Halogenated 2,5-pyrrolidinediones: synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E(LUMO)) and of the radical anion stability (DeltaH(f)(rad)-DeltaH(f)) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (lnM(m) vs. E(LUMO); lnM(m) vs. DeltaH(f)(rad)--DeltaH(f)) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E(LUMO) or DeltaH(f)(rad)-DeltaH(f) correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.     

Decarboxylation kinetics for the carbonatotris(pyridine)cobalt(III) ion in perchloric acid solutions

The kinetics of the decomposition reactions of the CO(py)₃(CO₃)(H₂O)⁺ ion have been investigated in aqueous perchloric acid solutions over a range of hydrogen ion concentrations (0.10 to 5.0 M) and at two ionic strengths (I = 1.0 and 5.0 M). At the lower ionic strength, plots of ln (A_t–A_∞ versus time show a nonlinearity that is consistent with that expected for consecutive first-order reactions. The rates of the faster reaction are similar to those reported for the spontaneous reduction of aquopyridine-cobalt(III) cations. At the higher ionic strength, the above noted curvature is not apparent and the decarboxylation kinetics of the title complex may be described by a pseudo-first-order rate constant: k_obs = k[H₃O⁺]. At 20°C, k = (1.75−+0.09) s⁻¹ M⁻¹ with activation parameters ofΔH^‡ = (97 −+ 4) kJ mol⁻¹ and ΔS^‡ = −(54 −+ 32) J deg⁻¹ mol⁻¹. These kinetic parameters are compared with those previously reported for the similar complexes, Co(py)₄CO₃⁺ and Co(py)₂(CO₃)(H₂O)₂⁺.     

Kinetics and mechanism of CO substitution of [η-CpFe(CO)3]PF6 with PPh3 in the presence of substituted pyridine N-oxide

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Isotopic fractionation factors of intermolecular hydrogen bonds in water

Association constants for N---H⁺…⁻O hydrogen bond formation between substituted ammonium dications and phenolate ion were measured in water and deuterium oxide at 25°C and 2.0 ionic strength. In combination with isotopic fractionation factors for phenol and the conjugate diacid of 1,2-ethanediamine determined by ¹³C NMR spectroscopy, these yield isotopic fractionation factors for amine dication-phenolate ion hydrogen bonds in water: φ_AB = 0.69 for 1,2-propanediamine dication with a pK difference between donor and acceptor, ΔpK_a = −0.45, φ_AB = 0.88 for 1,2-ethanediamine dication (ΔpK_a = −2.1), and φ_AB = 1.1 for piperizine dication (ΔpK_a = −3.5). The hydrogen bond association constants follow Brønsted correlations α = 0.19 in water and α = 0.27 in deuterium oxide. The results are consistent with a double-minimum potential with a significant barrier for motion across the hydrogen bond.     

Prostaglandin D2 metabolite stimulates collagen synthesis by human osteoblasts during calcification

We investigate the effect of the prostaglandin D₂ metabolite Δ¹²−PGD₂ (9−Deoxy−Δ⁹, Δ¹²−13,14-dihydroprostaglandin D₂) on collagen synthesis in human osteoblast. Δ¹²-PGJ₂ at 10⁻⁵M enhanced collagen synthesis in the presence of 2 mM α-glycerophosphate-2Na. The stimulative effect appeared as early as 3 days after addition and continued until 22 days. The enhancement of type I collagen synthesis was confirmed by polyacrylamide gel electrophoresis. The potency was the same as 10^1t-8M 1 α, 25 dihydroxy vitamine D₃ (1,25(OH)₂D₃). Northern blot analysis showed that 10⁻⁵M Δ ¹²-PGD₂ and 10⁻⁸M 1,25(OH)₂D₃ enhanced the transcribtion of type 1 procollagen (α₁) mRNA levels in osteoblasts.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

Key structural features in cis-carane, (+)-3-carene,cis-pinane, (+)-α-pinene,and (−)-β-pinene influencing red turpentine beetle primary attraction when released with ethanol

