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PubMed2002

Augmentation of hypoxia-induced nitric oxide generation in the rat carotid body adapted to chronic hypoxia: an involvement of constitutive and inducible nitric oxide synthases.

Acute hypoxia increases the endogenous release of nitric oxide (NO) in rat carotid body and the expression of nitric oxide synthases is modulated by chronic hypoxia. The aim of the study was to examine hypoxia-induced NO generation in rat carotid body adapted to chronic hypoxia with inspired oxygen at 10% for 4 weeks. The concentration of NO was measured electrochemically with a Pt/Nafion/Pd-IrOx/POAP modified electrode inserted into the isolated carotid body superfused with bicarbonate-buffer saline at 35 degrees C. Acute hypoxia increased the concentration of NO by 471.3+/-71.4 nM in the carotid body of chronically hypoxic (CH) rats. The amount of NO release induced by hypoxia was significantly augmented when compared with that of the normoxic control (87.6+/-15.9 nM). The hypoxia-induced NO generation was markedly attenuated by pretreatment with L- NG-nitroarginine methylester (L-NAME; 500 microM), a non-selective nitric oxide synthase (NOS) inhibitor and also by removal of extracellular calcium with the calcium chelator EGTA (5 mM). Additionally, NO generation during hypoxia was reduced by 30% in the CH carotid body treated with S-methylisothiourea (SMT; 50 microM), a specific blocker of inducible NOS (iNOS). Immunohistochemical study revealed that positive iNOS protein immunoreactivity was detected in clusters of glomus cells in the carotid bodies of CH rats, but not in the normoxic group. Thus, chronic hypoxia enhances hypoxia-induced NO generation mediated by calcium-dependent NOSs and iNOS in the carotid body. Extracellular recording of sinus nerve activity of CH carotid bodies showed that L-NAME treatment enhanced the afferent discharge in response to hypoxia, confirming that the generation of NO suppresses the activities of carotid chemoreceptors. Taken together, our results suggest that hypoxia-induced NO production increases in the rat carotid body adapted to chronic hypoxia and that constitutive and inducible NOSs are involved in the NO generation. The enhancement of NO generation may play a physiological role in blunting the hypoxic chemosensitivity during chronic hypoxia.

Animals↗
PubMed1998

Effect of various agonists on nitric oxide generation by human polymorphonuclear leukocytes.

Nitric oxide generation is involved in a range of diseases involving polymorphonuclear leukocytes. The aim of this study was to determine whether human polymorphonuclear leukocytes are able to generate nitric oxide and to investigate the time course of its generation after stimulation with 10(-7) M N-formyl-methionyl-leucyl-phenylalanine, 60 ng/ml phorbol myristate acetate, 10(-7) M concanavalin A, and 10(-7) M platelet activating factor. Stimulation of human polymorphonuclear leukocytes with N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate caused sustained nitric oxide generation, reaching maximal values of 1,105 +/- 361 nM (n = 32) and 628 +/- 119 nM (n = 30), respectively. Platelet activating factor did not affect nitric oxide production (maximal value 29 +/- 7 nM, n = 8), whereas concanavalin A caused only a slight increase (102 +/- 24 nM, n = 8) when compared with resting cells control (26 +/- 6 nM, n = 8). Human polymorphonuclear leukocytes were able to respond to both consecutive and alternate N-formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate stimulation with nitric oxide generation. Nitric oxide generation was inhibited by specific inhibitors (N omega-nitro-L-arginine and N omega-monomethyl-L-arginine) and restored with L-arginine. We provide, to our knowledge, the first direct evidence that human neutrophils generate nitric oxide.

Concanavalin A↗
PubMed1987

Evaluation of first-generation sulfonylureas and glipizide in non-insulin-dependent diabetes mellitus.

