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At least 793 records · Page 44Linked to original sources
PubMed1985

Secretion of phospholipase A1 by bone marrow-derived macrophages.

Bone marrow-derived macrophages contain phospholipase activity of the A1 and A2 types, active at acid or neutral pH and with different specificities for the fatty acid to be liberated. In contrast to this variety, only one single phospholipase could be detected in extracellular fluids of these cells. Surprisingly, this phospholipase was of the A1 type and active at about pH 8. It exhibited a restricted substrate specificity in that, of the various substrates tested, only phosphatidylcholine containing palmitic acid in position 2 was degraded. This total restriction was not detected with phosphatidylethanolamine substrates. In addition to phospholipase A1, extracellular fluids exhibited lipase activity. A modulation of enzyme secretion could not be achieved by lymphokines or phorbol esters. However, release could be blocked by treating cells with cycloheximide (5 micrograms/ml) or tunicamycin (0.5 micrograms/ml). Phospholipase A1 was also released by thioglycollate-induced peritoneal macrophages.

Animals↗
PubMed2000

Monomeric sarcosine oxidase: 2. Kinetic studies with sarcosine, alternate substrates, and a substrate analogue.

Monomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. MSOX can oxidize other secondary amino acids (N-methyl-L-alanine, N-ethylglycine, and L-proline), but N,N-dimethylglycine, a tertiary amine, is not a substrate. N-Methyl-L-alanine is a good alternate substrate, exhibiting a k(cat) value (8700 min(-)(1)) similar to sarcosine (7030 min(-)(1)). Turnover with L-proline (k(cat) = 25 min(-)(1)) at 25 degrees C occurs at less than 1% of the rate observed with sarcosine. MSOX is converted to a two-electron reduced form upon anaerobic reduction with sarcosine or L-proline. No evidence for a spectrally detectable intermediate was obtained in reductive half-reaction studies with L-proline. The reductive half-reaction with L-proline at 4 degrees C exhibited saturation kinetics (k(lim) = 6.0 min(-)(1), K(d) = 260 mM) and other features consistent with a mechanism in which a practically irreversible reduction step (E(ox). S --> E(red).P) with a rate constant, k(lim), is preceded by a rapidly attained equilibrium (K(d)) between free E and the E.S complex. Steady-state kinetic studies with sarcosine and N-methyl-L-alanine in the absence or presence of a dead-end inhibitor (pyrrole-2-carboxylate) indicate that catalysis proceeds via a "modified" ping pong mechanism in which oxygen reacts with E(red).P prior to the dissociation of the imino acid product. In this mechanism, double reciprocal plots will appear nearly parallel (as observed) if the reduction step is nearly irreversible. A polar mechanism, involving formation of a covalent 4a-flavin-substrate adduct is one of several plausible mechanisms for sarcosine oxidation. Thiols are known to form similar 4a-flavin adducts. MSOX does not form a 4a-adduct with thioglycolate but does form a charge-transfer complex that undergoes an unanticipated one-electron-transfer reaction to yield the anionic flavin radical.

Alanine↗
PubMed1998

Synthesis, DNA cleavage and cytotoxicity of some novel cyclic peptide-2,6-dimethoxyhydroquinone-3-mercaptoacetic acid conjugates containing D-amino acids.

This paper reports an ongoing study of the use of small-ring-size cyclic peptides as carriers of a potential antitumor agent: 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA). Three new cyclic tripeptide-DMQ-MA conjugates--cyclo[D-Val-Lys(DMQ-MA)-gamma-aminobutyric acid (GABA)-], cyclo[Val-Lys(DMQ-MA)-GABA-] and cyclo[D-Val-D-Lys(DMQ-MA)-GABA-]--were synthesized. The isomeric cyclic tripeptide-DMQ-MA conjugates were designed and synthesized to study the effect of stereoisomerism of the conjugates on cytotoxicity. The cyclic peptides were synthesized by coupling protected amino acids in solution and the final cyclization performed by the pentafluorophenyl ester method as described previously. After removing the lysyl-Z protecting group of the cyclic peptides the conjugation was achieved by reacting with the pentafluorophenyl ester of DMQ-MA. Electron spin resonance (ESR) studies of these three cyclic tripeptide-DMQ-MA conjugates showed that hydroxyl radicals were generated as a non-linear function of L-ascorbic acid (AH2) concentration. The IC50 of the cyclic tripeptide-DMQ-MA conjugates against a human pulmonary carcinoma cell line (PC-9 cells) under the synergistic activation of AH2 ranges from 0.4 to 1.6 microM, which is significantly lower than the parent compound DMQ-MA (6.1 microM). Agarose gel electrophoresis showed that DMQ-MA and these cyclic peptide-DMQ-MA conjugates are capable of cleaving supercoiled plasmid DNA to open circular and linear forms, even in the absence of AH2. The effects of enantiomeric and diastereomeric variations of these cyclic tripeptide-DMQ-MA conjugates on the cytotoxicity against PC-9 cells were discussed.

