Studies on the amino groups of myosin ATP-ase. I. Effect of ethyl-formyl-hydroxamate on the ATPase activity of myosin.
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Adenosine Triphosphatases↗
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Results for “THIOGLYCOLLIC ACID”
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Morphological and chemical studies of the spores and parasporal bodies of Bacillus laterosporus.
Spores of Bacillus laterosporus were studied to determine the chemical and morphological nature of their basophilic canoe-shaped parasporal bodies. An unusually high phosphorus content of these spores compared to other Bacillus species appeared to be associated with the parasporal body. Preparations of these "canoes" still attached to the spore coats were indeed high in phosphorus, but also in nitrogen. They were free of lipide-soluble and nucleic acid phosphorus and stained for protein. Some 50 per cent of the total nitrogen, but only 6 to 10 per cent of the total P were liberated by extraction with alkali-thioglycollate (pH 11.5) or alkali alone (pH 12.2-12.5). Proteinaceous material was recovered from these alkaline extracts and electron microscopy indicated that there had been a marked loss of "canoe" substance. Extraction with acid, removed some 80 per cent of the phosphorus associated with the "canoes" as orthophosphate. Chromatographic analyses for amino acids indicated some 14 ninhydrin-positive spots in the canoe-coat preparations whereas the whole spores contained at least 16.
Acids↗
Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile.
It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Akerlund, Microbiology 145:1683-1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n = 28) of C. difficile (>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in C. difficile and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C. difficile-associated diarrhea.
Amino Acid Sequence↗
CXC chemokine redundancy ensures local neutrophil recruitment during acute inflammation.
Previous publications demonstrated that elevated systemic levels of interleukin (IL)-8 decrease local neutrophil recruitment. We tested whether sustained, high plasma levels of IL-8 would prevent local inflammation after inflammatory insults. Mice carrying the transgene for human IL-8 were separated on the basis of their plasma levels of IL-8 into IL-8-positive (plasma levels >90 ng/ml) and IL-8-negative (IL-8 below detection). Presence of the IL-8 transgene did not improve survival or morbidity nor did it alter peritoneal neutrophil recruitment induced by the cecal ligation and puncture model of sepsis. In an acute lung injury model created by intratracheal injection of acid, IL-8-positive mice showed no reduction in alveolar neutrophil recruitment. There was no difference in the local recruitment of neutrophils when either thioglycollate or glycogen was injected intraperitoneally. We examined the chemotactic response to murine chemokines to test how neutrophil recruitment occurs in the setting of elevated plasma IL-8 and found that neutrophils from both IL-8-positive and -negative mice respond equally well to recombinant KC or macrophage inflammatory protein (MIP)-2. We measured KC and MIP-2 in the peritoneum after thioglycollate injection and demonstrated that IL-8-positive mice have significantly higher levels of the chemokines compared to the IL-8-negative mice. Antibody inhibition of KC and MIP-2 in the IL-8-positive mice significantly decreased peritoneal neutrophil recruitment in response to thioglycollate, clarifying their important role in the local neutrophil recruitment. Our data demonstrate that despite the presence of high plasma levels of IL-8, neutrophils may still be recruited to sites of local inflammation because of chemokine redundancy.
Acute Disease↗
Influence of plasminogen deficiency on the contribution of polymorphonuclear leucocytes to fibrin/ogenolysis: studies in plasminogen knock-out mice.