The current study examines the impact of glipizide, a second-generation sulfonylurea, on diabetes control in patients in whom adequate control was not achieved while receiving treatment with first-generation agents. The interim results of this multicenter study are presented in which patients in whom euglycemia was not achieved based on fasting and two-hour postprandial plasma glucose criteria with first-generation sulfonylureas were given glipizide therapy for 24 weeks. Twenty-three percent of the patients who had only fair results with first-generation sulfonylureas (fasting plasma glucose level of more than 140 mg/dl, postprandial plasma glucose level of more than 175 mg/dl) had good to excellent results with glipizide therapy alone (fasting plasma glucose level of less than 140 mg/dl or less than 115 mg/dl, respectively). Overall, 15.4 percent of patients with fair to poor control using first-generation agents had good to excellent control using glipizide. At least a 25-mg/dl decrease in fasting plasma glucose and postprandial plasma glucose levels was also seen in 47 and 46 percent of patients given treatment with glipizide who had poor control (fasting plasma glucose level of more than 200 mg/dl, postprandial plasma glucose level of more than 235 mg/dl) with first-generation agents. Glipizide was extremely well tolerated, with no significant side effects. Preliminary data indicate that glipizide may provide a significant advantage in terms of safety and efficacy over first-generation agents.

Adult↗
PubMed1990

Pathophysiologic basis for the use of third-generation cephalosporins.

After 10 years of use, the third-generation cephalosporins remain excellent antibiotics. They have superior activity against selected streptococcal species compared with other cephalosporins, and superior activity against Haemophilus, Neisseria, Branhamella, and other less common oral gram-negative aerobic species. Despite a very broad spectrum of activity, the third-generation cephalosporins, like all other cephalosporins, have only poor activity against enterococci, Listeria, Corynebacterium jekeium, and methicillin-resistant staphylococci. Over the past 10 years, the activity of the third-generation cephalosporins against Escherichia coli, Klebsiella, Proteus, Providencia, Serratia, Haemophilus, and Neisseria has remained excellent. Equally as important, though perhaps less well recognized, is the activity of some of these agents against mouth anaerobic species and the anaerobic Bacteroides and Clostridium spp. of the pelvic area. At present, there are two main threats to the continued use of the third-generation cephalosporins. These are the increasing number of infections due to Enterobacter spp., which constitutively produce large amounts of a beta-lactamase that hydrolyzes cephalosporins, and the recent appearance of Klebsiella spp. in many parts of the world that possess new plasmid-mediated beta-lactamases that destroy cefotaxime, ceftazidime, and related third-generation parenteral cephalosporins. Correlation of pharmacologic properties with in vitro activity provides information as to reasonable dosage regimens for the third-generation cephalosporins. For most serious infections cefotaxime, ceftizoxime, and ceftazidime should be given three times a day provided that the patient has relatively normal renal function. Ceftriaxone can be administered once daily in less severe infections. The use of lower doses or less frequent dosing with cefotaxime, ceftizoxime, or ceftazidime is recommended in aged patients whose renal function is impaired. The unique interaction of cefotaxime with its active metabolite, desacetylcefotaxime, allows cefotaxime to be administered less frequently than three times a day in selected anaerobic infections. Correlation of the antibacterial activity and pharmacology of cephalosporins will help us to tailor their use more appropriately, so that the third-generation cephalosporins will remain useful antimicrobial agents for a further decade.

Bacteria↗
PubMed1990

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron.

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of .OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for .OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of .OH scavengers was sensitive to catalase and competitive .OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H2O2; stimulation of H2O2 production may play a role in the increase in .OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or .OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize .OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.

Animals↗
PubMed1991

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.

Cyclic N-Oxides↗
PubMed1991

Metabolism of ethanol by rat hepatocytes results in generation of a lipid chemotactic factor: studies using a cell-free system and role of oxygen-derived free radicals.

We have reported that liver cells metabolizing ethanol release a lipid that has chemotactic activity for human polymorphonuclear leukocytes. The purpose of the present study was to investigate the role of ethanol and of oxygen-derived free radicals in generation of this activity. A cell-free, ethanol-metabolizing system consisting of rat liver cytosol was found to generate a polar lipid which possessed a chemotactic activity identical to the activity from intact cells. The importance of acetaldehyde in this process was established by the findings that (i) an inhibitor of the metabolism of ethanol to acetaldehyde, 4-methylpyrazole, blocked production of the chemotactic lipid; (ii) acetaldehyde could substitute for ethanol; and (iii) cytosol alone incubated with ethanol in the absence of mitochondrial acetaldehyde metabolism generated the activity. Acetaldehyde can serve as a substrate for cytosolic oxidases in reactions that generate radicals (e.g., superoxide anion) which have been shown to attack unsaturated lipids to yield lipid hydroperoxides. When scavengers of oxygen-derived free radicals (superoxide dismutase, catalase, and dimethylsulfoxide) were added to the ethanol-metabolizing system they prevented generation of the activity. The data are consistent with a mechanism of chemotactic factor generation by ethanol-metabolizing liver cytosol in which acetaldehyde causes formation of oxygen-derived free radicals that attack unsaturated cytosolic lipid to generate a polar chemotactic compound.

