Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Cyclic octapeptides containing thiazole. Effect of stereochemistry and degree of flexibility on calcium binding properties

Solution conformation and calcium binding properties have been investigated for the two cyclic octapeptides cyclo(-D-Thr-D-Val(Thz)-Ile-)(2) (4) and cyclo(-Thr-Gly(Thz)-Ile-Ser-Gly(Thz)-Ile-)(5) and the results are compared to those for the cyclic octapeptides previously studied; ascidiacyclamide (1), patellamide D (2), cyclo(-Thr-D-Val(Thz)-Ile-)(2) (3), and cyclo(-Thr-D-Val-alphaAbu-Ile-)2 (6). Both 4 and 5 contain two heterocyclic thiazole ring constraints but the latter has a larger degree of flexibility as a consequence of the glycine residues within the cyclic framework. The solution conformation of 4 and 5 was determined from H-1 NMR spectra and found to be a twisted figure of eight similar to that for 2. Complexation studies using H-1 NMR and CD spectroscopy yielded 1 : 1 calcium-peptide binding constants (logK) for the two peptides (2.3 (4) and 5.7 (5)). For 5 the magnitude of the binding constant was verified by a competition titration using CD. The different calcium-binding affinities of 3 (logK = 4.0) and 4 is attributed to the stereochemistry of the threonine residue. The magnitude of the binding constant for 5 compared to 3 and 4 (all peptides containing two thiazole ring constrains) demonstrates that the increase in flexibility of the cyclic peptide has a dramatic effect on the Ca2+ binding ability. The affinity for Ca2+ thus decreases in the order (6 similar to 5 > 3 > 2 similar to 1 > 4). The number of carbonyl donors available on each peptide has only a limited effect on calcium binding. The most important factor is the flexibility, which allows for a conformation of the peptide capable of binding calcium efficiently.

Parity measurement of one- and two-electron double well systems

We outline a scheme to accomplish measurements of a solid state double well system (DWS) with both one and two electrons in nonlocalized bases. We show that, for a single particle, measuring the local charge distribution at the midpoint of a DWS using a SET as a sensitive electrometer amounts to performing a projective measurement in the parity (symmetric/antisymmetric) eigenbasis. For two-electrons in a DWS, a similar configuration of SET results in close-to-projective measurement in the singlet/triplet basis. We analyze the sensitivity of the scheme to asymmetry in the SET position for some experimentally relevant parameter, and show that it is experimentally realizable.

Drinfield twists and alegebraic Bethe ansatz of the supersymmetric t-J model

We construct the Drinfeld twists (factorizing F-matrices) for the supersymmetric t-J model. Working in the basis provided by the F-matrix (i.e. the so-called F-basis), we obtain completely symmetric representations of the monodromy matrix and the pseudo-particle creation operators of the model. These enable us to resolve the hierarchy of the nested Bethe vectors for the gl(2\1) invariant t-J model.

Algebraic Bethe ansatz for integrable extended Hubbard models arising from supersymmetric group solutions

Integrable extended Hubbard models arising from symmetric group solutions are examined in the framework of the graded quantum inverse scattering method. The Bethe ansatz equations for all these models are derived by using the algebraic Bethe ansatz method.

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

Embryonic development of the nervous system of the temnocephalid flatworm Craspedella pedum

The nervous system of temnocephalid flatworms consists of the brain and three pairs of longitudinal connectives extending into the trunk and tail. The connectives are crosslinked by an invariant number of regularly spaced commissures. Branches of the connectives innervate the tentacles of the head and the sucker organ in the tail. A set of nerve rings encircling the pharynx and connected to the brain and connectives constitute the pharyngeal nervous system. The nervous system is formed during early embryogenesis when the embryo represents a multilayered mesenchymal mass of cells. Gastrulation and the formation of separate epithelial germ layers that characterize most other animal groups are absent. The brain arises as a bilaterally symmetric condensation of postmitotic cells in the deep layers of the anterior region of the embryonic mesenchyme. The pattern of axon outgrowth, visualized by labeling with anti-acetylated tubulin (acTub) antibody, shows marked differences from the pattern observed in other flatworm taxa. in regard to the number of neurons that express the acTub epitope. Acetylated tubulin is only expressed in neurons that form long axon tracts. In other flatworm species, such as the typhloplanoid Mesostoma and the polyclad Imogine, which were investigated by us with the acTub antibody (Hartenstein and Ehlers [2000] Dev. Genes Evol. 210:399-415; Younossi-Hartenstein and Hartenstein [2000] Dev. Genes Evol. 210:383-398), only a small number of pioneer neurons become acTub positive during the embryonic period. By contrast, in temnocephalids, most, if not all, neurons express acTub and form long, large-diameter axons. Initially, the brain commissure, pharyngeal nerve ring, and the connectives are laid down. Commissural tracts and tentacle nerves branching off the connectives appear later. We speculate that the precocious differentiation of the nervous system may be related to the fact that temnocephalids move by muscle action, and possess a massive and complex muscular system when they hatch. In addition, they have muscular specializations such as the anterior tentacles and the posterior sucker that are used as soon as they hatch. By contrast, juveniles of Mesostoma and larvae of polyclads move predominantly by ciliary action that may not require a complex neural circuitry for coordination. (C) 2001 Wiley-Liss, Inc.

