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PubMed2006

Detection of microbubble formation during radiofrequency ablation using phonocardiography.

AIMS: To detect and characterize the acoustic energy generated by microbubble (MB) formation in an isolated tissue preparation. MB formation during radiofrequency (RF) ablation indicates excessive tissue heating and may precede explosive 'pops'. Currently, MB formation can only be detected with echocardiography. We hypothesized that MB formation can be detected with high-sensitivity phonocardiography. METHODS AND RESULTS: In a saline bath, RF lesions were created in sections of porcine left ventricle, using a 4 mm tip irrigated catheter. MB formation was visualized with an echocardiography probe. In 20 preparations, RF energy was begun at 25 W and increased by 5 W every 20 s until a pop occurred. A high-sensitivity computerized phonocardiography transducer with frequency bandwidth of 2 kHz and system noise -90 dB (SonoMedica, Inc., Vienna, VA, USA) was coupled to the external glass wall of the bath. In 15 of 20 (75%) preparations, a characteristic acoustic signature corresponding to MB formation was noted before the pop. These signals were within the 600-2000 Hz range and had an intensity range of 10-40 dB. The earliest MB and acoustic signals occurred 51.3+/-51.5 s before the pop. The acoustic signals continued intermittently up to 10.3+/-12.9 s before the pop. CONCLUSION: The acoustic energy created by MB formation can be detected in an isolated tissue preparation, using a computer-based phonocardiography system. Characteristic acoustic signatures are present before pops and correspond to MB formation. Acoustic monitoring for MB formation may allow for the titration of cooled RF ablation without echocardiography.

Animals↗
PubMed1990

Effect of phospholipase A2 inhibitors on mouse T lymphocytes. II. Phospholipase A2 inhibitors induce T cell hybridomas and a T cell clone for the formation of glycosylation-inhibiting factor.

The mouse T cell hybridoma 12H5 cells constitutively form glycosylation-enhancing factor (GEF) and produce both IgE-potentiating factor and ovalbumin (OVA)-binding GEF upon antigenic stimulation with OVA-pulsed macrophages. Culture of the 12H5 cells either with nonspecific glycosylation inhibiting factor (GIF) or with a phospholipase A2 (PLA2) inhibitor, ONO-RS-082, stopped the formation of GEF and induced the same cells to form GIF. Induction of the GIF formation by a PLA2 inhibitor was observed even when the 12H5 cells had been treated with mitomycin C, indicating that the switching from the GEF formation to the GIF formation was not due to selective proliferation of a GIF-producing subclone. The OVA-binding GIF produced by the PLA2-inhibitor-treated, antigen-stimulated 12H5 cells binds to homologous antigen (ovalbumin), and shares both antigenic determinant recognized by the monoclonal antibody 14-30 and the lipomodulin-determinant with antigen-specific suppressor inducer factor (TsiF). The present experiments also showed that a typical helper T cell clone, D10, G4.1 cells, constitutively formed GEF and that preculture of the T cell clone with IL-2 and the PLA2 inhibitor switched the cells from the formation of GEF to the formation of GIF. Upon stimulation with antigen-pulsed macrophages, the inhibitor-treated D10.G4.1 cells formed GIF having affinity for conalbumin. The results indicated that the same T cells have the capacity to form either GIF or GEF under different conditions, and suggested that the GIF-producing suppressor T cells may be a phenotype of a subset of helper T cells. Switching of the same cells from the GEF formation to the GIF formation by the PLA2 inhibitor and the ability of the inhibitor to enhance GIF formation suggested that PLA2-inhibitory activity or GIF activity of TsiF is involved in the suppressor T cell cascade.

Aminobenzoates↗
PubMed2003

Biofilm formation by Pseudomonas aeruginosa: role of the C4-HSL cell-to-cell signal and inhibition by azithromycin.

