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Five procedures are presented for the determination of hydrogen peroxide and organic hydroperoxides (including organic peroxyacids) at concentrations of 1–10 μM in aqueous soltion. All five are spectrophotometric methods, based on formation of phenolphthalein or triiodide, catalytic dye bleaching, coupled oxidation of NADPH, and horseradish peroxidase-coupled oxidations. Each is evaluated in terms of sensitivity, selectivity and reliability. The method based on coupled oxidation of NADPH is the most widely applicable to peroxidic species, whereas the horseradish peroxidase method can be made highly selective for hydrogen peroxide.

The pathogenesis of acute myeloid leukemia (AML) involves the cooperation of mutations promoting proliferation/survival and those impairing differentiation. The RAS pathway has been implicated as a key component of the proliferative drive in AML. We have screened AML patients, predominantly younger than 60 years and treated within 2 clinical trials, for NRAS (n = 1106), KRAS (n = 739), and HRAS (n = 200) hot-spot mutations using denaturing high-performance liquid chromatography or restriction fragment length polymorphism (RFLP) analysis. NRAS mutations were confirmed in 11% of patients (126/1106) and KRAS mutations in 5% (39/739). No HRAS mutations were detected in 200 randomly selected samples. Codons most frequently mutated were N12 (43%), N13 (21%), and K12 (21%). KRAS mutations were relatively overrepresented in French-American-British (FAB) type M4 (P < .001). NRAS mutation was over-represented in the t(3;5)(q21 approximately 25;q31 approximately q35) subgroup (P < .001) and underrepresented in t(15;17)(q22;q21) (P < .001). KRAS mutation was overrepresented in inv(16)(p13q22) (P = .004). Twenty-three percent of KRAS mutations were within the inv(16) subgroup. RAS mutation and FLT3 ITD were rarely coexistent (14/768; P < .001). Median percentage of RAS mutant allele assayed by quantitative RFLP analysis was 28% (N12), 19% (N13), 25% (N61), and 21% (K12). RAS mutation did not influence clinical outcome (overall/disease-free survival, complete remission, relapse rate) either for the entire cohort or within cytogenetic risk groups.

The etiology of acute myeloid leukemia (AML) is largely unknown. Biologic and epidemiologic data implicate exogenous toxicants, including cytotoxic drugs, benzene, radiation, and cigarette smoking. Allelic variation in genes encoding enzymes such as NADP(H) quinone oxidoreductase (NQO1) and glutathione S-transferase T1 (GSTT1) that metabolize environmental toxicants predispose to subtypes of AML, including therapy-related AML. We assayed NRAS oncogene mutation and FLT3 internal tandem duplication in 447 AML patients with an abnormal karyotype treated in Medical Research Council (MRC) AML clinical trials. Functional allelic variant frequencies in genes encoding carcinogen-metabolizing enzymesGSTT1, GSTM1, CYP1A1,CYP2D6, CYP2C19, SULT1A1, and NQO1 were previously determined for this cohort. FLT3 internal tandem duplication (ITD) frequency was 17%, and NRAS mutation 12% for the entire cohort. The 2 mutations were found together in only 4 patients. No association was found between enzyme allelic variant frequencies and the presence of FLT3 ITD for the entire cohort or within cytogenetic subgroups. CYP1A1*2B (Val) high-inducibility variant allele was overrepresented in patients with NRAS mutation compared with no mutation, for (1) the entire AML cohort (n = 8/53 vs 26/371; odds ratio [OR] = 2.36; 95% confidence interval [CI] 1.01-5.53) and (2) the poor-risk karyotype group (n = 6/14 vs 4/89; OR = 15.94; 95% CI 3.71-68.52) comprising patients with partial/complete deletion of chromosome 5 or 7, or abnormalities of chromosome 3. The CYP1A1*2B allele may predispose to the development of these subgroups of AML by augmented phase 1 metabolism to highly reactive intermediates of CYP1A1 substrates, including polycyclic aromatic hydrocarbons, or by generation of oxidative stress as a metabolic by-product.

pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. In addition, they show a vestigial vacuole morphology and a sensitivity to growth on media containing excess divalent cations. This pleiotropic phenotype observed for pep5::TRP1 mutants is partially suppressed by the vps8-200 allele. pep5::TRP1 vps8-200 mutants show near wild-type levels of mature-sized soluble vacuolar hydrolases, growth on zinc-containing medium, and a more “wild-type” vacuolar morphology; however, aminopeptidase I and alkaline phosphatase accumulate as precursors. These data suggest that Pep5p is a bifunctional protein and that the TRP1 insertion does not eliminate function, but results in a shorter peptide that can interact with Vps8-200p, allowing for partial function. vps8 deletion/disruption mutants contain a single enlarged vacuole. This genetic interaction was unexpected, since Pep5p was thought to interact more directly with the vacuole, and Vps8p is thought to play a role in transport between the Golgi complex and the prevacuolar compartment. The data are consistent with Pep5p functioning both at the site of Vps8p function and more closely proximal to the vacuole. They also provide evidence that the three transport pathways to the vacuole either converge or share gene products at late step(s) in the pathway(s).

We present the first EPR and ENDOR examination of a catalase compound I (Cat I), the one formed by peracetic acid treatment of Micrococcus lysodeikticus catalase. The Cat I rapid-passage EPR signal (g perpendicular eff = 3.32; g parallel eff approximately 2) appears quite different from those reported previously for the compounds I from horseradish peroxidase (HRP I) and chloroperoxidase. Nonetheless, all three signals can be explained by the same model for exchange coupling between an S = 1 oxoferryl [Fe = O]2+ moiety and a porphyrin pi-cation radical (S' = 1/2) (Schulz, C. E., et al. (1979) FEBS Lett. 103, 102-105). The signal for Cat I is unlike those for the two peroxidases in that it reflects a ferromagnetic rather than antiferromagnetic exchange. Preliminary 1H ENDOR spectra for Cat I appear to differ from the proton (1H) ENDOR spectra of HRP I; the latter, along with the 14N ENDOR spectra, indicate that the porphyrin radical in HRP I exhibits a predominantly A2u-like state having large spin densities on porphyrin N and C(beta). The proton ENDOR spectrum of Cat I is insensitive to H/D exchange, which indicates that the [Fe = O]2+ moiety is not protonated. Consideration of the EPR results for a series of compounds I suggests that the sign and magnitude of the exchange parameter (J) is correlated with the nature of the proximal axial ligand.

Summary Chlamydia pneumoniae , a newly described Chlamydia species, has been shown to be a cause of acute respiratory tract infection in both adults and children, but its role in human infection is still under investigation. Here we present a family outbreak of C. pneumoniae infection where three members of a family presented with a ‘flu-like illness' and acute upper respiratory tract infection which did not improve despite penicillin or septrin therapy. No history of exposure to birds, pets or animals was obtained. As C. pneumoniae isolation from respiratory secretions is not without difficulty, diagnosis usually relies currently on serum-based tests. In this study C. pneumoniae specific IgM determined by the micro-immunofluorescence test was detected in the three clinical cases. All three cases had an elevated complement-fixing antibody titre to Psittacosis-LGV antigen, which may have suggested psittacosis, if type-specific tests had not been performed. In addition, three other members of the family had C. pneumoniae -specific IgG antibody although specific IgM was absent. These three younger members of the family had been symptomatic in the month preceding symptoms in their older sibling and their parents. All the symptomatic members of the family made a complete recovery on tetracycline therapy.

A series of 8-(ferrocenylalkyl)theophylline conjugates were synthesized for evaluation in a homogeneous, competitive electrochemical immunoassay for theophylline with amperometric detection of the ferrocene label at +320 mV. The electrical signal was amplified via redox cycling with the glucose oxidase/glucose system. The resulting catalytic current was strongly inhibited upon binding of the conjugates to anti-theophylline antibodies such that a large excess of theophylline was required to achieve complete reversal leading to an assay with poor sensitivity in the clinical range. A study of the nonspecific interaction of the antibodies with various ferrocene derivatives indicated that this was reduced when a charged functional group was present on the metallocene ring. Consequently, a conjugate was synthesized with a quaternary ammonium group which when incorporated into the assay resulted in improved sensitivity.

The electrochemistry of redox proteins is now well established. Conditions exist which allow electron-transfer reactions of all simple proteins to proceed rapidly and reversibly at electrodes. Coupling of the electrode reaction to enzymes, for which the redox proteins act as cofactors, allows exploitation of this good electrochemistry. This is well illustrated by the enzyme-catalysed electrochemical oxidation of p -cresol to p -hydroxybenzaldehyde, which has been shown to proceed along with coupling to the electrode via the copper protein, azurin, or the organometallic compound ferroceneboronic acid. Ferrocene derivatives, in general, show a degree of versatility, coupling the electron-transfer reactions of many enzymes. Thus derivatives of the ferricinium ion act as excellent electron-transfer reagents from the enzyme glucose oxidase. The system is capable of detecting glucose in blood. Similar procedures, in conjunction with the appropriate enzyme, have yielded assays for, among others, H 2 O 2 and cholesterol.

