[Principles of radiorace formation in yeasts. Report 8. Study of the role of heterozygosity of diploid yeasts in the formation of radiorace].
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Diploidy↗
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Evaluation of new multimedia formats for cancer communications.
BACKGROUND: Providing quality, current cancer information to cancer patients and their families is a key function of the National Cancer Institute (NCI) Web site. This information is now provided in predominantly-text format, but could be provided in formats using multimedia, including animation and sound. Since users have many choices about where to get their information, it is important to provide the information in a format that is helpful and that they prefer. OBJECTIVE: To pilot and evaluate multimedia strategies for future cancer-information program formats for lay users, the National Cancer Institute created new multimedia versions of existing text programs. We sought to evaluate user performance and preference on these 3 new formats and on the 2 existing text formats. METHODS: The National Cancer Institute's "What You Need to Know About Lung Cancer" program was the test vehicle. There were 5 testing sessions, 1 dedicated to each format. Each session lasted about 1 hour, with 9 participants per session and 45 users overall. Users were exposed to the assigned cancer program from beginning to end in 1 of 5 formats: text paperback booklet, paperback booklet formatted in HTML on the Web, spoken audio alone, spoken audio synchronized with a text Web page, and Flash multimedia (animation, spoken audio, and text). Immediately thereafter, the features and design of the 4 alternative formats were demonstrated in detail. A multiple-choice pre-test and post-test quiz on the cancer content was used to assess user learning (performance) before and after experiencing the assigned program. The quiz was administered using an Authorware software interface writing to an Access database. Users were asked to rank from 1 to 5 their preference for the 5 program formats, and provide structured and open-ended comments about usability of the 5 formats. RESULTS: Significant improvement in scores from pre-test to post-test was seen for the total study population. Average scores for users in each of the 5 format groups improved significantly. Increments in improvement, however, were not statistically different between any of the format groups. Significant improvements in quiz scores were seen irrespective of age group or education level. Of the users, 71.1% ranked the Flash program first among the 5 formats, and 84.4% rated Flash as their first or second choice. Audio was the least-preferred format, ranking fifth among 46.7% of users and first among none. Flash was ranked first among users regardless of education level, age group, or format group to which the user was assigned. CONCLUSIONS: Under the pilot study conditions, users overwhelmingly preferred the Flash format to the other 4 formats. Learning occurred equally in all formats. Use of multimedia should be considered as communication strategies are developed for updating cancer content and attracting new users.
Adult↗
Incidence, time course, and characteristics of microbubble formation during radiofrequency ablation of pulmonary veins with an 8-mm ablation catheter.
BACKGROUND: Microbubble formation during pulmonary vein (PV) radiofrequency (RF) ablation of atrial fibrillation (AF) occurs relatively frequently. Prior studies have shown that microbubble formation may be associated with an increased risk of complications. However, the incidence, time course, and temperature characteristics of microbubble formation during AF ablation with an 8-mm catheter have not been prospectively described in humans. METHODS: We studied 46 (30 men, age 56+/-10 years) patients with AF who underwent RF ablation of PVs between January 2005 and December 2005 using an 8F, 8-mm Biosensetrade mark ablation catheter (Biosense-Webster, Diamond Bar, CA, USA). All patients underwent continuous intracardiac echocardiography (ICE). Microbubble patterns were classified as either type 1 (intermittent, scattered microbubble formation) or type 2 (explosive shower of dense microbubbles). Formation of any microbubbles was detected by ICE and the time, PV location, and electrode temperature were recorded. RESULT: A total of 1,479 (32+/-13, range 12-73) RF lesions were delivered to 167 veins. Twenty (2%) lesions were classified as type 2. Since the number of lesions resulting in type 2 bubbles was very small, only type 1 lesions were included in the final analysis. Thirty-nine (85%) patients had at least one lesion associated with bubble formation during ablation (mean: 7+/-7 lesions, range 1-28 lesions). Twenty-three percent (327) of the RF lesions resulted in bubble formation. RF generator power setting during lesions resulting in bubble formation was lower than lesions which did not result in bubble formation (47.9+/-7.4 W vs 49.7+/-7.1 W, P<0.001). Logistic regression analysis revealed a significant negative correlation (P<0.001) between RF generator power settings and a positive correlation between the generator temperature settings and formation of bubbles (both P<0.02). However, the maximum temperature attained was not different between lesions resulting in bubble formation (n=327) and those which did not result in bubble formation (n=1,139). Fifty-three (16%) of the lesions associated with bubble formation occurred within 2-10 seconds after RF was begun. Bubble formation was significantly more frequent in left superior PVs compared to the other PVs (left superior PV 27.3% left inferior PV 18.6%, right superior PV 20.5%, and right inferior PV 18.8%, P=0.005, left superior PV vs other PVs, P<0.001) even after adjustment for the other factors including generator power settings and the temperature setting. CONCLUSION: Bubble formation is common during RF ablation of PV with 8-mm tip catheter and can occur as early as 2 seconds after starting RF. RF generator power is negatively correlated with bubble formation while generator temperature settings are positively correlated with formation of bubbles. Microbubble formation is also more frequent with ablation of the left superior PV probably due to better catheter contact in that area.