OBJECTIVES: In Pseudomonas aeruginosa, biofilm formation is controlled by a cell-to-cell signalling circuit relying on the secretion of 3-oxo-C12-HSL and C4-HSL. Previous studies suggested that C4-HSL plays no significant role in biofilm formation. However the wild-type PAO1 strain PAO-BI, used as a control in these studies is itself impaired in the production of C4-HSL. We wondered therefore whether the role of C4-HSL in biofilm formation might have been underestimated, and whether azithromycin inhibits biofilm formation by interfering with cell-to-cell signalling. METHODS: We used isogenic mutants of wild-type PAO1 strains PAO-BI and PT5 in a static biofilm model. Biofilm formation was quantified using Crystal Violet staining and exopolysaccharide measurements. RESULTS: Wild-type strain PAO-BI, as a result of its reduced C4-HSL secretion, produced 40% less biofilm compared with the wild-type PAO1 strain PT5. Using isogenic mutants of strain PT5 we have shown that whereas a lasI mutant (deficient in 3-oxo-C12-HSL) produced similar amounts of biofilm to the wild-type, a rhlI mutant (deficient in C4-HSL) produced 70% less biofilm. In the latter strain, biofilm formation could be restored by addition of exogenous C4-HSL. Azithromycin, known to reduce the production of both 3-oxo-C12-HSL and C4-HSL, inhibited biofilm formation of wild-type PT5 by 45%. This inhibition could be reversed by the addition of both cell-to-cell signals. CONCLUSIONS: Our results indicate that C4-HSL also plays a significant role in biofilm formation. Furthermore, we demonstrate the potential of using cell-to-cell signalling blocking agents such as azithromycin to interfere with biofilm formation.

4-Butyrolactone↗
PubMed1999

Promotion mechanism of triplex DNA formation by comb-type polycations: thermodynamic analyses of sequence specificity and ionic strength dependence.

We have previously reported that in the presence of poly (L-lysine)-graft-Dextran (PLL-g-Dex) copolymer, the binding constant of the pyrimidine-motif triplex formation at neutral pH is about 100-times higher than that observed without any triplex stabilizer. Here, to explore the mechanism of the promotion effect of the PLL-g-Dex copolymer at neutral pH, the sequence specificity and the ionic strength dependence of the pyrimidine-motif triplex formation was examined in the absence or presence of the copolymer. The sequence specificity of the pyrimidine-motif triplex formation at neutral pH in the presence of copolymer was almost similar to that at acidic pH without the copolymer. As the concentration of magnesium cation increased, the binding constant of the pyrimidine-motif triplex formation without the copolymer increased. On the other hand, the binding constant of the pyrimidine-motif triplex formation in the presence of the copolymer decreased upon the increase in the concentration of magnesium cation. Considering these results in light of counterion condensation (CC) theory, we conclude that the copolymer does not hinder the sequence specificity of the triplex formation, and isolates the triplex formation from the CC effect, which may lead to a net increase in entropy change upon the triplex formation, providing a favorable component to binding constant of the triplex formation.

Base Sequence↗
PubMed2000

CyA and OxLDL cause endothelial dysfunction in isolated arteries through endothelin-mediated stimulation of O(2)(-) formation.