Amino Acids↗
PubMed1975

Regulation of breakdown and synthesis of L-glutamate decarboxylase in Clostridium perfringens.

L-Glutamate decarboxylase (GAD) activity of Clostridium perfringens (ATCC 8009) cells grown in various culture conditions was investigated. Remarkable variations of GAD level occur during the growth cycle in thioglycollate broth. These changes are affected by the pH of the culture medium. Addition of alkali to the culture media results in decrease of cell GAD activity, whereas increase of enzyme level occurs only in cells growing in unbuffered media. The results indicate that the mechanism regulating the GAD levels is sensitive to the changes of pH (or buffering substances) rather than to the steady pH values. Neither repression by glucose nor induction by L-glutamate was observed. Moreover, high concentrations of the free amino acid substrate in the culture media considerably decrease cell GAD activity, owing to the buffering effect of the amino acid. The molecular mechanism supporting the variations of GAD activity during the growth cycle of the cells were investigated and tentatively related to the structural and functional properties of the pure enzyme. It is shown that the drop of GAD activity during the lag phase is due to protein breakdown. Evidence is presented suggesting a control of protein degradation by its quaternary structure. Data are also reported supporting de novo synthesis of GAD during the late logarithmic phase of cell growth. Finally, the possible role of GAD as part of the pH regulation system of C. perfringens cells is discussed in relation both to physiologic conditions of the bacterial cell and to the molecular mechanisms regulating the GAD activity in vivo.

Carboxy-Lyases↗
PubMed1997

Metabolic fuel privation in hibernating and awake ground squirrels.

The nature of metabolic fuel utilization during hibernation and periodic arousal is not completely understood. 2-Deoxy-D-glucose (2DG) and mercaptoacetate (MA) were administered to hibernating ground squirrels. These drugs disrupt glucose and fatty acid oxidation, respectively. Telemetrically recorded body temperature (Tb) was analyzed to determine rate of rewarming from hibernation, duration of euthermia during periodic arousal, and proportion of animals arousing after treatments. 2DG given during hibernation significantly increased latency to regain euthermia, especially during the initial phase of rewarming (from first Tb > 10 degrees C to first Tb > 15 degrees C), without affecting the duration or other features of the ensuing euthermic period; MA did not affect rate of rewarming. MA treatment during hibernation affected thermoregulation after the animals aroused, including an increased duration of euthermia and maintenance of erratic patterns of Tb. The percentage of animals that aroused from hibernation was increased in a dose-dependent fashion by each drug. 2DG and MA treatments had little or no impact on nonhibernating ground squirrels in the cold. We suggest that glucose oxidation is important for rewarming from deep torpor; limited glucose availability cannot, however, support normal levels of euthermia when fatty acid oxidation is compromised. On the other hand, fatty acid oxidation may be less necessary for normal arousal from torpor, but critical for the maintenance of euthermia during the arousal phase.

Animals↗
PubMed1988

Effect of pyridoxal isonicotinoyl hydrazone and other hydrazones on iron release from macrophages, reticulocytes and hepatocytes.