Animals↗
PubMed1992

Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides.

To clarify the nature of cytocidal molecular species among the radicals generated in the iron-catalyzed reactions of peroxides (ROOH), we examined the cytocidal effects of these radicals against gram-positive and gram-negative bacteria in the presence or absence of various radical scavengers. Three organic peroxides, t-butyl hydroperoxide (t-BuOOH), methyl ethyl ketone peroxide (MEKOOH), and cumene hydroperoxide, were used. Each radical generated from these peroxides was identified and quantitated by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The major cytotoxic radical species generated in the mixtures of various peroxides and heme iron, especially methemoglobin, metmyoglobin, or hemin, was the alkyl peroxyl radical (ROO.). Strong bactericidal action against gram-positive bacteria was observed in the peroxide-heme iron system, especially in the case of t-BuOOH and MEKOOH. Killing curves for gram-positive bacteria showed an initial lag period, which may indicate the multihit/multitarget kinetics of cell killing. When the diethylenetriamine pentaacetic acid (DTPA)-Fe2+ complex was used as a catalyst for decomposition of various peroxides, alkyl, alkoxyl, and alkyl peroxyl radicals were identified by spin-trapping analysis. However, study of the time course of alkyl peroxyl radical production in the DTPA-Fe2+ complex system revealed that radical species generated in this system were very short lived: a maximal level was achieved within 1 min and then declined sharply, and no bactericidal activity was observed after 10 min. In contrast, the alkyl peroxyl radical level generated by the organic peroxide-heme iron system remained high for 30 min or longer. The generation of alkyl peroxyl radicals quantified by ESR correlated quite well with the bactericidal effect of the system of peroxide plus iron. In addition, bactericidal activity was completely inhibited by the addition of the spin trap DMPO, as well as of other various radical scavengers (alpha-tocopherol and L-ascorbic acid), into the peroxide-heme iron system, but this effect was not observed with superoxide dismutase, beta-carotene, dimethyl sulfoxide, diphenylamine, or butylated hydroxyltoluene. In view of these results, it is assumed that alkyl peroxyl radicals are the potent molecular species that are cytotoxic against bacteria, whereas alkoxyl radicals (RO.) generated in this system do not affect bacterial viability.

Benzene Derivatives↗
PubMed1985

Peripheral influences on the central pattern-rhythm generator for tongue movements in the rat.

Rhythmic activity in the tongue and other oral muscles was evoked by mechanical stimulation of the hard palate in ketamine-anaesthetized rats. The relation between neural discharges of single hypoglossal motoneurones and activity in the masseter and anterior digastric muscle, and stimulus parameters was analysed. By varying the stimulus parameters, hypoglossal-motoneurone activity was modulated from phasic reflex activity into rhythmic activity. The tongue and other recorded muscles are likely to be controlled by the same pattern-rhythm generator, when rhythmic activity is induced by palatal stimulation. Exteroceptive oral stimuli can activate the tongue pattern-rhythm generator independently of proprioceptive feedback. Proprioceptive input from oral structures influenced the burst duration of rhythmic hypoglossal motoneurone activity. The cycle duration and the number of discharges/burst, however, was not affected by proprioceptive input. The hypoglossal-motoneurone pool may be influenced by more than one pattern-rhythm generator because the burst characteristics of rhythmically-firing hypoglossal motoneurones depend upon stimulus conditions. These hypoglossal pattern-rhythm generators have possible hierarchic relations because a shift from one burst pattern into another can be evoked by changing stimulus conditions. The pattern-rhythm generators for oral movements may be composed of a neural-pattern generator and a neural-rhythm generator, which can be modulated separately by peripheral inputs.

Action Potentials↗
PubMed1996

Integrin alpha v beta 3 and phospholipase C regulate prostacyclin formation of endothelial cells caused by ancrod-generated fibrin.