BACKGROUND: Cyclosporin A (CyA) and oxidized low-density lipoprotein (OxLDL) cause endothelial dysfunction, partly through stimulation of O(2)(-) formation (which can inactivate nitric oxide). We investigated whether CyA and OxLDL potentiate their influence on oxidative stress, whether endothelin (ET) is a mediator of CyA- and OxLDL-induced O(2)(-) formation, and whether enhanced oxidative stress results in further attenuation of endothelium-dependent vasodilation. METHODS AND RESULTS: Human LDL was oxidized by Cu(++). O(2)(-) formation of isolated rat aortic rings was measured using a chemiluminescence assay. Incubation (60 min) of aortic rings with CyA (10 ng-10 microg/ml) or with OxLDL (300 microg/ml) caused a significant, dosedependent increase of the basal O(2)(-) formation. Pretreatment of the aortic rings with CyA (10 ng/ml) further enhanced the OxLDL-induced O(2)(-) formation by factor 1.9. The enhancement of the OxLDL-induced stimulation of O(2)(-) formation by CyA could be completely blocked by BQ123, a selective endothelin-1 (ET-1) receptor antagonist. Likewise, exogenously applied ET-1 (1 nM) potentiated the OxLDL-induced O(2)(-) formation by factor 1.8. Endothelium-dependent dilation was measured in isolated rings of rabbit aorta superfused with physiological salt solution in an organ bath. Incubation of the aortic rings with CyA (10 microg/ml, 60 min) or with OxLDL (300 microg/ml, 60 min) alone did not attenuate endothelium-dependent dilations. However, coincubation of the aortic rings with CyA+OxLDL in the presence of diethyl-dithio-carbamate, an inhibitor of the endogenous superoxide dismutase, caused a 60% inhibition of acetylcholine-induced dilator responses. CONCLUSIONS: Coincubation of isolated aortic rings with CyA and OxLDL causes a potent enhancement of vascular O(2)(-) formation. ET-1 seems to be mediator of the CyA-induced O(2)(-) formation. Enhanced oxidative stress results in further attenuation of endothelium dependent vasodilation.

Animals↗
PubMed1998

Mechanistic aspects of carbon monoxide formation from volatile anesthetics.

BACKGROUND: Desflurane, enflurane and isoflurane can be degraded to carbon monoxide (CO) by carbon dioxide absorbents, whereas sevoflurane and halothane form negligible amounts of CO. Carbon monoxide formation is greater with drier absorbent, and with barium hydroxide, than with soda lime. The mechanism, role of absorbent composition and water, and anesthetic structures determining CO formation are unknown. This investigation examined sequential steps in anesthetic degradation to CO. METHODS: Carbon monoxide formation from anesthetics and desiccated barium hydroxide lime or soda lime was determined at equimole and equiMAC concentrations. Carbon monoxide formation from deuterium-substituted anesthetics was also quantified. Proton abstraction from anesthetics by strong base was determined by deuterium isotope exchange. A reactive chemical intermediate was trapped and identified by gas chromatography-mass spectrometry. The source of the oxygen in CO was identified by 18O incorporation. RESULTS: Desflurane,enflurane,andisoflurane(difluoromethylethyl ethers), but not sevoflurane (monofluoromethyl ether), methoxyflurane (methy-ethyl ether), or halothane (alkane) were degraded to CO. The amount of CO formed was desflurane > or = enflurane > isoflurane at equiMAC and enflurane > desflurane > isoflurane at equimole concentrations. Proton abstraction from the difluoromethoxy carbon was greater with potassium than with sodium hydroxide, but unmeasurable with barium hydroxide. Carbon monoxide formation was correlated (r = 0.95-1.00) with difluoromethoxy (enflurane > desflurane > isoflurane > or = methoxyflurane = sevoflurane = 0) but not ethyl carbon proton abstraction. Deuterium substitution on enflurane and desflurane diminished CO formation. Chemical trapping showed formation of a difluorocarbene intermediate from enflurane and desflurane. Incorporation of H2(18)O in barium hydroxide lime resulted in C18O formation from unlabeled enflurane and desflurane. CONCLUSIONS: A difluoromethoxy group is a structural requirement for haloether degradation to CO. Results are consistent with initial base-catalyzed difluoromethoxy proton abstraction (potassium > sodium hydroxide, thus greater CO formation with barium hydroxide lime vs. soda lime) forming a carbanion (reprotonated by water to regenerate the anesthetic, hence requirements for relatively dry absorbent), carbanion decomposition to a difluorocarbene, and subsequent difluorocarbene reaction to form CO.

Absorption↗
PubMed1994

Reduction in perineural scar formation after laminectomy with Polyactive membrane sheets.

