulation than those from mice fed the LF or HCO diets. There was no effect of dietary lipid manipulation upon the production of interleukin-1 by LPS-stimulated macrophages. Macrophages from mice fed the MO diet produced more superoxide and hydrogen peroxide in response to phorbol ester stimulation than those from mice fed each of the other diets. In response to unopsonized zymosan, macrophages from mice fed the SO or MO diets produced more hydrogen peroxide than macrophages from mice fed the other diets. LPS-stimulated nitric oxide production was greater from macrophages from OO-, SO-, or MO-fed mice than from those fed the LF or HCO diets. Thus, the nature of the lipid consumed in the diet has significant effects upon the production of a variety of inflammatory mediators by macrophages. The most potent effect is caused by fish oil consumption. Possible mechanisms by which dietary fatty acids, particularly the n-3 polyunsaturated fatty acids found in fish oils, could affect mediator production by macrophages are described. The clinical relevance of such effects is discussed.

Animals↗