1. In US Pacific Northwest ponderosa pine forests the primary attraction order shown previously for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae), is (−)-β-pinene+ethanol > (+)-3-carene+ethanol > (+)-α-pinene+ethanol. The monoterpenes are bicyclic C₁₀H₁₆ isomers containing one 6-carbon ring with one double bond. Both pinenes have a 4-carbon second ring and differ only by their endocyclic or exocyclic double bond. The (+)-3-carene second ring has 3-carbons; its double bond is endocyclic like (+)-α-pinene.
2. Ring system and double bond influences on primary attraction were evaluated by hydrogenating (+)-3-carene and (+)-α-pinene to cis-carane and cis-pinane, respectively. Field test primary attraction strengths were (−)-β-pinene+ethanol > cis-carane+ethanol > cis-pinane+ethanol > ethanol.
3. In combination with ethanol (i) a double bond is not required in either ring system to attract D. valens, (ii) the cis-carane bicyclic 3, 6-carbon ring system provides stronger beetle attraction than the cis-pinane 4, 6-carbon bicyclic ring system, and likely structural basis for stronger (+)-3-carene attraction over (+)-α-pinene, (iii) adding an exocyclic double bond to the 4, 6-carbon ring system elevates attraction above the 3, 6-carbon ring system with no double bond, and (iv) the 4, 6-carbon ring system is a much stronger attractant with an exocyclic rather than endocyclic double bond.

     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Adaptative response to enhanced basal oxidative damage in sod mutants from Saccharomyces cerevisiae

We investigated the adaptative response of S. cerevisiae in sod mutants (sod1Δ, sod2Δ and sod1Δsod2Δ) after H₂O₂ treatment in the stationary phase. sod2Δ and sod1Δsod2Δ demonstrated the highest levels of GSH in the control, suggesting that pathways which include GSH protect these double mutants against oxidative stress. In addition, sod1Δ and sod1Δsod2Δ had higher iron levels than the wild-type, independently of H₂O₂ stress. Fe levels were increased in sod2Δ following H₂O₂ In addition, the sod2Δ mutant was more sensitive to H₂O₂ treatment than the wild-type. These results suggest that sod2Δ sensibility may be associated with •OH production by the Fenton reaction. This increased iron demand in the sod2Δ mutant may be a reflection of the cells’ efforts to reconstitute proteins that are inactivated in conditions of excess superoxide. MDA levels were assayed by HPLC in these mutants. The highest MDA levels could be observed after 10mM H₂O₂ treatment in the sod1Δsod2Δ double mutant. After treatment with a GSH inhibitor, the MDA level was still higher in the same strain. Thus, both direct and indirect GSH pathways are involved in the protection of lipid membranes and proteins in these mutants and may constitute an adaptative response to enhanced basal oxidative damage produced by superoxide.     

Quantification of hydrogen peroxide and glucose using 3-methyl-2-benzothiazolinonehydrazone hydrochloride with 10,11-dihydro-5H-benz(b,f)azepine as chromogenic probe

A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H₂O₂) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H₂O₂ by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H₂O₂ is found between 5 × 10⁻⁶ and 45 × 10⁻⁶ mol L⁻¹ at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 10⁶ M⁻¹ min⁻¹ and 9.81 × 10⁻⁴ min⁻¹, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the cosubstrates were 163.2 min⁻¹ and 4.156 × 10⁶ L mol⁻¹ min⁻¹, respectively. This method can be incorporated into biochemical analysis where H₂O₂ undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum.     