STUDY DESIGN: The effectiveness of Polyactive (Osteotech, Inc., Shrewsbury, NJ), an elastomeric segmental copolymer, as a barrier material for the prevention of perineural scar formation was evaluated in eight adult mongrel dogs. Two animals each were killed at 2, 4, 8, and 12 weeks postoperation. OBJECTIVE: This study determined the ability of a Polyactive membrane to prevent perineural scar and compared the results to those obtained using free fat graft and nonimplanted control subjects. SUMMARY OF BACKGROUND DATA: Perineural scar formation after laminectomy presents considerable morbidity in lumbar surgery. To date, a wide variety of materials has been evaluated to prevent scar formation including free fat grafts, mechanical barrier devices, hemostatic agents, and anti-inflammatory drugs. However, all studies indicate scar formation is present, and the search for an effective barrier continues. METHODS: Gross dissection, radiographic studies, including computed tomography and magnetic resonance imaging, and histologic sections were used to evaluate the presence and degree of perineural scarring. If scarring was evident in each of the evaluations, the degree was graded on a 0-3 scale where: 0 = no scarring; 1 = mild scarring; 2 = moderate scarring; and 3 = extensive scar formation. RESULTS: Polyactive membranes were an effective barrier with only minimal scar formation to the dura observed at any time period. Free fat graft was an effective barrier to scar formation at early time periods; however, progressive fat graft degradation with some increased scarring was observed at latter time periods. Nonimplanted control defect sites resulted in moderate to severe scar formation to the dura as early as 2 weeks postimplantation. CONCLUSIONS: The results indicate Polyactive is a reliable and effective means of reducing perineural scar formation. The effectiveness of the Polyactive membrane could be improved markedly if techniques or methods to ensure retention of the membrane in the proper position were developed.

Adipose Tissue↗
PubMed2001

The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein.

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.

Animals↗
PubMed2006

Differential in vitro inhibition of M3G and M6G formation from morphine by (R)- and (S)-methadone and structurally related opioids.

AIMS: To determine the in vitro kinetics of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) formation and the inhibition potential by methadone enantiomers and structurally related opioids. METHODS: M3G and M6G formation kinetics from morphine were determined using microsomes from five human livers. Inhibition of glucuronide formation was investigated with eight inhibitors (100 microm) and the mechanism of inhibition determined for (R)- and (S)-methadone (70-500 microm) using three microsomal samples. RESULTS: Glucuronide formation displayed single enzyme kinetics. The M3G Vmax (mean+/-SD) was 4.8-fold greater than M6G Vmax (555+/-110 vs. 115+/-19 nmol mg-1 protein h-1; P=0.006, mean of difference 439; 95% confidence interval 313, 565 nmol mg-1 protein h-1). Km values for M3G and M6G formation were not significantly different (1.12+/-0.37 vs. 1.11+/-0.31 mm; P=0.89, 0.02; -0.29, 0.32 mm). M3G and M6G formation was inhibited (P<0.01) with a significant increase in the M3G/M6G ratio (P<0.01) for all compounds tested. Detailed analysis with (R)- and (S)-methadone revealed noncompetitive inhibition with (R)-methadone Ki of 320+/-42 microm and 192+/-12 microm for M3G and M6G, respectively, and (S)-methadone Ki of 226+/-30 microm and 152+/-20 microm for M3G and M6G, respectively. Ki values for M3G inhibition were significantly greater than for M6G for (R)-methadone (P=0.017, 128; 55, 202 microm) and (S)-methadone (P=0.026, 75; 22, 128 microm). CONCLUSIONS: Both methadone enantiomers noncompetitively inhibited the formation of morphine's primary metabolites, with greater inhibition of M6G formation compared with M3G. These findings indicate a mechanism for reduced morphine clearance in methadone-maintained patients and reduced relative formation of the opioid active M6G compared with M3G.

Alamethicin↗
PubMed1998

ATP is a potent stimulator of the activation and formation of rodent osteoclasts.