Plasminogen knock-out (PG(-/-)) mice provide an unique opportunity for the study of alternative mediators of fibrinolysis. Polymorpho-nuclear leucocytes (PMNs) contain non-plasmin fibrinolytic proteases, however the degree to which these cells contribute to fibrin(ogen) degradation in these animals is not known. Thrombi were generated in carotid arteries and jugular veins of PG(-/-) and wild type (PG(+/+)) mice following adventitial application of a 20% ferric chloride solution. PMNs, identified histologically on H&E staining and by immunohistochemistry using anti-mouse PMN RB6-8C5 antibody, accumulated within the thrombus by 6 h after the injury and peaked at 24 h. There was significantly greater retention of PMNs within the thrombi of PG(-/-) mice from 48 to 72 h than in the PG(+/+) controls (at 72 h: PG(-/-)255 +/- 41 cell/mm(2) (n = 5), PG(+/+) 61 +/- 10 cell/mm(2) (n = 5), p<0.01 in the arterial thrombi; PG(-/-) 252 +/- 50 cell/mm(2) (n = 5), PG(+/+) 100 +/- 36 cell/mm(2) (n = 5), p<0.05 in the venous thrombi), providing potential for more PMN derived fibrinolytic enzymes to be present at late times after a thrombotic challenge in PG(-/-) mice relative to the PG(+/+) controls. Intact PMNs were elicited from the peritoneal cavities of PG(-/-) and PG(+/+) mice following 4% thioglycolate stimulation. In vitro studies showed PMNs from PG(-/-) mice to release greater quantities of 10% trichloroacetic acid (TCA)-soluble fibrinopeptides from I(125)-labeled fibrinogen, than cells from PG(+/+) controls although these differences did not become apparent until after 24 h of incubation (at 72 h incubation: PG(-/-) 918 n/10 x 10(6) cells/0.5 ml, PG(+/+) 589 ng/10 10(6) cells/0.5 ml p = 0.005). Furthermore, autoradiographic analysis of the I(125)-labeled fibrinogen degradation products showed the cleavage pattern by PG(-/-) PMNs to be distinct from that produced by PG(+/+) PMNs. These data suggest that a relatively greater role for PMNs-initiated fibrinolysis exists in the setting of plasminogen deficiency, although this prominence only becomes evident more than 24 h after the thrombotic insult. In addition, mechanisms responsible for the process in PG(-/-) mice may be distinct from those primarily responsible for the process in PMNs from PG(+/+) mice.
Animals↗
In vitro metabolism of inorganic arsenic by the gastro-intestinal microflora of the rat.
The contents of the rat small intestine or caecum reduced sodium [74As] arsenate to arsenite over a period of 2-3 days. In the presence of caecal, but not small intestine, contents, small amounts of methyl arsonic acid and dimethylarsinic acid were formed. Stomach contents reduced arsenic very slowly. The metabolism of arsenate was inhibited by autoclaving the gut contents or by adding antibiotics, thus indicating that bacteria were involved. In prereduced media containing thioglycollate or cysteine in the absence of intestinal bacteria, sodium arsenate was reduced to arsenite. The rate of reduction of arsenate by the gut flora was increased by the presence of hydrogen sulphide or bile acids.
Animals↗
Comparative cell wall analyses of morphological forms within the genus Actinomyces.
Comparative cell wall analyses were made of mycelial and smooth forms of Actinomyces bovis and A. israelii to determine the changes which occur in the cell wall composition concurrent with a change in morphology, and to evaluate cell wall analyses as a criterion for taxonomic identification within the genus Actinomyces. Cell walls of the spider forms of A. boyis had little or no aspartic acid and a high hexosamine concentration; cell walls of the smooth forms had a high aspartic acid content and low concentrations of hexosamine. Both forms had large amounts of glutamic acid, alanine, and lysine, as previously reported. A strain of Actinomyces, previously identified as A. naeslundii on the basis of morphology and aerobic growth characteristics, was found to have the basic cell wall composition of A. israelii. When transferred from the Actinomyces maintenance broth to a thioglycolate broth, the cells of this strain passed from a mycelial form through a transient filamentous morphology to become diphtheroidal with continued incubation. Concomitantly, the concentrations of glutamic acid relative to alanine decreased, and the hexosamine content increased. Variation in morphology within the species A. israelii and A. bovis could not be related to any mutual chemical change of their cell walls.
Actinomyces↗
Evidence that macrophages transfer arachidonic acid and cholesterol to tissues in vivo.
Our previous studies have shown that [(14)C]-labelled cholesterol (CHOL) and arachidonic acid (AA) are transferred from macrophages (Mphi) to lymphocytes (LY) when these cells are co-cultured. In this study, we investigated whether these lipids can be transferred from control and thioglycollate-elicited Mphi (THIO-elicited Mphi) to various tissues and organs in vivo. For this purpose, control and THIO-elicited Mphi were pre-treated with [(14)C]-AA and [(3)H]-CHOL and then injected into the jugular vein of adult rats. More than 75% of the radioactivity injected was found in the liver of rats treated with [(14)C]-AA labelled-Mphi either control and THIO-stimulated. The radioactivity of [(3)H]-CHOL labelled Mphi was transferred mainly to the liver (51% in the control Mphi and 23% in the thioglycollate Mphi7) but it was also found in the kidney, lung and spleen. These results support the proposition that the transfer of lipids between cells also occurs in vivo. The full significance of this phenomenon however remains to be elucidated.