Atrial Fibrillation↗
Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus.
Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2. Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis. The ability to metabolize formate at high rates was observed only in cells previously grown on formate. Two such strains were distinguished: strain F and strain HF. Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2. Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate. Initial adaptation of strain H to growth on formate was preceded by a long lag. The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings. Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium. High levels of formate dehydrogenase were also constitutive in strain F. Only low formate dehydrogenase activities were observed in strain H. High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2. In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase.
Aldehyde Oxidoreductases↗
Enhancing and suppressive effects of immunosuppressants cyclosporin A, FK506, and KM2210 on the colony formation of murine bone marrow cells.
Immunosuppressants cyclosporin A (CsA), FK506, and KM2210 modulated colony formations of murine hematopoietic progenitor cells. In a 4-h treatment with CsA, 10 micrograms/ml increased the formation of colony-forming units of mixed lineages (CFU-Mix) but decreased the formation of highly proliferative potential colony-forming units (CFU-HPP); 1 microgram/ml of CsA increased the formations of CFU-HPP, CFU-Mix, and colony-forming units of granulocytes/macrophages (CFU-GM); 0.1 microgram of CsA increased the formation of CFU-Mix and burst-forming units of erythroid lineage (BFU-E). Lower doses of CsA appeared to induce an increase in various colony formations. FK506 increased CFU-HPP and CFU-Mix formations at lower doses. Another immunosuppressant, KM2210, increased CFU-HPP and CFU-GM formations but decreased CFU-Mix and BFU-E formations. In a 24-h treatment, 10 micrograms/ml and 1 microgram/ml of CsA inhibited all the colony formations, but 0.1 microgram/ml of CsA increased CFU-Mix, CFU-GM, and BFU-E formations. Similarly, 100 ng/ml and 10 ng/ml of FK506 decreased all the colony formations but 1 ng/ml of FK506 increased CFU-HPP and CFU-GM formations. KM2210 inhibited all the colony formations. These findings showed that lower doses of CsA and FK506 appeared to increase the colony formations, although higher doses of these drugs decreased the colony formations, similar to the findings in a 4-h treatment. On the other hand, KM2210 showed opposing effects on colony formation with 4-h and 24-h treatments.
Animals↗
Pharmacokinetics of methanol and formate in female cynomolgus monkeys exposed to methanol vapors.
