Polarization-based non-staining cell detection.

Polarization is an important characteristic of electromagnetic waves, which can not be detected by either the human visual system or traditional image sensors. Motivated by various animal species with polarization vision as well as by the prospect of improving the image quality of the imaging systems, we are exploring the potential of polarization for microscope imaging. The most powerful techniques for molecule monitoring requires complex preprocessing for labeling the sample with different dyes. In this paper, we propose a cell detection method using polarization imaging without any need for staining target cell samples with any chemical dye. The motivation for this work is to develop an optical imaging technique that is simple and that can be used on live cells. The polarization sensitivity of cell samples is studied in this paper. A definition for the quantity called "polarization deviation" is proposed in order to identify clearer the difference between target cells and the background. Based on the polarization deviation detection method, a three-parameter polarization imaging method is employed to further simplify the image capture procedure for the proposed label-free cell detection. A color imaging methodology based on the well-known color space is utilized in order to represent the captured polarization information using computer graphics.

Enzyme-mediated dialdehyde formation: an alternative pathway for benzo[a]pyrene 7,8-dihydrodiol bioactivation.

Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are widespread environmental carcinogens of human concern. Several enzymatic systems have been shown to activate benzo[a]pyrene 7, 8-dihydrodiol, the proximate carcinogenic metabolite of benzo[a]pyrene, to a reactive species which produces both a chemiluminescence response and genotoxic lesions. The chemiluminescence response has been proposed to be the result of the formation of a dioxetane which upon ring opening forms a reactive dialdehyde intermediate. In in vitro incubations involving phorbol ester-stimulated human polymorphonuclear leukocytes or an isolated enzyme system consisting of myeloperoxidase, taurine, and hydrogen peroxide, a prolonged (>60 min) chemiluminescence response was observed from benzo[a]pyrene 7,8-dihydrodiol. HPLC analysis of the reaction mixture revealed the existence of a product which is dependent upon both taurine and the hydrocarbon. Characterization of this product using UV, NMR, and MS indicated that the product is a pyrene with two side chains resulting from bond breakage of a ring, yielding a dialdehyde. These side chains contain a portion of taurine covalently attached through imine formation with the aldehydes resulting from dioxetane ring opening. Replacement of taurine with either protein or DNA also produced a prolonged chemiluminescence response. These results demonstrate for the first time the formation of a novel electrophilic species from benzo[a]pyrene 7,8-dihydrodiol which along with an increased production of photons from this activation mechanism may lead to DNA and/or protein damage that is different from that elicited by diol epoxides.

Identification and characterization of chlorin e(4) ethyl ester in sera of individuals participating in the chlorophyllin chemoprevention trial.

Chlorophyllin (CHL), a mixture of water soluble derivatives of chlorophyll, has been shown to be an effective inhibitor of aflatoxin B(1) (AFB(1)) carcinogenesis and AFB(1)-DNA adduct formation in rainbow trout and rats [Breinholt, V., Hendricks, J., Pereira, C., Arbogast, D., and Bailey, G. (1995) Cancer Res. 55, 57-62; Kensler, T. W., Groopman, J. D., and Roebuck, B. D. (1998) Mutat. Res. 402, 165-172]. The chemopreventive action of CHL has been previously attributed to molecular complexing. In 1997, a randomized, double-blind clinical trial of CHL was conducted in Qidong, Jiangsu Province, People's Republic of China. At the completion of the study, when serum samples were regrouped by subject identification number, it was noted that many of the participant samples were green in color. Using HPLC, ESI/MS, and MS/MS techniques, serum samples from individuals receiving CHL were found to contain previously unreported copper chlorin e(4) ethyl ester (CuCle(4) ethyl ester) as well as copper chlorin e(4) (CuCle(4)). Both chlorins originated in the study tablet, were absorbed into the bloodstream, and conferred a green color to the sera. This initial finding of in vivo absorption and bioavailability of two chlorin derivatives suggests that the mechanism of CHL chemoprevention may lie in the actions of these two components in vivo in addition to preventing carcinogen absorption from the gut.