Ancrod-generated fibrin has been shown to stimulate prostacyclin synthesis of human umbilical vein endothelial cells (Chang et al., 1994, Biochem. Biophys. Res. Commun. 203, 1920). We further investigated its mechanism of action. The increment of 6-keto prostaglandin F1 alpha stimulated by ancrod-generated fibrin was almost completely inhibited when endothelial cells were either pretreated with 50 microM 8-(N,N'-diethylamino)octyl-3,4,5- trimethoxybenzoate (TMB-8) or preloaded with 15 microM 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). 6-Keto prostaglandin F1 alpha production during 2 and 10 h incubation period was also inhibited by 1.2 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid (EGTA) (41 +/- 12 and 53 +/- 17% inhibition, respectively). Further, ancrod-generated fibrin caused a rapid-onset increase in [3h]inositol monophosphate (IP1) formation in endothelial cells. This increase in IP1 was significantly inhibited by 1 mM Gly-Pro-Arg-Pro, 1 mM neomycin or 100 ng/ml pertussis toxin. At the same time, neomycin and pertussis toxin also significantly inhibited 6-keto prostaglandin F1 alpha synthesis of endothelial cells stimulated by ancrod-generated fibrin. Additionally, the increment of IP1 production as well as prostacyclin production were significantly inhibited by monoclonal antibodies directed against alpha v beta 3. These results suggest that intra- and extra-cellular Ca2+ participate in prostacyclin synthesis stimulated by ancrod-generated fibrin. Ancrod-generated fibrin stimulates pertussis toxin-sensitive G-protein regulated phosphoinositide breakdown, which is responsible for prostacyclin synthesis. This augmentation in prostacyclin synthesis and phosphoinositide breakdown caused by ancrod-generated fibrin area, at least in part, mediated by fibrin binding to integrin alpha v beta 3 on endothelial cells.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Fluoride activates diradylglycerol and superoxide generation in human neutrophils via PLD/PA phosphohydrolase-dependent and -independent pathways.

In contrast to the rapid, ethanol-inhibited superoxide generation by the receptor-linked agonist formyl-methionyl-leucyl-phenylalanine (fMLP), fluoride-activated superoxide generation occurs after a prolonged lag, and as shown herein is relatively ethanol-insensitive. We have investigated fluoride-activation of diradylglycerol generation and phospholipase D activity. Fluoride induces a very large increase in diradylglycerol mass (both 1,2-diacylglycerol (DAG) and 1-O-alkyl,2-acylglycerol (EAG)), with kinetics similar to superoxide generation. Unlike fMLP-activated diglyceride generation which is completely inhibited by ethanol, that produced by fluoride is only partially (30%) blocked. When the phosphatidylcholine pool is 3H-prelabeled, fluoride activates both [3H]phosphatidic acid (PA) and [3H]diglyceride generation with similar kinetics. Partial inhibition of the production of these species by ethanol was seen, coincident with the appearance of [3H]phosphatidylethanol, indicating phospholipase D-dependent transphosphatidylation had occurred. The data are consistent with the fluoride activation of PA and diglyceride generation by both phospholipase D-dependent and -independent (presumably phospholipase C) mechanisms.

Diglycerides↗
PubMedDate not supplied

Effects of hip center location on the moment-generating capacity of the muscles.

We have developed a three-dimensional biomechanical model of the human lower extremity to study how the location of the hip center affects the moment-generating capacity of four muscle groups: the hip abductors, adductors, flexors, and extensors. The model computes the maximum isometric force and the resulting joint moments that each of 25 muscle-tendon complexes develops at any body position. Abduction, adduction, flexion, and extension moments calculated with the model correspond closely with isometric joint moments measured during maximum voluntary contractions. We used the model to determine (1) the hip center locations that maximize and minimize the moment-generating capacity of each muscle group and (2) the effects of superior-inferior, anterior-posterior, and medial-lateral displacement of the hip center on the moment arms, maximum isometric muscle forces, and maximum isometric moments generated by each muscle group. We found that superior-inferior displacement of the hip center has the greatest effect on the force- and moment-generating capacity of the muscles. A 2 cm superior displacement decreases abduction force (44%), moment arm (12%), and moment (49%), while a 2 cm inferior displacement increases abduction force (20%), moment arm (7%) and moment (26%). Similarly, a 2 cm superior displacement decreases flexion force (27%), moment arm (6%), and moment (22%), while inferior displacement increases all three variables. Anterior-posterior displacement alters the moment-generating capacity of the flexors and extensors considerably, primarily due to moment arm changes. Medial-lateral displacement has a large effect on the moment-generating capacity of the adductors only. A 2 cm medial displacement decreases adduction moment arm (20%), force (26%) and moment (40%). These results demonstrate that the force- and moment-generating capacities of the muscles are sensitive to the location of the hip center.