The 1990 Clean Air Act Amendments contain mandates for reduced automotive emissions and add new requirements for the use of alternative fuels such as methanol to reduce certain automotive pollutants. Methanol is acutely toxic in humans at relatively low doses, and the potential for exposure to methanol will be increased if it is used in automotive fuel. Formate is the metabolite responsible for neurotoxic effects of acute methanol exposure. Since formate metabolism is dependent on folate, potentially sensitive folate-deficient subpopulations, such as pregnant women, may accumulate formate and be at higher risk from low-level methanol exposure. Our objective was to determine the pharmacokinetics of 14C-methanol and 14C-formate in normal and folate-deficient monkeys after exposure to 14C-methanol vapors at environmentally relevant concentrations: below the threshold limit value (TLV), at the TLV of 200 parts per million (ppm), and above the TLV. Four normal adult female cynomolgus monkeys were individually anesthetized with isoflurane, and each was exposed by endotracheal intubation to 10, 45, 200, or 900 ppm 14C-methanol for 2 hours. Concentrations of the inhaled and exhaled 14C-methanol, blood concentrations of 14C-methanol and 14C-formate, exhaled 14C-carbon dioxide (14CO2), and respiratory parameters were measured during exposure. After exposure, 14C-methanol and 14CO2 exhaled, 14C-methanol and 14C-formate excreted in urine, and 14C-methanol and 14C-formate in blood were quantified. The amounts of exhaled 14C-methanol and 14CO2, blood concentrations of 14C-methanol and 14C-formate, and 14C-methanol and 14C-formate excreted in urine were linearly related to methanol exposure concentration. For all exposures, blood concentrations of 14C-methanol-derived formate were 10 to 1000 times lower than endogenous blood formate concentrations (100 to 200 mM) reported for monkeys and were several orders of magnitude lower than levels of formate known to be toxic. Since the metabolism of formate in primates depends on the availability of tetrahydrofolate, the same four monkeys were next placed on a folate-deficient diet until folate concentrations in red blood cells consistent with moderate folate deficiency (29 to 107 ng/mL) were achieved. Monkeys were then reexposed to the highest exposure concentration, 900 ppm 14C-methanol, for a similar 2-hour period, and again the pharmacokinetic data described above were obtained. Even with a reduced folate status, monkeys exposed to 900 ppm methanol for 2 hours had peak concentrations of methanol-derived formate that were well below the endogenous levels of formate. Although these results represent only a single exposure and therefore preclude broad generalizations, they do suggest the body contains sufficient folate stores to effectively detoxify small doses of methanol-derived formate from exogenous sources, such as those that might occur during normal use of automotive fuel.
Adult↗
Response formats and satisfaction surveys for elders.
PURPOSE: A factor common to the results of many satisfaction surveys of elders is a lack of response variability. Increasing response variability may be useful if satisfaction surveys of elders are to be productively used in the future. In this paper, we first examine elders' preferences between five response formats and then examine the response variability of these five commonly used formats. DESIGN AND METHODS: Satisfaction, demographic, and Short-Form 36 Health Survey data were self-reported by patients in four outpatient surgery centers during 1998 and 1999. We used six different survey instruments randomly given to 3,122 elders. Five instruments varied in response format (5-item Likert format, 5-item satisfaction format, 5-item valuation format, 4-item Chernoff faces, and 10-item visual analogue format). The sixth survey used all five different response sets, and then it asked for the respondents' preferences among the different response sets. RESULTS: A total of 2,450 questionnaires were examined (response rate of 78.5%). The response format using four Chernoff faces was liked the least, with only 5% of the respondents preferring this format. The 10-item visual analogue format (10VAF) was liked the most, with 39% of the respondents preferring this format. In addition, 10% more elders thought this format was easier to use than the second-place choice (i.e., 32% vs. 22%). The coefficient of variation for the 10VAF was also higher than those in identical domains using the other response formats. This would seem to indicate that the 10VAF is less prone to a ceiling effect than the other response formats. IMPLICATIONS: Our results show that elders have a preference for some response formats, and from the choices we gave them a 10VAF was preferred. The 10VAF also had more response variability then the other formats we tested.
Aged↗
Structure-activity study with bioreductive benzoquinone alkylating agents: effects on DT-diaphorase-mediated DNA crosslink and strand break formation in relation to mechanisms of cytotoxicity.