Responses of preschoolers in two frustrating episodes: emergence of complex strategies for emotion regulation.

Although research on young children's abilities to organize emotional states has increased in recent years, little is known about the emergence of complex strategies for emotion regulation in preschoolers. In the present study, emotion-regulation strategies used by 52 normally developing 3- and 4-year-olds were examined. Children and their primary caregivers (50 mothers, 2 fathers) participated in 2 controlled frustration episodes that were videotaped. Four types of strategies were coded: comforting behaviors, instrumental behaviors, distraction behaviors, and cognitive reappraisals. Results indicated that 3-year-olds used proportionately more instrumental strategies than 4-year-olds, and parents of 3-year-olds showed the same pattern, whereas parents of 4-year-olds did not. Moreover, 3-year-olds used a variety of strategies when frustrated, including cognitive reappraisals. Significant positive correlations were found between the types of strategies used by the children and by the parents to help their children. It is suggested that children may be using strategies to organize their emotional states before they are able to accurately report on them.

Individual differences in stress reactions during a peer entry episode: effects of age, temperament, approach behavior, and self-perceived peer competence.

The purpose of the current study was to determine whether a standardized peer entry paradigm would produce stress responses in 3- and 4-year-olds and how such stress responses would relate to temperament, observed approach to peers, and self-perceived peer competence. Physiological stress reactions were measured by activity of the hypothalamic-pituitary-adrenal (HPA) system. The 4-year-old group showed significantly less avoidance of the new peers and was rated higher on approach temperament. This older group also showed larger HPA stress responses to the new peer situation. Finally, discrepancy between self-reported peer competence and behavior in the peer entry situation was associated with larger stress responses on average. These findings support the notion that investment in a salient episode plays a role in determining the magnitude of HPA stress responses. The importance of examining discrepancies between self-perception and action in research on stress is discussed.

Biochemical characterization of lucigenin (Bis-N-methylacridinium) as a chemiluminescent probe for detecting intramitochondrial superoxide anion radical production.

Direct detection of intramitochondrial superoxide anion radical (O(-*)(2)) production is of critical importance for investigating the pathophysiological consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize the biochemical basis for lucigenin as a chemiluminescent probe to detect intramitochondrial O(-*)(2) production. Incubation of isolated mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial substrates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. With an ion-pair HPLC method, a concentration-dependent accumulation of lucigenin was detected within mitochondria. The accumulation of lucigenin by mitochondria was reduced markedly in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyhyldrazone, an uncoupler known to dissipate the mitochondrial membrane potential. With submitochondrial particles, we observed that both complexes I and III of the mitochondrial electron transport chain appear to be able to catalyze the one electron reduction of lucigenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylacridone, the proposed end product of the reaction pathway leading to LDCL, within the mitochondrial fraction was also detected. In addition, a significant linear correlation was observed between the LDCL and either the lucigenin accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly used LDCL can reliably detect intramitochondrial O(-*)(2) production.

Relations among child language skills, maternal socialization of emotion regulation, and child behavior problems.

Research has linked language delays in young children to behavior problems and risk for psychopathology. We hypothesized that low language skill would affect normal socialization of emotion regulation, which in turn would affect the development of behavior problems. Seventy-eight mother/preschool-age child pairs participated in two mildly frustrating situations. Parents of children with low verbal comprehension used more unexplained compliance demands than other parents. Further, children whose parents used more unexplained compliance demands used fewer cognitive and distraction strategies, and more instrumental strategies. Children's use of physical self-comforting was positively related to overall, internalizing, and externalizing behavior problems. Findings supported the original hypothesis.

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1.

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.

Familiar and novel contexts yield different associations between cortisol and behavior among 2-year-old children.