1. There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non-selective cation channels and Ca2+-dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation. 2. We found that ATP stimulated resorption pit formation up to 5.6-fold when osteoclast-containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2-2 microM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9-7.0). Pit formation by acid-activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto-ATPase and by suramin, an antagonist of P2 receptors. 3. Over the same concentration range at which rat osteoclast activation occurred (0.2-2 microM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3-fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20-200 microM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation. 4. These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.

Adenosine Triphosphate↗
PubMed1996

The effect of a tachykinin NK1 receptor antagonist, SR140333, on oedema formation induced in rat skin by venom from the Phoneutria nigriventer spider.

1. The possibility that tachykinin NK1 receptors are involved in the plasma extravasation evoked by intradermal (i.d.) injection of Phoneutria nigriventer venom (PNV) in rat dorsal skin in vivo has been investigated. 2. Local oedema formation induced by the i.d. injection of test agents was measured by the extravascular accumulation of intravenously (i.v.) injected 125I-labelled human serum albumin over a 30 min period. 3. The tachykinin NK1 agonist, GR73632 (30 pmol per site), induced local oedema formation which was potentiated by co-injection with the neuropeptide vasodilator, calcitonin gene-related peptide (CGRP, 10 pmol per site). The non-peptide tachykinin NK1 receptor antagonist, SR140333 (0.03-1 nmol per site co-injected, i.d.) significantly inhibited (0.3 nmol per site, P < 0.05; 1 nmol per site, P < 0.001) local oedema formation induced by GR73632 with CGRP but not that induced by histamine (10 nmol per site) with CGRP. 4. PNV (0.03-0.3 microgram per site) injected i.d. induced dose-dependent local oedema formation. SR140333 (1 nmol per site, co-injected i.d.) inhibited oedema formation; with complete inhibition observed at doses of 0.03 microgram (P < 0.05) and 0.1 microgram (P < 0.001); and partial inhibition (50%) observed with the highest dose of PNV, 0.3 microgram (P < 0.05). 5. Local oedema formation induced by PNV was not affected by systemic pretreatment with the bradykinin B2 receptor antagonist, Hoe 140 (80 nmol kg-1, i.v.), which was used at a dose which significantly inhibited oedema formation by bradykinin (1 nmol per site). 6. Local oedema formation induced by PNV was significantly inhibited (P < 0.01) by co-injection of the histamine H1 receptor antagonist, mepyramine (2.5 nmol per site), together with the 5-hydroxytryptamine (5-HT) antagonist, methysergide (2.8 nmol per site). 7. In the presence of all three antagonists (mepyramine 2.5 nmol per site; methysergide, 2.8 nmol per site and SR140333 1 nmol per site), the plasma extravasation induced by PNV was further significantly inhibited (P < 0.001, when compared with PNV injected i.d. alone; P < 0.05 when compared with PNV co-injected with mepyramine and methysergide and P < 0.01, when compared with PNV co-injected with SR140333). 8. These results suggest that oedema formation evoked by i.d. PNV in rat skin may be partially mediated via a mechanism involving tachykinin NK1 receptors and that this effect is independent of histamine and 5-HT.

Animals↗
PubMed2005

Hippocampal formation involvement in a language-activation task in patients with mesial temporal lobe epilepsy.

PURPOSE: The study aims to explore the contribution of the hippocampal formation to the retained language-comprehension network in patients with unilateral mesial temporal lobe epilepsy (TLE). METHODS: We performed a functional magnetic resonance (MRI) study based on a language comprehension paradigm in 45 right-handed patients with unilateral mesial TLE and 35 healthy control subjects. Activations in the hippocampal formations in both hemispheres were analyzed for each subject as well as for groups of left TLE, right TLE, and controls. RESULTS: In sum, 82% of TLE patients displayed hippocampal activations. A significant difference in hippocampal activation between left and right TLE was found: Right TLE patients showed increased activity in the left hippocampal formation compared with left TLE patients. In contrast, patients with left TLE did not show increased activity in the right hippocampal formation compared with right TLE patients. In comparison with a healthy control group, right TLE patients activated the left hippocampal formation to a greater extent, whereas patients with left TLE did not activate the right hippocampal formation to a greater degree. These findings point to an increased involvement of the left hippocampal formation during a language-comprehension task in right TLE patients. In contrast, left TLE in right-handed patients seems not associated with an enhanced involvement of the right hippocampal formation in retained language comprehension. CONCLUSIONS: These findings suggest that effective language comprehension in right-handed subjects with TLE depends on the involvement of the left hippocampal formation and underline the risks of postoperative language decline in patients with left TLE.