PURPOSE: Structure-activity studies were carried out with the model bioreductive alkylating agent benzoquinone mustard (BM) and its structural analogs. The specific objectives were: (1) to investigate the effects of functional group substitutions to the benzoquinone ring on DNA crosslink and strand break formation subsequent to reduction of the analogs by DT-diaphorase (DTD) in vitro, (2) to correlate DNA crosslink and strand break formation by the analogs with anaerobic reduction of the BM analogs by DTD and their redox cycling in vitro, and (3) to correlate DNA crosslink and strand break formation by the BM analogs with their cytotoxic effects in cancer cells. METHODS: DNA interstrand crosslink and single-strand break formation were assessed using agarose gel assays. To determine DNA interstrand crosslinks or single-strand breaks, linearized or supercoiled plasmid DNA, respectively, were incubated with purified human DTD and increasing concentrations of each BM analog. Subsequently, DNA was electrophoresed on an agarose gel and DNA crosslink and strand break formation were quantified using densitometry. The rates of reduction of the BM analogs by purified human DTD were measured in vitro under hypoxic conditions, and the redox cycling potential was determined under aerobic conditions using HPLC analysis. The cytotoxic activities of these agents in human tumor cell lines were measured by the MTT assay, with and without the DTD inhibitor, dicoumarol. RESULTS: BM analogs with electron-donating groups (MeBM, MBM, m-MeBM), electron-withdrawing groups (CBM, FBM), sterically bulky groups (PBM, m-PBM, m-TBM) and positional isomers (MeBM, m-MeBM, PBM, m-PBM) were synthesized. After reduction by DTD, the BM analogs produced a concentration-dependent increase in DNA crosslink and DNA strand break formation. The E(10) (extent of DNA crosslink formation produced by 10 micro M BM analog) for DNA crosslink formation displayed the rank order MeBM approximately MBM>m-MeBM approximately PBM approximately BM>CBM>FBM>m-PBM approximately m-TBM. For DNA strand break formation, the E(10) values (extent of DNA strand break formation produced by 10 micro M BM analog) displayed the rank order MeBM>MBM>m-MeBM>PBM>BM approximately CBM>FBM>m-PBM approximately m-TBM. Importantly, the cytotoxic activity of the BM analogs in SK-Mel-28 human melanoma cells correlated positively with the E(10) values for DTD-mediated DNA crosslink formation ( r(s)=0.87, P<0.05) and DNA strand break formation ( r(s)=0.95, P<0.05). Similar correlations were observed in NCI-H661 human lung carcinoma cells. Furthermore, the D(10) values (concentration of BM analog that decreased the surviving cell fraction to 0.1) for cytotoxic activity of the BM analogs correlated with the maximum levels of DNA crosslinks formed with each BM analog, with r(s) values of -0.85 ( P<0.05) for the NCI-H661 cell line, and -0.81 ( P<0.05) for the SK-MEL-28 cell line. The half-time of reduction (t(1/2)) of the BM analogs by DTD did not correlate with DNA crosslink formation, DNA strand break formation, or cytotoxic potency of the analogs. CONCLUSIONS: Functional groups on the benzoquinone ring affect the ability of BM to produce DNA crosslinks and strand breaks following reduction by DTD. Electron-donating groups increased DNA damage, whereas electron-withdrawing groups and sterically bulky groups at the C6 position had no effect or decreased the ability of the compounds to produce DNA damage compared to BM. Moreover, both DNA crosslink and strand break formation appear to have an important impact on the cytotoxicity of the BM analogs. These results may have significance for optimal use of BM-based antitumor agents and for rationalization of the development of novel therapeutic compounds that require bioactivation by DTD.
Antineoplastic Agents, Alkylating↗
Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei.
The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K(m) and V(max) values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 muM. The K(m) and V(max) values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 muM. The apparent K(m) for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K(m) for H(2) uptake by cultures of Methanobacterium formicicum was 6 muM dissolved H(2). Formate and H(2) were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H(2) uptake was severely depressed when formate was above saturation and the dissolved H(2) was below 6 muM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.
Journal Article↗
Functional analysis of structural genes for NAD(+)-dependent formate dehydrogenase in Saccharomyces cerevisiae.
