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.

The role of affect and affective congruence in perceptions of leaders: an experimental study

Based on the idea that emotional exchanges determine symbolic meanings in interpersonal exchanges, we hypothesized that displays of positive and congruent affect determine members' ratings of leaders in a simulated performance appraisal context. To test the hypotheses, 537 participants viewed videotapes of four male and female leaders giving positive and negative feedback, and with facial expressions of affect that were either congruent or incongruent with the verbal message that they were delivering. Results supported hypotheses that positive and message-congruent leader affect results in more positive member ratings of the leader, assessed using a seven-item measure of members' perceptions of the leader's negotiating latitude. The least positive ratings of negotiating latitude were given when positive feedback was delivered with negative facial affect. (C) 2002 Elsevier Science Inc. All rights reserved.

Experimental evidence that partner choice is a driving force in the payoff distribution among cooperators or mutualists: the cleaner fish case

Supply and demand largely determine the price of goods on human markets. It has been proposed that in animals, similar forces influence the payoff distribution between trading partners in Sexual selection, intraspecific cooperation and interspecific mutualism. Here we present the first experimental evidence supporting biological market theory in it study on cleaner fish, Labroides dimidiatus. Cleaners interact with two classes of clients: choosy client species with access to several cleaners usually do not queue for service and do not return if ignored, while resident client species with access to only one cleaning station do queue or return. We used plexiglas plates with equal amounts of food to stimulate these behaviours of the two client classes. Cleaners soon inspected 'choosy' plates before 'resident' plates. This supports previous field observations that suggest that client species with access to several cleaners exert choice to receive better(immediate) service.

Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human

By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/similar tothanaraj/altExtron/.

Development of preferences for the human body shape in infancy

Two studies investigated the development of infants' visual preferences for the human body shape. In Study 1, infants of 12,15 and 18 months were tested in a standard preferential looking experiment, in which they were shown paired line drawings of typical and scrambled bodies. Results indicated that the 18-month-olds had a reliable preference for the scrambled body shapes over typical body shapes, while the younger infants did not show differential responding. In Study 2, 12- and 18-month-olds were tested with the same procedure, except that the typical and scrambled body stimuli were photographic images. The results of Study 2 again indicated that only the 18-month-olds had a reliable preference for the scrambled body shapes. This finding contrasts sharply with infants' precocious preferences for human faces, suggesting that infants' learning about human faces and human bodies follow different developmental trajectories. (C) 2002 Elsevier Science B.V. All rights reserved.

Probing the time course of nonlinear discriminations during human electrodermal conditioning

Animal-based theories of Pavlovian conditioning propose that patterning discriminations are solved using unique cues or immediate configuring. Recent studies with humans, however, provided evidence that in positive and negative patterning two different rules are utilized. The present experiment was designed to provide further support for this proposal by tracking the time course of the allocation of cognitive resources. One group was trained in a positive patterning; schedule (A-, B-, AB+) and a second in a negative patterning schedule (A+, B+, AB-). Electrodermal responses and secondary task probe reaction time were measured. In negative patterning, reaction times were slower during reinforced stimuli than during non-reinforced stimuli at both probe positions while there were no differences in positive patterning. These results support the assumption that negative patterning is solved using a rule that is more complex and requires more resources than does the rule employed to solve positive patterning. (C) 2001 Elsevier Science (USA).

Are transitions in human gait determined by mechanical, kinetic or energetic factors?

It is currently unclear whether it is the need to maintain metabolic efficiency, the need to keep skeletal loading below critical force levels, or simple mechanical factors that drive the walk-to-run (W R) and run-to-walk (R-W) transitions in human gait. Eighteen adults (9 males and 9 females) locomoted on an instrumented treadmill using their preferred gait. Each completed 2 ascending (W-R) and 2 descending (R-W) series of trials under three levels of loading (0%, 15% and 30% body weight). For each trial, participants locomoted for 60 s at each of 9 different speeds -4 speeds both above and below their preferred transition speed (PTS) plus their PTS. Evidence was sought for critical levels of key kinetic (maximum vertical force, impulse, first peak force, time to first peak force and maximum loading rate), energetic (oxygen consumption, transport cost) and mechanical variables (limb lengths, strength) predictive of the gait transition. Analyses suggested the kinetic variables of time to first peak force and loading rate as the most likely determinants of the W-R and R-W transitions. (C) 2003 Elsevier Science B.V. All rights reserved.

Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K-l of 0.9 muM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Chapter 16: Prophylactic Human Papillomavirus Vaccines

Candidate prophylactic vaccines based on papillomavirus L1 virus-like particles (VLPs) are currently in human clinical trials. The main long-term goal of the vaccine is to reduce the incidence of cervical cancer and its precursors. In animal papillomavirus models, systemic immunization with L1 VLPs can induce high titers of neutralizing antibodies that confer protection against high-dose experimental papillomavirus challenge. In humans, systemic vaccination with L1 VLPs has been well tolerated and induced high serum antibody titers (at least 40 times higher than titers seen following natural infection). A recent proof of principle HPV16 L1 VLP efficacy trial has shown excellent protection against persistent HPV16 infection and associated cytological abnormalities. Large scale efficacy trials of L1 VLPs from HPV16 and 18 (the HPV types found most frequently in cervical cancer), with or without HPV6 and 11 (the HPV types responsible for most genital warts), are planned. If the results of these large trials support the encouraging results of the early trials, they should lead to a commercial prophylactic HPV vaccine. Implementation issues may include how to make the vaccine available in the developing world, where the majority of cervical cancer cases occur, the appropriate age of vaccination, and the role of male vaccination. Because a VLP vaccine is likely to provide type-specific protection, increasing the number of cancer-associated HPV types in the vaccine is a likely approach to broadening the protection to additional types. There will probably also be efforts to develop alternative vaccine formulations better suited to implementation in developing countries as well as attempts to develop vaccines with a therapeutic activity against established HPV infection because a combined prophylactic/therapeutic vaccine may be expected to have an even greater impact than a purely prophylactic vaccine on HPV induced disease.

Effects of rosiglitazone and linoleic acid on human preadipocyte differentiation

Background Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro . Materials and methods Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters. Results Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect. Conclusions These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro , they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.

On the induced electric field gradients in the human body for magnetic stimulation by gradient coils in MRI

Prior theoretical studies indicate that the negative spatial derivative of the electric field induced by magnetic stimulation may he one of the main factors contributing to depolarization of the nerve fiber. This paper studies this parameter for peripheral nerve stimulation (PNS) induced by time.-varying gradient fields during MRI scans. The numerical calculations are based on an efficient, quasi-static, finite-difference scheme and an anatomically realistic human, full-body model. Whole-body cylindrical and planar gradient sets in MRI systems and various input signals have been explored. The spatial distributions of the induced electric field and their gradients are calculated and attempts are made to correlate these areas with reported experimental stimulation data. The induced electrical field pattern is similar for both the planar coils and cylindrical coils. This study provides some insight into the spatial characteristics of the induced field gradients for PNS in MRI, which may be used to further evaluate the sites where magnetic stimulation is likely to occur and to optimize gradient coil design.

Experimental muscle pain changes feedforward postural responses of the trunk muscles

Many studies have identified changes in trunk muscle recruitment in clinical low back pain (LBP). However, due to the heterogeneity of the LBP population these changes have been variable and it has been impossible to identify a cause-effect relationship. Several studies have identified a consistent change in the feed-forward postural response of transversus abdominis (TrA), the deepest abdominal muscle, in association with arm movements in chronic LBP. This study aimed to determine whether the feedforward recruitment of the trunk muscles in a postural task could be altered by acute experimentally induced LBP. Electromyographic (EMG) recordings of the abdominal and paraspinal muscles were made during arm movements in a control trial, following the injection of isotonic (non-painful) and hypertonic (painful) saline into the longissimus muscle at L4, and during a 1-h follow-up. Movements included rapid arm flexion in response to a light and repetitive arm flexion-extension. Temporal and spatial EMG parameters were measured. The onset and amplitude of EMG of most muscles was changed in a variable manner during the period of experimentally induced pain. However, across movement trials and subjects the activation of TrA was consistently reduced in amplitude or delayed. Analyses in the time and frequency domain were used to confirm these findings. The results suggest that acute experimentally induced pain may affect feedforward postural activity of the trunk muscles. Although the response was variable, pain produced differential changes in the motor control of the trunk muscles, with consistent impairment of TrA activity.