The development of a sensitive electrochemical assay for low levels of hydrogen peroxide is described. The system is based on the enzymic reduction of substrate (H2O2) by peroxidase and subsequent electron transfer from a gold or pyrolytic graphite electrode to the enzyme, via a redox mediator. Cyclic voltammetry has been employed to assess the ability of certain redox-active couples to act as mediator. A linear response to hydrogen peroxide is observed in the concentration range 5 × 10−8 –6 × 10−2M.

The kinetics of the reduction by aniline and a series of substituted anilines of a peroxidatically active intermediate, formed by oxidation of deuteroferriheme with hydrogen peroxide, have been studied by stopped-flow spectrophotometry. The reaction with aniline was first order with respect to [intermediate] and showed first-order saturation kinetics with respect to [aniline]. The second-order rate constant was 2.0 ± 0.2 × 10 5 M −1 sec −1 at 25°C (independent of pH in the range 6.60–9.68) compared with the value of 2.4 × 10 5 M −1 sec −1 for the reaction of aniline with horseradish peroxidase Compound I. The effect of aniline substituents upon reactivity towards the heme intermediate closely paralled those reported for reaction with the enzymic intermediate. Anilines bearing electron-donating substituents reacted more rapidly and those bearing electron-withdrawing substituents more slowly than the unsubstituted amine. The rate constants for the heme intermediate reactions ( k dfh )found to be related to those for the enzymic reactions ( k hrp ) by the equation: log k DFH = 0.65log k HRP + 1.96 with a correlation coefficient of 0. 98.

Dc cyclic voltammetry and chronoamperometry have been used to examine the electrochemistry of a series of phosphate esters that are substrates for calf intestinal alkaline phosphatase. p-Aminophenyl phosphate was selected as the most suitable substrate for the electrochemical measurement of alkaline phosphatase activity. Picomolar levels of enzyme can be detected, which enables the design of electrochemical immunoassays using this system. Theophylline and phenytoin have been used as model analytes in competition and displacement assay formats.

The kinetics of formation of Compound I of yeast cytochrome c peroxidase (ferrocytochrome c :hydrogen-peroxide oxidoreductase, EC 1.11.1.5) with a series of peroxybenzoic acids were studied. Reactivity is affected not only by protein ionization, as in the reaction with H 2 O 2 , but also by substrate ionization. The reactivity of negatively charged substrates is markedly lower than that of uncharged species, implying that electrostatic factors profoundly influence substrate binding. The rate constants for neutral peroxybenzoic acids carrying electron-withdrawing substituents increase with increasing peroxy acid p K a . This behaviour suggests that, as previously discussed for reactions of turnip peroxidases, formation of peroxy anion by ionization of substrate within the active site is kinetically important. The results support the mechanism of cytochrome c peroxidase Compound I formation which has been proposed by Poulos and Kraut (J. Biol. Chem. 225 (1980), 8199–8205) on the basis of enzyme structural studies.

1. The resting nuclei of Cucurbita pepo and Cucurbita maxima contain a single, spherical nucleolus that is relatively very large. Part, at least, of this nucleolus persists during nearly the entire process of mitosis. During the metaphase it lies in the equatorial plate and becomes elongated in the direction of the long axis of the spindle, to form first a cylinder and then, with further elongation, a dumb-bell-shaped structure, which finally separates into two fragments that migrate to the opposite poles of the spindle. This entire movement occurs prior to any anaphase migration of the chromosomes. The nucleolar fragments apparently are not included in the daughter nuclei during the telophasic reconstruction, but degenerate in the cytoplasm. 2. Examination of the literature suggests that a similar process of nucleolar division probably occurs in a wide variety of plant-cells. 3. The bearing of this type of nucleolar division on theories of the dynamics of anaphase migration of the chromosomes is briefly discussed.

Modification of glucose oxidase by the covalent coupling of ferrocenecarboxylic acids gives mediator modified enzymes which undergo direct oxidation at an electrode; the use of ferroceneacetic acid instead of ferrocenecarboxylic acid is an improvement in terms of the overpotential for oxidation, the reactivity towards glucose, and the stability on storage.