Co-consumption of formate by aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK 113-7D led to an increased biomass yield relative to cultures grown on glucose as the sole carbon and energy substrate. In this respect, this strain differed from two previously investigated S. cerevisiae strains, in which formate oxidation did not lead to an increased biomass yield on glucose. Enzyme assays confirmed the presence of a formate-inducible, cytosolic and NAD(+)-dependent formate dehydrogenase. To investigate whether this enzyme activity was entirely encoded by the previously reported FDH1 gene, an fdh1Delta null mutant was constructed. This mutant strain still contained formate dehydrogenase activity and remained capable of co-consumption of formate. The formate dehydrogenase activity in the mutant was demonstrated to be encoded by a second structural gene for formate dehydrogenase (FDH2) in S. cerevisiae CEN.PK 113-7D. FDH2 was highly homologous to FDH1 and consisted of a fusion of two open reading frames (ORFs) (YPL275w and YPL276w) reported in the S. cerevisiae genome databases. Sequence analysis confirmed that, in the database genetic background, the presence of two single-nucleotide differences led to two truncated ORFs rather than the full-length FDH2 gene present in strain CEN.PK 113-7D. In the latter strain background an fdh1Deltafdh2Delta double mutant lacked formate dehydrogenase activity and was unable to co-consume formate. Absence of formate dehydrogenase activity did not affect growth on glucose as sole carbon source, but led to a reduced biomass yield on glucose-formate mixtures. These findings are consistent with a role of formate dehydrogenase in the detoxification of exogenous formate.
Base Sequence↗
Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes.
Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (Mr 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (Em = -224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.
Cytochrome b Group↗
Metabolism of formate in Methanobacterium formicicum.
Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H(2) concentration of the culture medium was below 1 muM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37 degrees C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56 degrees C. H(2) production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 +/- 0.06 g (dry weight) per mol of formate and 4.8 +/- 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H(2)-CO(2) as the sole energy source at 37 degrees C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H(2)-CO(2) cultures was 3.5 g (dry weight) per mol. Both formate and H(2)-CO(2) cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F(420) than did H(2)-CO(2)-grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F(342), and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.
Carbon Dioxide↗
Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.
The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.
Animals↗
Antipyrine as a probe for human oxidative drug metabolism: identification of the cytochrome P450 enzymes catalyzing 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norantipyrine formation.
BACKGROUND AND OBJECTIVE: Antipyrine has been widely used as a probe drug for human oxidative drug metabolism. To evaluate the role of antipyrine as a model drug, we have identified the cytochrome P450 enzymes involved in 4-hydroxyantipyrine, 3-hydroxymethylantipyrine, and norantipyrine formation. METHODS: We used the following methods for this study: (1) determination of enzyme kinetics for antipyrine metabolite formation in human liver microsomes, (2) inhibition studies with antibodies and inhibitors, and (3) formation of metabolites by stable expressed human P450 enzymes. RESULTS: Antipyrine biotransformation could be described by Michaelis-Menten kinetics: norantipyrine: maximum rate of metabolite formation (Vmax), 0.91 +/- 0.04 nmol . mg-1 . min-1; Michaelis-Menten constant (Km), 19.0 +/- 0.8 mmol/L; 4-hydroxyantipyrine: Vmax, 1.54 +/- 0.08 nmol . mg-1 . min-1;Km,39.6 +/- 2.5 mmol/L. Antibodies against CYP3A4 inhibited the formation of 4-hydroxyantipyrine by 25% to 65%. LKM-2 antibodies (anti-CYP2C) caused a 75% to 100% inhibition of norantipyrine and a 58% to 80% inhibition of 3-hydroxymethylantipyrine formation. Sulfaphenazole inhibited the formation of 3-hydroxymethylantipyrine and norantipyrine by about 50%. Furafylline and fluvoxamine inhibited norantipyrine, 4-hydroxyantipyrine, and 3-hydroxymethylantipyrine formation by about 30%, 30%, and 50%, respectively. Ketoconazole reduced formation of norantipyrine, 3-hydroxymethylantipyrine, and 4-hydroxyantipyrine by up to 80%. Formation in stable expressed enzymes indicated involvement of CYP1A2, CYP2B6, CYP2C, and CYP3A4 in metabolite formation. CONCLUSION: Antipyrine metabolites are formed by at least six hepatic cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, and CYP3A4). 4-Hydroxylation is mainly catalyzed by CYP3A4 and, to a lesser extent, by CYP1A2. The CYP2C subfamily contains the predominant enzymes for norantipyrine formation, and CYP1A2 is also involved. Formation of 3-hydroxymethylantipyrine is mediated by CYP1A2 and CYP2C9. Because several cytochrome P450 enzymes are involved in the formation of each metabolite, antipyrine is not well suited as a probe for distinct human cytochrome P450 enzymes.