Adult↗
PubMed1987

Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells.

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.

Calcimycin↗
PubMed1984

An aerosol generator for the resuspension of cotton dust.

An aerosol generator, the Pitt 3 model, was designed, fabricated, and characterized for the resuspension of inhalable particles from bulk cotton dust. The generator was constructed around a loudspeaker whose energy is transferred into an air column through latex rubber dams. This action tumbles the bulk dust, and small particles are loosened which can then be carried out of the column with the air passing through it. Thirty to forty grams of bulk cotton dust produced a stable aerosol concentration for at least 90 min. The maximum output of about 100 mg/m3 can be reduced to lower concentrations by adding dilution air. In one application, the generator produced a stable aerosol cloud in the range of 2 to 30 mg/m3 with a mass median aerodynamic diameter (MMAD) of about 3 microns and a geometric standard deviation (sigma g) of about 1.5. In another application the concentration in an animal exposure chamber was kept at 20.8 mg/m3 with an MMAD = 2.5 microns and a sigma g = 1.8 for over 6 months. The Pitt 3 generator proved to be trouble-free and produced large amounts of inhalable particles from bulk cotton dust. The generator was also used to generate dust clouds from silica powder, fly ash, and cellulose dust. The only requirement for successful resuspension of any dust with this generator is the presence of small particles in the bulk feed dust.

Aerosols↗
PubMed1992

Generation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene by murine splenic macrophages.

Studies have demonstrated that macrophages are the cell types which metabolize benzo[a]pyrene (B[a]P) within the murine spleen. B and T cells, polymorphonuclear cells, or the splenic capsule did not produce amounts of B[a]P metabolites above those of background. Exposure of mice to B[a]P, a known inducer of isozymes of cytochrome P450, resulted in an increase in the amounts of some B[a]P metabolites generated by macrophages. Evaluation of the in vitro plaque-forming-cell response to several T-cell and macrophage-dependent antigens following in vivo exposure to B[a]P indicated that the macrophage is the cell type responsible for B[a]P-induced immunosuppression. While suggestive, the reported data have not definitively established that an enriched splenic macrophage population can generate 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (BPDE) from B[a]P-7,8,-dihydroxy-7,8-dihydrodiol (B[a]P-7,8-diol). These data are critical to our hypothesis that the splenic cell type(s) which form BPDE will be the primary target cell responsible for B[a]P-induced immunosuppression. The first objective was to determine if splenic macrophages could generate BPDE. Enriched (80-90% purity) populations of macrophages were incubated with [3H]B[a]P for 24 hr. BPDE generated was quantitated by analysis of the cis- and trans-tetrol hydrolysis products of BPDE via HPLC procedures. Splenic macrophages generated BPDE from B[a]P. The cis syn was the predominate tetrol detected. Exposure of mice to B[a]P increased the amounts of the trans-anti-tetrols 2.2-fold, the trans-syn-tetrols 2.0-fold, the cis-anti-tetrols 1.8-fold, and the cis-syn-tetrols 2.6-fold above those formed by macrophages of vehicle-exposed mice. Both cytochrome P450- and peroxyl radical-dependent pathways are known to oxidize B[a]P-7,8-diol to BPDE. Since macrophages were found to generate BPDE, the second objective was to investigate which enzymatic pathway was responsible for its formation. The B[a]P-(+)-7,8-diol isomer has been shown to produce different specific BPDE isomers via the cytochrome P450 and peroxyl radical pathways. Macrophage populations were incubated with [3H]B[a]P-(+)-7,8-diol for 24 hr and the contribution of the cytochrome P450 and peroxyl radical pathways to BPDE formation determined by detection of syn-BPDE-hydrolysis and anti-BPDE-hydrolysis products, respectively. Based on the ratio of anti/syn BPDE-derived tetrol products, the results demonstrate that splenic macrophages can generate the BPDE by both a cytochrome P450-dependent and -independent (peroxyl radical) pathway. Macrophages are the cells which metabolize B[a]P within the murine spleen and may be the cell type responsible for B[a]P-induced suppression of humoral immune responses.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1984

Mechanisms for the generation of src-deletion mutants and recovered sarcoma viruses: identification of viral sequences involved in src deletions and in recombination with c-src sequences.