Aging↗
Polyphenols inhibit promotional phase of tumorigenesis: relevance of superoxide radicals.
Ellagic acid (EA), tannic acid (TA), caffeic acid (CA), and ferulic acid (FA) offer considerable promise as anticarcinogens. The role of these dietary polyphenols was investigated in the promotional phase of carcinogenesis. Topical application of polyphenols simultaneously with phorbol-12-myristate-13-acetate (PMA) or mezerein resulted in significant protection against 7,12-dimethyl-benz[a]anthracene-induced skin tumors in mice. Caffeic acid was the most effective inhibitor of tumor promotion. In vivo and in vitro treatment of murine peritoneal macrophages with the tumor promoters resulted in stimulation of superoxide anion radical formation. Tannic acid, caffeic acid, and ferulic acid were stronger inhibitors of PMA- and mezerein-induced superoxide anion radical than ellagic acid in in vivo and in vitro conditions. Treatment of [1(3)-14C]glycerol- or [methyl-14C]choline chloride-labeled resident or thioglycollate-elicited macrophages with PMA and mezerein led to accumulation of radioactive diacylglycerol equivalents. The polyphenols were capable of inhibiting these releases.
9,10-Dimethyl-1,2-benzanthracene↗
A new acid hydrolysis method for determining tryptophan in peptides and proteins.
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Alkanesulfonates↗
Inhibitory effect of 2-mercaptoacetate on fatty acid oxidation in the liver.
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Acetyl Coenzyme A↗
Differential responses to adjuvants of macrophages from young virgin, aging virgin and aging breeder mice.
Age related differences in macrophage responsiveness to adjuvants seen previously with thioglycollate induced cells were also found with resident macrophages. Thus, activation of phagocytosis by lipopolysaccharide, polyadenylic-polyuridylic acid complexes and muramyl dipeptides occurred when exposed to macrophages removed from young but not aging C58 and C3H/He mice. However, breeding status differences were observed in that aging virgin mice were unresponsive to these adjuvants, while aging breeder mice responded similarly to young virgin mice. Analysis of any changes in membrane phospholipids was carried out to determine their association with the age related and breeding status differences observed. Significant differences occurred in 32P labelling of phosphatidyl inositol between unstimulated macrophages from young and aging C58 mice. No differences were evident, however, after LPS stimulation. Analysis of macrophage plasma membranes from young and aging mice for cholesterol revealed significant age related differences in their ability to undergo increases in cholesterol content after LPS activation.
Adjuvants, Immunologic↗
Vagal afferents mediate the feeding response to mercaptoacetate but not to the beta (3) adrenergic receptor agonist CL 316,243.
To evaluate the role of subdiaphragmatic vagal afferents in the anorectic response to peripheral administration of the highly selective beta(3)-adrenergic receptor agonist CL 316,243 (CL), we tested the ability of intraperitoneal (IP) injections of CL to inhibit feeding in rats with subdiaphragmatic vagal deafferentation (SDA, n=13) or sham surgeries (SHAM, n=13). Doses of 10, 100 and 1000 ng/kg CL significantly reduced feeding by statistically similar amounts in SHAM and SDA rats. One hour after IP injection, each dose of CL also significantly increased plasma concentrations of free fatty acids and beta-hydroxybutyrate, an indicator of hepatic fatty acid oxidation (FAO), whereas 6h after injection only the two highest CL doses increased plasma beta-hydroxybutyrate. In contrast, peripheral administration of the FAO inhibitor mercaptoacetate (MA, 45.6 mg/kg IP) stimulated feeding in SHAM but not in SDA rats, extending previous data suggesting a necessary role of vagal afferents in the feeding-stimulatory effect of FAO inhibition. We conclude that subdiaphragmatic vagal afferents are essential for the feeding-stimulatory action of MA but not for the anorectic action of peripheral CL and that CL-induced increase in hepatic FAO is not essential for its feeding-inhibitory effect.
3-Hydroxybutyric Acid↗
Medial versus lateral parabrachial nucleus lesions in the rat: effects on mercaptoacetate-induced feeding and conditioned taste aversion.