We examined 10:30 a.m. salivary cortisol levels in twenty-four 2-year-old children at home, and then at several points during transition into preschool: Week 1, Weeks 6-9, and the 1st week following a month-long holiday break. Cortisol levels did not increase when the children first started school as compared to either home or later school levels. Cortisol levels were correlated across similar, but not across dissimilar, psychosocial contexts. Home levels were correlated wit more shy, anxious, internalizing behavior while the response to starting school was correlated with more assertive, angry, and aggressive behavior. Behavior was assessed using parent temperament reports, teacher reports, and observational measures. We conclude that HPA activity as indexed by salivary cortisol measures is differentially associated with behavior in familiar and novel contexts. Consistent with our prior work, shy/anxious behavior is not significantly associated with elevations in cortisol when young children enter new social situations.

Temperament, social competence, and adrenocortical activity in preschoolers.

The relations among temperament, social competence, and levels of a stress-sensitive hormone (salivary cortisol) were examined in two studies of preschoolers children (Study 1, N = 29; Study 2, N = 46). In both studies, we sampled cortisol daily for the initial weeks of school year (Group Formation period) and for several weeks later in the year (Familiar Group period). For each child, we examined two measures of cortisol activity (separately for each period) based on the distribution of cortisol levels across days: (a) median cortisol (50th percentile) and (b) cortisol reactivity (the difference between the 75th and 50th percentile). Median cortisol was modestly stable across periods, but cortisol reactivity was not. Children who showed high cortisol reactivity (75th minus 50th percentile > or = 0.10 micrograms/dl) during the Group Formation period but low-to-normal cortisol reactivity during the Familiar Group period were outgoing, competent, and well liked by their peers. In contrast, children who changed from low/normal to high cortisol reactivity and those who maintained high cortisol reactivity from the Group Formation to Familiar Group period were affectively negative and solitary. Children who showed high median cortisol during the Familiar Group period or over both periods scored lower on a measure of attentional and inhibitory control. Together, these results suggest that relations among temperament, social competence, and neuroendocrine reactivity reflect both individual and contextual differences.

Mechanism of aralkyl-DNA adduct formation from benzo[a]pyrene in vivo.

Three different pathways have been proposed for the metabolic activation of the ubiquitous polycyclic aromatic hydrocarbon, benzo[a]pyrene (BP). The most widely accepted activation mechanism is based on ring oxidation to diol epoxides; the other two relatively less studied pathways involve radical cation formation and benzylic electrophilic ester formation arising from a chain of substitution reactions. The present study was undertaken to test for the existence of the latter mechanism in vivo. Female Sprague-Dawley weanling rats were injected subcutaneously with 320 mumol of BP/kg body weight, and the formation of DNA adducts was examined. 32P-Postlabeling analysis of the subcutaneous tissue DNA under newly developed chromatography conditions exhibited two sets of adduct profiles: one resulting from alkyl substitution and the other from ring oxidation. One major and two minor aralkyl-DNA adducts were detected. The relative adduct labeling (adducts/10(10) nucleotides) remained constant at around 2 during the first 5 days of treatment and then increased to 6.4 +/- 2.6 at day 7. The corresponding total values of the known ring oxidation (e.g., diol epoxide) adducts were 15-50 times higher. When animals were injected with 6-methyl-BP, 6-(hydroxymethyl)-BP, and 6-(acetoxymethyl)-BP, the known or proposed intermediates in the benzylic ester pathway, each of these and the parent compound showed chromatographically identical profiles of aralkyl-DNA adducts. Cochromatography in multiple solvents of these in vivo adducts with standards prepared by reaction of 6-(bromomethyl)-BP with individual nucleotides showed that the predominant in vivo aralkyl-DNA adduct was derived from guanine while the second major adduct was from adenine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocortical activity and emotion regulation.