A model consisting of 59Fe-labelled macrophages was developed for screening potential iron-chelating drugs. Mouse peritoneal macrophages, induced by previous intraperitoneal injections of 3% thioglycollate, were labelled in vitro by their exposure to immune complexes of 59Fe-transferrin-antitransferrin antibody. Optimal conditions for macrophage labelling and subsequent 59Fe release were established. Sixty-two aromatic hydrazones, the majority of which had iron binding structures similar to pyridoxal isonicotinoyl hydrazone, were synthesized by condensation of aromatic aldehydes (pyridoxal, salicylaldehyde, 2-hydroxy-1-naphthylaldehyde and 2-furaldehyde) with various acid hydrazides prepared by systematic substitutions on the benzene ring. These compounds were examined for their potential to stimulate 59Fe release from 59Fe-labelled macrophages and also from reticulocytes and hepatocytes loaded with non-heme 59Fe. The majority of hydrazones derived from pyridoxal, salicylaldehyde and 2-hydroxy-1-naphthylaldehyde seemed to be equally effective in both the macrophage and reticulocyte testing systems. However, the pyridoxal hydrazones were much more active in hepatocytes than the other groups of hydrazones. Several compounds proved to be very potent in mobilizing 59Fe. These included hydrazones derived from 2-hydroxy-1-naphthylaldehyde and benzoic acid hydrazide, p-hydroxybenzoic acid hydrazide, 2-thiophenecarboxylic acid hydrazide, and also pyridoxal benzoyl hydrazone, pyridoxal m-fluorobenzoyl hydrazone and pyridoxal 2-thiophenecarboxyl hydrazone.

Animals↗
PubMed2006

Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase ST6Gal I.

Elevation of serum sialic acid and the ST6Gal-1 sialyltransferase is part of the hepatic system inflammatory response, but the contribution of ST6Gal-1 has remained unclear. Hepatic ST6Gal-1 elevation is mediated by P1, 1 of 6 promoters regulating the ST6Gal1 gene. We report that the P1-ablated mouse, Siat1DeltaP1, and a globally ST6Gal-1-deficient mouse had significantly increased peritoneal leukocytosis after intraperitoneal challenge with thioglycollate. Exaggerated peritonitis was accompanied by only a modest increase in neutrophil viability, and transferred bone marrow-derived neutrophils from Siat1DeltaP1 mice migrated to the peritonea of recipients with normal efficiency after thioglycollate challenge. Siat1DeltaP1 mice exhibited 3-fold greater neutrophilia by thioglycollate, greater pools of epinephrine-releasable marginated neutrophils, greater sensitivity to G-CSF, elevated bone marrow CFU-G and proliferative-stage myeloid cells, and a more robust recovery from cyclophosphamide-induced myelosuppression. Bone marrow leukocytes from Siat1DeltaP1 are indistinguishable from those of wild-type mice in alpha2,6-sialylation, as revealed by the Sambucus nigra lectin, and in the expression of total ST6Gal-1 mRNA. Together, our study demonstrated a role for ST6Gal-1, possibly from extramedullary sources (eg, produced in liver) in regulating inflammation, circulating neutrophil homeostasis, and replenishing granulocyte numbers.

Acute Disease↗
PubMed1994

Endocytosis, partial degradation and release of heparan sulfate by elicited mouse peritoneal macrophages.

Interactions between glycosaminoglycans (GAGs) and low density lipoprotein (LDL) are thought to influence the progression of atherogenesis. In an effort to gauge whether macrophages mediate GAG-LDL interaction by GAG modification, we have investigated the endocytosis, degradation and retro-endocytosis of the GAG heparan sulfate (HS) by mouse peritoneal macrophages. Radiolabelled HS was produced by derivatization with sulfosuccinimidyl-3-(4-hydroxyphenyl) propionate and radio-iodination by the chloramine T method. The amount of 125I-HS internalized by cultures of thioglycollate-elicited macrophages rose over a 24 h time period in proportion to the amount of tracer added to the wells (2-2500 ng ml-1). Analysis of GAG molecular weight was performed using gel filtration chromatography and polyacrylamide gel electrophoresis. After a 24 h pulse period, the 125I-HS in the intracellular fraction of the cultured cells was of smaller molecular weight than for control material. During a 24 h cold chase, fragments of 125I-HS were released into the medium. These fragments had lower affinity for Polybrene-Sepharose but did not appear significantly N-desulfated as determined by low pH nitrous acid treatment. The NADPH oxidase inhibitor diphenylene iodonium, although minimizing basal and phorbol ester-triggered radical output, did not inhibit 125I-HS depolymerization. These data indicate that elicited macrophages can interact with and reduce the polymer length of HS without extensively desulfating the molecule. They are consistent with a mechanism by which the macrophage internalizes and partially degrades HS by endoglucuronidase activity rather than NADPH oxidase-generated free radicals, followed by release of the products into the extracellular milieu.