The two experiments of the present study examined the influence of bilateral electrophysiologically-guided ibotenic acid lesions of the medial (gustatory) and lateral (viscerosensory) subdivisions of the parabrachial nucleus (PBN) on lipoprivic feeding and on the acquisition of a conditioned taste aversion. In Experiment 1, mercaptoacetate (0, 400, 600, or 800 micromol/kg) failed to enhance food intake in normal rats maintained and tested on standard laboratory chow. In the same procedure, rats with lesions of the medial or lateral PBN consumed less food during baseline but nonetheless were sensitive to the orexigenic action of mercaptoacetate. In Experiment 2, both types of PBN lesions prevented acquisition of a conditioned taste aversion induced by the oral self administration of lithium chloride. The results suggest that PBN neurons essential for conditioned taste aversion are not involved in the mercaptoacetate-induced feeding of rats maintained and tested on standard laboratory chow.
Animals↗
The effects of amino acids and metals on the infectivity of poliovirus ribonucleic acid.
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Amino Acids↗
Metabolic influences on satiety in rats receiving parenteral nutrition.
Food intake is reduced during parenteral nutrition (PN) proportionally to the amount of calories or composition of the solution infused. The relative importance of infused glucose and lipid, 50 and 30% of PN kilocalories, respectively, in reducing food intake during PN was examined. Glycolysis, fatty acid oxidation, or both were acutely disrupted with 2-deoxy-D-glucose (2-DG) and mercaptoacetate (MA). Rats receiving intravenous infusions of saline or a PN solution providing 100% of total daily calories (PN-100) received a single intraperitoneal injection of saline, 2-DG, and/or MA during the early light phase. 2-DG (1.4 or 2.2 mmol/kg) did not initiate feeding in PN-100 rats, although hyperglycemia was evident in all rats 1 h after 2-DG. Food intake of PN-100 rats after MA (0.4 mmol/kg) was approximately 50% that of control rats. When 2-DG (1.4 mmol/kg) and MA (0.4 mmol/kg) were administered concomitantly, PN-100 and control rats consumed the same amount of food. During PN-100, rats appeared to be more sensitive to losing metabolic energy derived from lipid than from glucose.
Animals↗
Reduced glucose availability induces torpor in Siberian hamsters.
Siberian hamsters kept in long photoperiods (16 h light/day) at 10 degrees C do not display torpor when provided with food ad libitum but do reduce their body temperatures (Tb) from 37 degrees C to as low as 15 degrees C for several hours each day in response to food restriction. Female hamsters maintained in a long photoperiod and fed ad libitum were injected with 2-deoxy-D-glucose (2-DG), a glucose analogue that reduces cellular glucose oxidation, or mercaptoacetate (MA), a drug that disrupts fatty acid oxidation. Dosages of 2-DG in excess of 1,500 mg/kg body mass reliably induced torpor with latencies of approximately 50 min from time of injection to a decrease in Tb below 30 degrees C; 79% of females displayed torpor at a dosage of 2,500 mg/kg. MA was uniformly ineffective in inducing torpor and did not increase incidence of torpor when combined with 2-DG treatment or food restriction. Decreased availability of glucose may be the major metabolic signal for inducing torpor in Siberian hamsters; availability of fatty acids appears to play little if any role in regulating this behavior.
Animals↗
Macrophage oxidation of low density lipoprotein generates a modified form recognized by the scavenger receptor.
Incubation of low density lipoprotein (LDL) with endothelial cells or smooth muscle cells overnight has resulted in an oxidative modification of LDL that results in its recognition by macrophages by way of the acetyl LDL receptor. In the present study, we examined whether macrophages themselves can oxidize and modify LDL in a manner similar to that of endothelial cells. Incubation of 125I-labeled LDL with resident or thioglycollate-elicited macrophages for 24 hours in Ham's F-10 medium resulted in the appearance of thiobarbituric acid (TBA) reactive materials and trichloroacetic acid (TCA) soluble radioactivity in the medium. The LDL harvested from these incubations showed increased electrophoretic mobility and was degraded rapidly when added to fresh macrophages as compared to LDL previously incubated in the absence of cells. These macrophage-induced modifications could be prevented if the first incubation was carried out in the presence of the antioxidant butylated hydroxytoluene (BHT) or in Dulbecco's modified Eagle's medium (DMEM). The degradation of 125I-labeled macrophage-modified LDL by macrophages was competitively inhibited by unlabeled acetyl LDL or unlabeled endothelial cell-modified LDL but not by native LDL, indicating that the degradation was mediated by the acetyl LDL receptor.
Animals↗