Adolescent↗
PubMedDate not supplied

Pyruvate formate lyase (PFL) and PFL activating enzyme in the chytrid fungus Neocallimastix frontalis: a free-radical enzyme system conserved across divergent eukaryotic lineages.

Fermentative formate production involves the activity of pyruvate formate lyase, an oxygen-sensitive enzyme that employs a glycyl radical in its reaction mechanism. While common among anaerobic prokaryotes, this enzyme has so far been found in only two distantly related eukaryotic lineages, anaerobic chytridiomycetes and chlorophytes. Sequence comparisons of homologues from the chytridiomycetes Piromyces and Neocallimastix, the chlorophyte Chlamydomonas, and numerous prokaryotes suggest a single, eubacterial origin of eukaryotic pyruvate formate lyases. Pyruvate formate lyase activating enzyme introduces the glycyl radical into the pyruvate formate lyase protein chain. We discovered this enzyme, which had not previously been reported from eukaryotes, in the same two eukaryotic lineages and show that it shares a similar evolutionary history to pyruvate formate lyase. Sequences with high homology to pyruvate formate lyase activating enzyme were identified in the genomes of the anaerobic protozoan parasites Trichomonas vaginalis, Entamoeba histolytica, and Giardia intestinalis. While the occurrence of pyruvate formate lyase activating enzyme together with pyruvate formate lyase in fungi and chlorophytes was to be expected, the target protein of a glycyl radical enzyme-activating enzyme in these protozoa remains to be identified.

Acetyltransferases↗
PubMedDate not supplied

Megamitochondria formation - physiology and pathology.

Mitochondria undergo structural changes simultaneously with their functional changes in both physiological and pathological conditions. These structural changes of mitochondria are classified into two categories: simple swelling and the formation of megamitochondria (MG). Data have been accumulated to indicate that free radicals play a crucial role in the mechanism of the MG formation induced by various experimental conditions which are apparently various. These include ethanol-, chloramphenicol- and hydrazine-induced MG formation. Involvement of free radicals in the mechanism of MG formation is showed by the fact that MG formation is successfully suppressed by free radical scavengers such as alpha-tocopherol, coenzyme Q(10), and 4-OH-TEMPO. Detailed mechanisms and pathophysiological meanings of MG formation still remain to be investigated. However, a body of evidence strongly suggests that enormous changes in physicochemical and biochemical properties of the mitochondrial membranes during MG formation take place and these changes are favorable for membrane fusion. A recent report showed that continous exposure of cells with MG to free radicals induces apoptosis, finding which suggests that MG formation is an adaptative process to unfavorable environments at the level of intracellular organelles. Mitochondria try to decrease intracellular reactive oxygen species (ROS) levels by decreasing the consume of oxygen via MG formation. If mitochondria succeed to suppress intracellular ROS levels, MG return to normal both structurally and functionally, and they restore the ability to actively synthesize ATP. If cells are additionally exposed to excess amounts of free radicals, MG become swollen, membrane potential of mitochondria (DeltaPsim) decreases, cytochrome c is released from mitochondria, leading to activation of caspases and apoptosis is induced.

Animals↗
PubMed2002

Enzyme kinetics for the formation of 3-hydroxyquinine and three new metabolites of quinine in vitro; 3-hydroxylation by CYP3A4 is indeed the major metabolic pathway.