Formation of cumulenes, triple-bonded, and related compounds by flash vacuum thermolysis of five-membered heterocycles

Flash vacuum thermolysis of a large variety of heterocyclic compounds is a useful means of production of ketenes, ketenimines, thioketenes, allenes, iminopropadienones, bis(imino)propadienes, iminopropadienethiones, carbodiimides, isothiocyanates, acetylenes, fulminic acid, nitrile imines and nitrile ylides, nitriles, cyanamides, cyanates, and other compounds, often in preparatively useful yields.

A comparison of low-storage strategies for spectral analysis in dissipative systems: filter diagonalisation in the Lanczos representation and harmonic inversion of the Chebychev-order-domain autocorrelation function

We compare the performance of two different low-storage filter diagonalisation (LSFD) strategies in the calculation of complex resonance energies of the HO2, radical. The first is carried out within a complex-symmetric Lanczos subspace representation [H. Zhang, S.C. Smith, Phys. Chem. Chem. Phys. 3 (2001) 2281]. The second involves harmonic inversion of a real autocorrelation function obtained via a damped Chebychev recursion [V.A. Mandelshtam, H.S. Taylor, J. Chem. Phys. 107 (1997) 6756]. We find that while the Chebychev approach has the advantage of utilizing real algebra in the time-consuming process of generating the vector recursion, the Lanczos, method (using complex vectors) requires fewer iterations, especially for low-energy part of the spectrum. The overall efficiency in calculating resonances for these two methods is comparable for this challenging system. (C) 2001 Elsevier Science B.V. All rights reserved.

The solution structure of C1-T1, a two-domain proteinase inhibitor derived from a circular precursor protein from Nicotiana alata

A two-domain portion of the proteinase inhibitor precursor from Nicotiana alata (NaProPI) has been expressed and its structure determined by NMR spectroscopy. NaProPI contains six almost identical 53 amino acid repeats that fold into six highly similar domains; however, the sequence repeats do nut coincide with the structural domains. Five of the structural domains comprise the C-terminal portion of one repeat and the N-terminal portion of the next. The sixth domain contains the C-terminal portion of the sixth repeat and the N-terminal portion of the first repeat. Disulphide bonds link these C and N-terminal fragments to generate the clasped-bracelet fold of NaProPI. The three-dimensional structure of NaProPI is not known, but it is conceivable that adjacent domains in NaProPI interact to generate the circular bracelet with the N and C termini in close enough proximity to facilitate formation of the disulphide bonds that form the clasp The expressed protein, examined in the current study, comprises residues 25-135 of NaProPI and encompasses the first two contiguous structural domains, namely the chymotrypsin inhibitor C1 and the trypsin inhibitor T1, joined by a five-residue linker, and is referred to as C1-T1. The tertiary structure of each domain in C1-T1 is identical to that found in the isolated inhibitors. However, no nuclear Overhauser effect contacts are observed between the two domains and the five-residue linker adopts an extended conformation. The absence of interactions between the domains indicates that adjacent domains do not specifically interact to drive the circularisation of NaProPI. These results are in agreement with recent data which describe similar PI precursors from other members of the Solanaceae having two, three, or four repeats. The lack of strong interdomain association is likely to be important for the function of individual inhibitors by ensuring that there is no masking of reactive sites upon release from the precursor. (C) 2001 Academic Press.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

A flexible method for rapid force computation in elliptical magnets

The design of open-access elliptical cross-section magnet systems has recently come under consideration. Obtaining values for the forces generated within these unusual magnets is important to progress the designs towards feasible instruments. This paper presents a novel and flexible method for the rapid computation of forces within elliptical magnets. The method is demonstrated by the analysis of a clinical magnetic resonance imaging magnet of elliptical cross-section and open design. The analysis reveals the non-symmetric nature of the generated Maxwell forces, which are an important consideration, particularly in the design of superconducting systems.

An inverse design method for elliptical MRI magnets

An inverse, current density mapping (CDM) method has been developed for the design of elliptical cross-section MRI magnets. The method provides a rapid prototyping system for unusual magnet designs, as it generates a 3D current density in response to a set of target field and geometric constraints. The emphasis of this work is on the investigation of new elliptical coil structures for clinical MRI magnets. The effect of the elliptical aspect ratio on magnet performance is investigated. Viable designs are generated for symmetric, asymmetric and open architecture elliptical magnets using the new method. Clinically relevant attributes such as reduced stray field and large homogeneous regions relative to total magnet length are included in the design process and investigated in detail. The preliminary magnet designs have several novel features.