A review on imaging techniques and quantitative measurements for dynamic imaging of cerebral aneurysm pulsations.

Measurement of intracranial aneurysm wall motion may refine the current rupture risk estimation. A golden standard for measuring aneurysm pulsation is lacking. The aim is to evaluate magnitudes of aneurysm pulsation as published in current literature. Embase and PubMed were searched for publications containing quantitative measures of cardiac-cycle related cerebral aneurysm pulsation (no date or language restrictions). Eleven studies were included, covering 197 unruptured and untreated cerebral aneurysms. Quantitative pulsation measurements were extracted from the studies. Characteristics of the study population and aneurysms were taken into account, as well as the imaging modality, scanning technique and data processing methods used. A meta-analysis was performed of studies with similar methodologies and individual IA measures and locations. The magnitude of the absolute volume pulsations varied between 14 ± 9 mm3 and 106 ± 123 mm3 and the mean relative volume change varied between 5 and 36%. The meta-analysis revealed a positive correlation between size and absolute volume change. The relative volume change in Basilar artery aneurysms seems smaller. No authors were contacted for original study data and articles only describing visual pulsations were excluded. The variation in methodologies impedes an accurate estimation of the magnitude of IA pulsations. Validation of aneurysm pulsation measurement is crucial prior to clinical studies evaluating IA pulsatility in relation to IA rupture risk. Prerequisite is a reliable and robust imaging method with high spatial and temporal resolution and standardization of the image analysis methods.

The normal evolution of the cranium in three dimensions.

Insight into the growth and development of the normal newborn cranial shape is essential to monitor cranial development, to detect and diagnose abnormal skull shapes, and for the long-term follow-up of craniosynostosis surgery. The aim of this study was to analyse the growth pattern of the cranial shape of infants during the first years of life using 3D stereophotogrammetry and 3D computed tomography (CT) with advanced 3D evaluation techniques. A large set of 3D photographs (n=199) and CT scans (n=183), taken between ages 0 and 54 months, was collected. Cranial shapes with artefacts and asymmetries were removed. Total volumes and intracranial volumes were obtained, as well as 3D and 2D measurements, including the cranial width, cranial length, cranial index, and suture lengths. Growth maps were created for all modalities to indicate 3D growth over time. For the final analysis, a total of 130 3D photographs, 94 hard tissue CT scans, and 76 soft tissue CT scans were used. 3D and 2D measures, volumes, growth maps, and growth animations were obtained. A non-uniform growth was revealed by the 3D growth maps. This study addresses the need for normative cranial evolution data to monitor healthy cranial development and for detection, follow-up, and treatment planning in craniosynostosis.

A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

BACKGROUND: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. OBJECTIVES: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. METHODS: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. RESULTS: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. CONCLUSIONS: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

A new method for three-dimensional evaluation of the cranial shape and the automatic identification of craniosynostosis using 3D stereophotogrammetry.

Craniosynostosis is a congenital defect which can result in abnormal cranial morphology. Three dimensional (3D) stereophotogrammetry is potentially an ideal technique for the evaluation of cranial morphology and diagnosis of craniosynostosis because it is fast and harmless. This study presents a new method for objective characterization of the morphological abnormalities of scaphocephaly and trigonocephaly patients using 3D photographs of patients and healthy controls. Sixty 3D photographs of healthy controls in the age range of 3-6 months were superimposed and scaled. Principal component analysis (PCA) was applied to find the mean cranial shape and the cranial shape variation in this normal population. 3D photographs of 20 scaphocephaly and 20 trigonocephaly patients were analysed by this PCA model to test whether cranial deformities of scaphocephaly and trigonocephaly patients could be objectively identified. PCA was used to find the mean cranial shape and the cranial shape variation in the normal population. The PCA model was able to significantly distinguish scaphocephaly and trigonocephaly patients from the normal population. 3D stereophotogrammetry in combination with the presented method can be used to objectively identify and classify the cranial shape of healthy newborns, scaphocephaly and trigonocephaly patients.

The computed cranial focal point.

INTRODUCTION: Stereophotogrammetry is a radiation-free method for monitoring skull development after craniosynostosis repair. Lack of clear fixed reference points complicate longitudinal comparison of 3D photographs. Therefore we developed the 'computed cranial focal point' (CCFP). METHODS: The CCFP was calculated in segmented 3D CT-scans of 36 adult subjects using Matlab. The robustness of the CCFP calculation was evaluated in predefined hemi-ellipsoid shapes. Finally we used the CCFP in two clinical cases to correlate CT data with 3D-photographic data. RESULTS: The CCFP calculation was found to be hardly influenced by incomplete or deformed surface data which resulted in small deviations (<2.5 mm). The average position of the CCFP of the skin relative to the sella turcica was at (0.0, 27.1, 19.4) mm, with CCFPσ (0.6, 4.6, 3.9) mm. The mean difference between the CCFP for the skull and skin was (-0.1, 1.9, -1.4) mm, with CCFPσ (0.5, 1.4, 1.0) mm. Using the CCFP in two cases to correlate the skin from a 3D-photo and the segmented skin from a CT-scan resulted in absolute mean differences of 0.7 and 2.3 mm, with a standard deviation of 1.1 mm in both cases. CONCLUSION: The CCFP calculation is a robust method to define a reference point relative to the sella turcica based on the skin or cranial bone surfaces. The CCFP can be used to correlate 3D photographs with CT-scan data or for longitudinal radiation-free comparison of 3D-photos.

Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ = 0.8 ng ml(-1) in extracts, corresponding to 0.02 µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25 mg g(-1), while the skins contained considerably less, 0.4 mg g(-1). Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5 µg g(-1), or 0.5 ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02 µg g(-1) parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15 µg g(-1) of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.

Enhancing the in vitro Fe(2+) bio-accessibility using ascorbate and cold-set whey protein gel particles.

This paper investigates the possibility for iron fortification of food using a new preparation method for protein gel particles in which iron is entrapped in the presence of ascorbate using cold-set gelation. The effect of ascorbate on the iron-induced cold-set gelation process of whey protein was studied in order to optimize the ratio of iron/ascorbate. Subsequently, the effect of ascorbate on iron bio-accessibility was assessed in vitro. Rheology was used to study the protein gel formation, and the stability of the gel particles was determined by measuring the iron and protein content at different pH. In vitro studies were performed with the TNO Intestinal Model (TIM). Ascorbate appeared to affect the gel formation process and increased the gel strength of the iron-induced cold-set gels at specific iron/ascorbate ratio. With the Fe-protein gel particles being stable at a broad pH range, the release of iron from the particles was studied as a function of time. The low release of iron indicated a good encapsulation efficiency and the capability of whey protein to keep iron bound at different conditions (pH and presence of calcium). Results obtained with the TIM showed that ascorbate, when added to the protein gel particles, was very successful in enhancing the recovery and absorption of iron. The in vitro Fe(2+) bio-accessibility in the presence of ascorbate in iron-protein particles increased from 10% to almost 80%. This suggests that the concept of using protein particles with iron and ascorbate can effectively be used to fortify food products with iron for human consumption.

The effect of the food matrix on in vivo immune responses to purified peanut allergens.

There is little knowledge about the factors that determine the allergenicity of food proteins. One aspect that remains to be elucidated is the effect of the food matrix on immune responses to food proteins. To study the intrinsic immunogenicity of allergens and the influence of the food matrix, purified peanut allergens (Ara h 1, Ara h 2, Ara h 3, or Ara h 6) and a whole peanut extract (PE) were tested in the popliteal lymph node assay (PLNA) and in an oral model of peanut hypersensitivity. In the PLNA, peanut proteins were injected into the hind footpad of BALB/c mice; in the oral exposure experiments C3H/HeOuJ mice were gavaged weekly with PE or allergens in the presence of cholera toxin (CT). Upon footpad injection, none of the allergens induced significant immune activation. In contrast, PE induced an increase in cell number, cytokine production, and activation of antigen-presenting cells. Furthermore, the presence of a food matrix enhanced the immune response to the individual allergens. Oral exposure to the purified allergens in the presence of CT induced specific IgE responses, irrespective of the presence of a food matrix. These results suggest that purified peanut allergens possess little intrinsic immune-stimulating capacity in contrast to a whole PE. Moreover, the data indicate that the food matrix can influence responses to individual proteins and, therefore, the food matrix must be taken into account when developing models for allergenic potential assessment.

Purification and immunoglobulin E-binding properties of peanut allergen Ara h 6: evidence for cross-reactivity with Ara h 2.

BACKGROUND: IgE-binding peanut proteins smaller than 15 kDa were previously identified as potential allergens in the majority of our peanut allergic population. OBJECTIVE: To characterize the novel allergen in order to determine whether it was similar to one of the thus far identified recombinant peanut allergens (Ara h 1-7). METHODS: An IgE-binding protein of <15 kDa was purified and identified via N-terminal sequencing. Its IgE-binding properties were investigated using immunoblotting, basophil degranulation, and skin prick testing. Possible cross-reacting epitopes with other peanut allergens were studied using IgE-immunoblotting inhibition. RESULTS: The purified protein is a monomeric protein with a molecular weight of 14,981 Da as determined using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The amino acid sequence of the first 39 N-terminal residues is identical to that of Ara h 6, indicating that the allergen is Ara h 6. It is recognized by 20 out of 29 peanut-allergic patients on IgE-immunoblot, and its potent biological functionality is demonstrated by the degranulation of basophils, even at concentrations below 10 pg/mL, and by positive skin prick reactions. Ara h 6 has homology to Ara h 2, especially in the middle part and at the C-terminal part of the protein. Almost complete inhibition of IgE-Ara h 6 interaction with Ara h 2 demonstrates that at least part of the epitopes of Ara h 6 are cross-reactive with epitopes on Ara h 2. CONCLUSIONS: Peanut-derived Ara h 6 is a biologically active allergen recognized by the majority of our peanut-allergic patient population and can be considered a clinically relevant peanut allergen.

[Information provision for the biomedical sciences in the Netherlands remains safe guarded]. / Informatievoorziening voor de biomedische wetenschappen in Nederland blijft behouden.