Aromatase inactivation by 2-substituted derivatives of the suicide substrate androsta-1,4-diene-3,17-dione

To gain the structure–activity relationship of Δ¹-androstenediones (Δ¹-ADs) as mechanism-based inactivator of aromatase, series of 2-alkyl- and 2-alkoxy-substitiuted Δ¹-ADs (6 and 9) as well as 2-bromo-Δ¹-AD (14) were synthesized and tested. All of the inhibitors examined blocked aromatase in human placental microsomes in a competitive manner. In a series of 2-alkyl-Δ¹-ADs (6), n-hexyl compound 6f was the most powerful inhibitor with an apparent K_i value of 31 nM. The inhibitory activities of 2-alkoxy steroids 9 decreased in relation to length of the alkyl chain up to n-hexyloxy group (K_i: 95 nM for methoxy 9a). All of the alkyl steroids 6 along with the alkoxy steroid 9, except for the ethyl and n-propyl compounds 6b and 6c, caused a time-dependent inactivation of aromatase. The inactivation rates (k_inact: 0.020–0.084 min⁻¹) were comparable to that of the parent compound Δ¹-AD. The inactivation was prevented by the substrate AD, and no significant effect of l-cysteine on the inactivation was observed in each case. The results indicate that the 2-hexyl compound 6f act as the most powerful mechanism-based inactivator of aromatase among Δ¹-AD analogs and may be submitted to the preclinical study in estrogen-dependent breast cancer.     

Dissociation of hyperreninemia and renal prostaglandin synthesis in the adrenalectomized rat

The aim of this study was to determine whether hyperreninemia in the adrenalectomized (ADX) rat is dependent on renal prostaglandin synthesis, as has been suggested for two other hyperreninemic conditions, Bartter's syndrome and chronic liver disease.Plasma renin concentration (PRC) in anesthetized, ADX rats was significantly increased (Δ +480%; p < 0.001) compared to sham-operated controls. , indomethacin (10 mg/kg i.v.) significantly reduced PRC of anesthetized, ADX rats after both 45 min (Δ −34%; p < 0.05) and 90 min (Δ −47%; p < 0.05). renin release from renal cortical slices of ADX rats was also significantly greater (Δ +130%; p < 0.05) than from sham-operated control cortical slices. Renin release from cortical slices of ADX rats given dexamethasone (10 μg/kg/day) for 4 days prior to sacrifice did not differ from sham-operated control values.Prostaglandin E₂ (PGE₂) release from cortical slices of ADX rats did not differ significantly from controls. However, PGE₂ synthesis in glomeruli microdissected from ADX rats was significantly increased (Δ +110%; p < 0.001) compared to controls. PGE₂ synthesis in glomeruli of dexamethasone-treated ADX rats remained significantly elevated compared to controls. Ibuprofen (10⁻⁶ M) decreased PGE₂ synthesis in cortical slices by 80%. However, prostaglandin synthesis inhibition had no effect on renin release from either ADX or control renal cortical slices.These results suggest that despite increased glomerular synthesis, prostaglandins do not directly influence renin release in the ADX rat.     

Specific growth rate as a determinant of the carbon isotope composition of the temperate seagrass Zostera marina

The seasonal variability of specific growth rate and the carbon stable isotope ratio (δ¹³C) of leaf blades (δ¹³C_leaf) of a temperate seagrass, Zostera marina (within 10 days old) were measured simultaneously, together with the δ¹³C of dissolved inorganic carbon (δ¹³C_DIC) at three sites in the semi-closed Akkeshi estuary system, northeastern Japan, in June, September, and November 2004. The δ¹³C_leaf ranged from −16.2 to −6.3‰ and decreased from summer to winter. The simultaneous measurement of the δ¹³C_leaf, growth rate, and morphological parameters (mean leaf length and width, mean number of leaves per shoot, and sheath length) of the seagrass and δ¹³C_DIC in the surrounding water allowed us to compare directly the δ¹³C_leaf and specific growth rate of seagrass. The difference in the δ¹³C of seagrass leaves relative to the source DIC (Δδ¹³C_{leaf − DIC}) was the least negative (−11 to −7‰) in June at all three sites and became more negative (−17 to −8‰) as the specific growth rate decreased. This positive correlation between Δδ¹³C_{leaf − DIC} and specific growth rate can be used to diagnose the growth of seagrasses. Δδ¹³C_{leaf − DIC} changed by −1.7 ± 0.2‰ when the leaf specific growth rate decreased by 1% d⁻¹.     