Activity of the HPA system does appear to be related to emotion regulation processes in children. The conditions known to modulate HPA activity in animals, adults, and children correspond well to the behavioral strategies often discussed in the domain of emotion regulation. Individual differences in emotion processes related to negative emotion temperaments appear to be associated with individual differences in HPA reactivity among normally developing children, with both fearful, inhibited temperaments and distressed, angry temperaments being associated with greater HPA reactivity. Among children exhibiting behavior problems in the clinical range, however, it may be the "internalizing" patterns that are associated with greater HPA reactivity. The body of research concerning the psychobiology of the HPA system strongly suggests that associations between emotion regulation styles and HPA activity are not merely correlations, that they do indeed reflect potential causal connections. HPA activation and regulation has been shown in animals both to influence and to be influenced by emotions and their corresponding behavioral and psychological processes. Despite a reasonable body of research that now exists on children, many questions regarding the relations between HPA activity and emotion processes remain to be examined. In addressing these questions, it may be useful to consider several periods of the HPA response. Most of the work on children involves the interrelations between emotion and adrenocortical systems during the first 10-15 min of the stress response. This would include the initial activation of the system and the subsequent emotion and physiological processes involved in continuing or terminating the response. Little attention has been paid to more slowly developing effects of HPA activity on the central nervous system in children, particularly with regard to its influence on children's memories for stressful events and the emotion regulation strategies that they employed during the event. Studies of change or continuity of these interconnections over several exposures to a stressor, as well as between earlier and later points in the activation and regulation process, will be especially important to our understanding of the regulation of affective behavior.

The site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.

The major product of the S-adenosyl-L-methionine methyltransferase dependent substitution reaction (bioalkylation) of benzo[a]pyrene in preparations of rat liver cytosol was isolated by solvent extraction and high performance liquid chromatography and its structure determined by GC/MS and NMR. The results indicate that the methyl group is introduced at the center of highest chemical reactivity (or carbon atom at which it is easiest to localize pi electrons) in the anthracene nucleus, giving rise to 6-methylbenzo[a]pyrene. These data, unequivocally demonstrate the site of substitution of the methyl group in the bioalkylation of benzo[a]pyrene.

Beta-oxidized N-nitrosoalkylcarbamates as models for DNA alkylation by N-nitrosobis(2-oxopropyl)amine in Syrian hamsters.

A single dose of N-nitrosobis(2-oxopropyl)amine (NDOPA) can selectively induce pancreatic-duct adenocarcinomas in Syrian hamsters. Multiple doses or a higher single dose can induce tumours of the liver and other organs. Our earlier studies employing NDOPA systematically labelled with 14C in the three-carbon chain showed that hamster pancreatic DNA is almost exclusively methylated and that the sole source of the methyl group is the alpha carbon of NDOPA. Hamster liver DNA was equally methylated and alkylated by a three-carbon chain. Current studies using generally labelled tritiated NDOPA with a very high specific activity have shown that the three-carbon alkylation is 2-hydroxypropylation. We have identified two adducts isolated from hamster liver DNA, N7-(2-hydroxypropyl)-guanine and O6-(2-hydroxypropyl)guanine, which contain this group, and we have also isolated and identified N7-methylguanine and O6-methylguanine in DNA from hamster liver and pancreas. beta-Oxidized N-nitrosocarbamates, ethyl N-nitroso-2-oxopropylcarbamate (NOPC) and ethyl N-nitroso-2-hydroxypropylcarbamate (NHPC), are useful models for predicting the DNA adducts observed in vivo following NDOPA treatment. Base-catalysed decomposition of NOPC in the presence of exogenous DNA yields five methylated purines (N3-, N7- and O6-methylguanines and N1- and N3-methyladenines). NHPC, a model for N-nitrosamines containing the 2-hydroxypropyl group, reacts with guanosine to yield N7- and O6-(2-hydroxypropyl)guanines.(ABSTRACT TRUNCATED AT 250 WORDS)

S-Adenosyl-L-methionine is a carbon donor in the conversion of benzo[alpha]pyrene to 6-hydroxymethylbenzo[alpha]pyrene by rat liver S-9.

In earlier studies we reported that 6-hydroxymethylbenzo[alpha]pyrene is a metabolite of benzo[alpha]pyrene and of 6-methylbenzo[alpha]pyrene when these substrates are incubated with homogenates or microsomal preparations of liver and microsomal preparations of subcutaneous tissue from Sprague--Dawley rats. However, the origin of the carbon donor was not established. We now report that S-adenosyl-L-methionine (SAM) can serve as a carbon donor in the metabolic formation of 6-hydroxymethylbenzo[alpha]pyrene from benzo[alpha]pyrene by the S-9 fraction of rat liver.