In view of the rapid development of electronic journals covering the biomedical sciences, the Netherlands Institute for Scientific Information Services has decided to review its document delivery services. The Institute will cancel all of its journal subscriptions at the end of 2002. This will not affect services provided to users of the library. Requests for documents from the collection will continue to be honoured. Cooperation with other European libraries, in particular with the German Central Library of Medicine in Cologne, will ensure continued access to a broad collection of journals. However, users will need to ensure that they prepare themselves for the world of digital journals. Document delivery is seen as a substitute for fully-fledged digital access.

Crystallization of quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni: crystals with unique optical properties.

Quinohaemoprotein alcohol dehydrogenase from Comamonas testosteroni is a functional electron-transfer protein containing both a haem c and a pyrroloquinoline quinone cofactor. The enzyme has been crystallized at 277 K using polyethylene glycol 6000 as precipitant. The crystals belong to space group C2, with unit-cell parameters a = 98.1, b = 74.3, c = 92.2 A, beta = 105.9 degrees. A native data set with a resolution of 2.44 A resolution has been collected. The approximate orientation of the haem group with respect to the unit-cell axes has been determined from the optical properties of the crystals.

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.

Anaphylaxis caused by the unexpected presence of casein in salmon.

A new process for restructured meat and fish has been introduced to the market recently. Its main compound is casein, and it may therefore endanger patients with a milk allergy.

Purification and characterization of sulfide dehydrogenase from alkaliphilic chemolithoautotrophic sulfur-oxidizing bacteria.

Extracts of the alkaliphilic sulfur-oxidizing autotroph strain AL3 contained sulfide:cytochrome c oxidoreductase. This was active above pH 8, and was associated with the cell membranes. Although up to 60% of the initial activity was lost during Triton X-100 extraction, further purification resulted in an enzyme that catalyzed sulfide oxidation with horse heart cytochrome c. This enzyme was a 41 kDa protein containing heme c554. The optimum pH of the membrane bound enzyme was 9.0, but after extraction this fell to 8.0. The enzyme catalyzed a single electron oxidation of HS-. Hydrosulfide radical is therefore the most probable product.

Isolation of the tetrathionate hydrolase from Thiobacillus acidophilus.

An enzyme capable of hydrolysing tetrathionate was purified from cell-free extracts of Thiobacillus acidophilus. The purified enzyme converts tetrathionate into thiosulfate, sulfur and sulfate. In addition, pentathionate could also be converted by the same enzyme. Measurement of the enzyme activity during purification is based on the absorbance of the initial intermediates formed from tetrathionate in the ultraviolet region, which have not been identified. Enzyme activity could also be measured by the scattering of insoluble sulfur in the visible region. The purified enzyme has a pH optimum of 2.5 and a temperature optimum of 65 degrees C. Enzyme activity is strongly stimulated by the presence of sulfate ions. The purified enzyme is a dimer with two identical subunits of 48 kDa. The ultraviolet-visible absorption spectra and denaturation experiments indicate the presence of an organic cofactor.

Purification and characterization of a periplasmic Thiosulfate dehydrogenase from the obligately autotrophic Thiobacillus sp. W5.

A periplasmic thiosulfate dehydrogenase (EC 1.8.2.2) was purified to homogeneity from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5. A five-step procedure resulted in an approximately 2,300-fold purification. The purified protein had a molecular mass of 120 +/- 3 kDa, as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 33 +/- 1 kDa and 27 +/- 0.5 kDa, as determined by SDS-PAGE. UV/visible spectroscopy revealed that the enzyme contained haem c; haem staining showed that both subunits contained haem c. A haem c content of 4 mol per mol of enzyme was calculated using the pyridine haemochrome test. The pH optimum of the enzyme was 5.5. At pH 7.5, the Km and Vmax were 120 +/- 10 microM and 1,160 +/- 30 U mg-1, respectively. The absence of 2-heptyl-4-hydroquinoline-N-oxide (HQNO) inhibition for the oxidation of thiosulfate by whole cells suggested that the electrons enter the respiratory chain at the level of cytochrome c. Comparison with thiosulfate dehydrogenases from other Thiobacillus species showed that the enzyme was structurally similar to the thiosulfate dehydrogenase of the acidophilic, facultatively chemolithoautotrophic Thiobacillus acidophilus, but not to the thiosulfate dehydrogenases published for the obligately chemolithoautotrophic Thiobacillus tepidarius and Thiobacillus thioparus.

The copper-topaquinone-phenylhydrazine-adduct geometry in Escherichia coli amine oxidase derivatized with phenylhydrazines substituted with 19F-NMR relaxation measurements.

The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazine, substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of CU(II) to the transversal-relaxation time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 +/- 0.06 nm from the C2-O, 1.30 +/- 0.04 nm from the C4-O, and 1.26 +/- 0.04 nm from the C5-N). However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F-NMR data obtained for porcine-plasma marine oxidase [Williams, T J. & Falk, M.C.(1986) J. Biol. Chem. 261, 15949- 15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco.

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.

Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni.

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.