High-performance affinity capture-removal of bacterial pyrogen from solutions

Synthetic peptide S3Δ has high affinity for bacterial endotoxin or lipopolysaccharide (LPS). Under tested conditions of pH 5–9 and 0–0.4 M NaCl, the affinity constant, K_D ranged from 2·10⁻⁶ to 2·10⁻⁹ M⁻¹. A novel affinity matrix based on peptide S3Δ was developed for removal of LPS from solutions such as: water; buffers with a wide range of ionic strength and pH; medium for cell culture; and protein solutions under optimized conditions. At a starting LPS of ≈100 EU/ml, a post-purification level below 0.005 EU/ml was achieved.     

Conformational stabilities of guinea pig OCP1 and OCP2

OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 — previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins. The high concentrations of OCP1 and OCP2 in the cochlea suggest that the OCP1–OCP2 heterodimer may serve an additional function independent of its role in a canonical SCF complex. At 25 °C, urea-induced denaturation of OCP1 is slow, but reversible. The data suggest that the protein possesses one or more disordered regions, a conclusion supported by analysis of the far-UV circular dichroism spectrum and the appearance of the ¹H, ¹⁵N-HSQC spectrum. Thermal denaturation of OCP1 is irreversible, evidently due to formation of high molecular weight aggregates. Analysis with a kinetic model yields an estimate for the activation energy for unfolding of 49 kcal/mol. Urea denaturation data for OCP2 returns ΔG_o and m values of 6.2 kcal/mol and 1.5 kcal mol^− 1 M^− 1, respectively. In contrast to OCP1, thermal denaturation of OCP2 is reversible. In phosphate-buffered saline, at pH 7.40, the protein exhibits a ΔH_vH/ΔH_cal ratio of 1.69, suggesting that denaturation proceeds largely from the native dimer directly to the unfolded state. OCP1 and OCP2 associate tightly at room temperature. However, DSC data for the complex suggest that they denature independently, consistent with the highly exothermic enthalpy of complex formation reported previously.     

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1 mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Effect of microtubule-disrupting agents on superoxide production in human polymorphonuclear leukocytes

We explored the effects of compounds known or proposed to affect microtubule functions on superoxide (O₂⁻) production in human polymorphonuclear leukocytes stimulated by N-formyl-methionyl-phenylalanine (f-Met-Phe), calcium ionophore A23187 and phorbol myristate acetate. F-Met-Phe-induced O₂⁻ production was markedly potentiated not only by microtubule-disrupting agents, including colchicine, vincristine, vinblastine, nocodazole, podophyllotoxin and griseofulvin, but also deuterium oxide (²H₂O), which is proposed to stabilize microtubules, and not affected by lumicolchicine. Ionophore A23187-induced O₂⁻ production was not influenced by colchicine, and markedly enhanced by ²H₂O, whereas phorbol myristate acetate-induced O₂⁻ production was not influenced by colchicine, and slightly inhibited by ²H₂O. ²H₂O did not counteract the effects of colchicine and vice versa. Dibutyryl cyclic AMP and prostaglandin E₁ inhibited O₂⁻ production stimulated by f-Met-Phe and ionophore A23187, whereas phorbol myristate acetate-induced O₂⁻ production was strongly resistant to the inhibitory effect of these agents. The enhancing effect of colchicine and ²H₂O on f-Met-Phe-induced O₂⁻ production was abolished by dibutyryl cyclic AMP. Colchicine promoted concanavalin A cap formation, and ²H₂O produced cancanavalin A patch formation, whereas dibutyryl cyclic AMP did not affect the distribution of concanavalin A receptors. In addition, ²H₂O and dibutyryl cyclic AMP did not interfere with the colchicine-induced concanavalin A cap formation. These findings suggest that f-Met-Phe, ionophore A23187 and phorbol myristate acetate may activate the oxidative metabolism of human polymorphonuclear leukocytes through different mechanisms, and that microtubule-disrupting agents, ²H₂O and cyclic AMP agonists may affect the different steps of the activating system of NAD(P)H oxidase.