Anti-Inflammatory Agents, Non-Steroidal↗
Patterns of protein production in Myxococcus xanthus during spore formation induced by glycerol, dimethyl sulfoxide, and phenethyl alcohol.
Spore formation of Myxococcus xanthus can occur not only on agar plates during fruiting body formation, but also in a liquid culture by simply adding glycerol, dimethyl sulfoxide, or phenethyl alcohol to the culture. This chemically-induced spore formation occurs synchronously and much faster than that occurring during fruiting body formation. Dramatic changes in patterns of protein synthesis were observed during chemically-induced spore formation, as had previously been observed during fruiting body formation (Inouye et al., Dev. Biol. 68:579-591, 1979). However, the production of protein S, one of the major development-specific proteins during fruiting body formation, was not detected at all, although protein U, another development-specific protein, was produced in a late stage of spore formation as in the case of fruiting body formation. This indicates that the control of the gene expression during chemically-induced spore formation is significantly different from that during fruiting body formation. It was also found that during spore formation, every cell seems to have a potential to form a spore regardless of its age, since smaller cells as well as larger cells separated by sucrose density gradient centrifugation could equally form spores upon the addition of glycerol. Patterns of protein synthesis were almost identical for all the three chemicals. However, the final yield of spores was significantly different depending upon the chemicals used. When phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals added. This suggests that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation.
Bacterial Proteins↗
Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.
Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.
Acetates↗
The effect of formate on cytochrome aa3 and on electron transport in the intact respiratory chain.
1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species. 2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome aa3 (a3 + a33+) and in the half-reduced species (a2 + a33+). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a32+ minus a33+, free of the difficulties with cyanide (which induces marked high leads to low spin spectral shifts in cytochrome a33+) and azide (which induces peak shifts of cytochrome a2+ towards the blue in both alpha- and Soret regions). 3. The rate of formate dissociation from cytochrome a2+ a33+ -HCOOH is faster than its rate of dissociation from a3+ a33+ -HCOOH, especially in the presence of cytochrome c. The Ki for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 degrees C. 4. Succinate-cytochrome c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2. 5. Formate inhibition of ascorbate plus N, N, N', N'-tetramethyl-p-phenylenediamine oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in 'on' or 'off' inhibition rates were observed when intact mitochondria were compared with submitochondrial particles. 6. NADH-cytochrome c reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome aa3 level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.
Animals↗
Effects of apical membrane Cl(-)-formate exchange on cell volume in rabbit proximal tubule.
We used real-time recordings of cell volume changes to test for the role of the Cl(-)-formate exchanger in mediating NaCl entry across the apical membrane of rabbit proximal tubule cells. In the absence of extracellular Cl-, 0.5 and 5 mM formate in the tubule lumen induced an increase in cell volume of 1 and 9%, respectively. Formate-induced cell swelling was reduced by alkalinizing the tubule lumen or by addition of luminal amiloride (2 mM), indicating that the increase in cell volume results from the intracellular accumulation of Na-formate via nonionic diffusion of formic acid in parallel with Na(+)-H+ exchange. The cell volume increase induced by 0.5 mM formate was potentiated (from 1 to 4%) by Cl-, as expected for a formate-mediated stimulation of NaCl uptake via parallel Cl(-)-formate exchange and Na(+)-H+ exchange across the apical membrane. By contrast, the cell volume increase induced by 5 mM formate was attenuated (from 9 to 4%) by Cl-. The attenuating effect of Cl- on formate-induced cell swelling required the operation of the apical membrane Cl(-)-formate exchanger. The effect of 1:1 Cl(-)-formate exchange to attenuate formate-induced cell swelling can be explained if the cell possesses a volume-activated anion exit pathway, most likely at the basolateral cell membrane, that is capable of mediating the efflux of Cl- but not formate from the cell.
Animals↗