The formation kinetics of 3-hydroxyquinine, 2'-quininone, (10S)-11-dihydroxydihydroquinine, and (10R)-11-dihydroxydihydroquinine were investigated in human liver microsomes and in human recombinant-expressed CYP3A4. The inhibition profile was studied by the use of different concentrations of ketoconazole, troleandomycin, and fluvoxamine. In addition, formation rates of the metabolites were correlated to different enzyme probe activities of CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in microsomes from 20 human livers. Formation of 3-hydroxyquinine had the highest intrinsic clearance in human liver microsomes (mean +/- S.D.) of 11.0 +/- 4.6 micro l/min/mg. A markedly lower intrinsic clearance, 1.4 +/- 0.7, 0.5 +/- 0.1, and 1.1 +/- 0.2 micro l/min/mg was measured for 2'-quininone, (10R)-11-dihydroxydihydroquinine and (10S)-11-dihydroxydihydroquinine, respectively. Incubation with human recombinant CYP3A4 resulted in a 20-fold higher intrinsic clearance for 3-hydroxyquinine compared with 2'-quininone formation whereas no other metabolites were detected. The formation rate of 3-hydroxyquinine was completely inhibited by ketoconazole (1 micro M) and troleandomycin (80 micro M). Strong inhibition was observed on the formation of 2'-quininone whereas the formation of (10S)-11-dihydroxydihydroquinine was partly inhibited by these two inhibitors. No inhibition on the formation of (10R)-11-dihydroxydihydroquinine was observed. There was a significant correlation between the formation rates of quinine metabolites and activities of the CYP3A4 selected marker probes. This in vitro study demonstrates that 3-hydroxyquinine is the principal metabolite of quinine and CYP3A4 is the major enzyme involved in this metabolic pathway.

Cytochrome P-450 CYP3A↗
PubMed2000

Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites.

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.

Antineoplastic Agents↗
PubMed1996

Evidence for Isotopic Exchange during Metabolism of Stable-Isotope-Labeled Formate in a Methanogenic Sediment.

The disappearance of stable-isotope-labeled formate in freshwater sediment from Lake Vechten was studied by gas chromatography-mass spectrometry. Both deuterium- and (sup13)C-labeled formate were removed from pore water at a high rate (400 (plusmn) 35 [standard deviation] and 1,700 (plusmn) 600 (mu)M h(sup-1), respectively), with concomitant formation of nonlabeled formate. The removal rate of labeled and nonlabeled formate amounted to 240 (plusmn) 15 and 400 (plusmn) 40 (mu)M h(sup-1), respectively. The formation of nonlabeled formate is explained by isotopic exchange due to the activity of formate dehydrogenase rather than by the turnover of formate.

Journal Article↗
PubMed1997

Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts.

Methyl formate synthase, which catalyzes methyl formate formation during the growth of methylotrophic yeasts, was purified to homogeneity from methanol-grown Candida boidinii and Pichia methanolica cells. Both purified enzymes were tetrameric, with identical subunits with molecular masses of 42 to 45 kDa, containing two atoms of zinc per subunit. The enzymes catalyze NAD(+)-linked dehydrogenation of the hydroxyl group of the hemiacetal adduct [CH2(OH)OCH3] of methanol and formaldehyde, leading to the formation of a stoichiometric amount of methyl formate. Although neither methanol nor formaldehyde alone acted as a substrate for the enzymes, they showed simple NAD(+)-linked alcohol dehydrogenase activity toward aliphatic long-chain alcohols such as octanol, showing that they belong to the class III alcohol dehydrogenase family. The methyl formate synthase activity of C. boidinii was found in the mitochondrial fraction in subcellular fractionation experiments, suggesting that methyl formate synthase is a homolog of Saccharomyces cerevisiae Adh3p. These results indicate that formaldehyde could be oxidized in a glutathione-independent manner by methyl formate synthase in methylotrophic yeasts. The significance of methyl formate synthase in both formaldehyde resistance and energy metabolism is also discussed.

Alcohol Oxidoreductases↗