The precise src deletions in six transformation-defective (td) deletion mutants derived from the Schmidt-Ruppin strain of Rous sarcoma virus were determined by sequence analysis. Examination of the parental viral sequences neighboring the junctions of deletions in these td mutants revealed that these regions contained either directly repeated or inverted complementary sequences ranging from 9 to 28 nucleotides. Five td mutants were found to contain deletions flanked by directly repeated sequences, of which the 3' direct repeat was retained whereas the 5' direct repeat was deleted in the resulting td viral RNA. In the deletions of two td mutants where inverted complementary sequences were present at junctions of the deletions, both copies of the inverted complementary sequence were deleted in the td viral RNA. It is proposed from these observations that deletions of these mutants have been generated during the synthesis of minus-strand viral DNA by reverse transcriptase by jumping over a sequence flanked by direct repeats or by skipping a stem-and-loop structure formed via inverted complementary sequences on the viral RNA template. Data provide further information on the sequences in the td viral genome that are required for the generation of recovered sarcoma viruses (rASVs) by recombination with c-src. Sequence data of td viruses revealed that retaining as few as 82 nucleotides of the 3' src coding sequence is sufficient, whereas retaining as much as one-third of the 5' src but none of the 3' src coding sequences is not sufficient, for the generation of rASVs. Those that generate replication-competent rASVs retain, in addition to the 3' src region, a portion of the 5' src and/or its immediate upstream sequence that is homologous to exon 1 of the c-src DNA. These two sequence domains apparently provided 5' and 3' homologous regions for recombination between td viral genome and c-src DNA resulting in nondefective rASVs. Td109, which was shown previously to generate only replication-defective rASVs, retains 296 nucleotides of the 3' src sequence but lacks all the 5' src and 316 nucleotides of its immediate upstream region. It is concluded that the 5' src coding sequence and its immediate upstream region are not essential for the generation of rASVs. However, retaining a portion of those sequences is required for the generation of replication-competent rASVs.

Animals↗
PubMed1994

Reduction in human neutrophil superoxide anion generation by n-3 polyunsaturated fatty acids: role of cyclooxygenase products and endothelium-derived relaxing factor.

Dietary supplementation with n-3 polyunsaturated acids (PUFAs) results in augumented vasorelaxation and reduction in superoxide anion generation. Augmented vasorelaxation may be mediated by enhanced generation of vasodilator prostaglandins and/or endothelium-derived relaxing factor (EDRF), now thought to be nitric oxide (NO). To determine the importance of enhanced vasodilator prostaglandins or EDRF-NO in reduction in superoxide anion generation during n-3 PUFAs intake, human polymorphonuclear leukocytes (PMNs) were incubated with n-6 PUFA arachidonic acid (AA), or n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (each 10(-7) M) for 1 hr at 37 degrees C. Parallel sets of PMNs were treated with the cyclooxygenase inhibitors indomethacin (10(-5) M), or aspirin (10(-5) M), or the EDRF-NO synthase inhibitor L-NMMA (10(-3) M) prior to incubation with PUFAs. Superoxide anion generation by PMNs was determined by measuring the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C. PMNs incubated with EPA or DHA, but not AA, demonstrated marked reduction in superoxide anion generation. This reduction in superoxide anion generation by n-3 PUFAs was abolished by treatment of PMNs with indomethacin or aspirin, but not by L-NMMA. These observations suggest that n-3 PUFAs decrease superoxide anion generation primarily by a prostaglandin-dependent pathway.

Arachidonic Acid↗
PubMed1994

Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro--modulatory action of glycosaminoglycans.

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC50 255 ng/ml) was more pronounced than that of factor Xa (IC50 684 ng/ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 micrograms/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3-40 micrograms/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40-50% was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.

Animals↗