Animals↗
PubMed1977

Rapid identification of Bacteroides fragilis with bile and antibiotic disks.

A simple screening test is described for separating Bacteroides fragilis from other anaerobic gram-negative bacilli. The test utilizes filter paper disks impregnated with 25 mg of oxgall (Difco), tested in conjunction with antibiotic identification disks. The bile disks and antibiotic disks are placed on a supplemented brucella blood agar plate which has been inoculated by swabbing with a standardized cell suspension. After 24 h at 35 degrees C in a GasPak jar, resistance to kanamycin and bile is taken as a presumptive identification of B. fragilis. Susceptibility to one or both disks indicates the need for further identification and additional biochemical tests are required. Those strains that produce insufficient growth within 24 h are not likely to be B. fragilis. The reliability of the bile disk method was tested by comparing results with 100 clinical isolates versus results with bile in thioglycolate broth, peptone-yeast extract-glucose broth, and tryptic soy agar. All four bile test methods gave equilvalent results, but the broth media required much longer periods of incubation.

Animals↗
PubMed1975

Pharmacological effects of urotensins. II. Renal effects of urotensin I in the rat.

Urotensin I decreased urine flow, glomerular filtration rate and renal plasma flow in intact and hypophysectomized rats. The antidiuretic effect of urotensin I differed from the that caused by AVT in that (a) the urine conductivity was not usually increased by the former, and (b) the rates of excretion of Na and K were different. The use of hypophysectomized rats and of Na-thioglycollate-treated urotensin I preparations indicated that the antidiuretic effect of urotensin I was not due to the release of endogenous ADH, nor was it caused by presence of AVT in the urotensin I samples.

Aminohippuric Acids↗
PubMed1960

Enteroviral ribonucleic acid. II. Biological, physical, and chemical studies.

Chromatographic behavior of whole type 1 poliovirus and phenol-extracted viral RNA on diethylaminoethyl cellulose columns, as revealed by assay of plaque-forming capacity, indicated that infectious RNA had surface properties markedly different from those of the intact virus. Infectious RNA of type 1 poliovirus and Coxsackie B1 virus was relatively resistant to heat inactivation as compared to intact virus. Kinetics of inactivation at elevated temperatures were multi-hit in character. The structure of infectious enterovirus RNA was investigated by treatment with chemical inactivating agents. Urea and guanidine as hydrogen bond-disrupting agents, and mercaptoethanol and thioglycolate as disulfide bond-disrupting agents, and combinations of these did not destroy RNA infectivity whereas hydrogen bond-disrupting treatment inactivated intact virus rapidly. RNA infectivity was not reduced by chloroform extraction alone, or by octanol extraction alone, but was reduced by chloroform-octanol extraction which failed to depolymerize RNA to an extent detectable by ultracentrifugal analysis. Infectivity of type 1 poliovirus and Coxsackie B1 virus RNA was destroyed in accordance with first order kinetics by very dilute solutions of pancreatic ribonuclease, and by purified snake venom phosphodiesterase, but not at all by bacterial alkaline phosphatase. Inactivation by venom diesterase was not accelerated by prior treatment of RNA with bacterial alkaline phosphatase. These results indicated that infectivity of enteroviral RNA resided in a single stranded structure, that a single break of a phosphodiester bond anywhere along the structure was sufficient to destroy infectivity, and that infectivity did not require a terminal phosphate group. Hydroxylamine, but not other carbonyl reagents, rapidly destroyed infectivity of intact type 1 poliovirus viral RNA without depolymerization of RNA-detectable by behavior in the analytical ultracentrifuge. With S(35)-methionine-labeled poliovirus a very small fraction of radioactivity remained in RNA preparations following phenol extraction. No evidence could be obtained to indicate that infectious enteroviral RNA was composed of subunits. RNA extracted with phenol during the course of infection of HeLa cells with type 1 poliovirus resembled RNA obtained from purified whole virus with respect to heat inactivation, hydroxylamine inactivation, chromatographic separation, susceptibility to protein denaturing agents, and ability to infect productively both naturally susceptible HeLa cells and naturally insusceptible L strain mouse cells. Intracellular production of infectious RNA paralleled intracellular maturation of whole virus and preceded it by a very short interval.

Animals↗