RECEIVED DATETITLE RUNNING HEAD: QMR representation of asphaltenes and MD simulation of aggregation. CORRESPONDING AUTHOR FOOTNOTE: EBoek@slb.com; Tel +44 1223 325222 2 ABSTRACT We have developed a computer algorithm to generate Quantitative Molecular Representations (QMR) of asphaltenes based on experimental data. First, we generate molecular representations using a Monte Carlo method. For this purpose, we use an extensive set of aromatic and aliphatic building blocks, which are sampled randomly from the corresponding distribution and then linked together using a connection algorithm. The building blocks can be taken from a pre-defined inventory or generated during run-time.Manually pre-fabricated blocks ensure model flexibility while automatically generated blocks allow us to build large aromatic sheets. We allow for both archipelago and peri-condensed structures to be generated. Then, we use a non-linear optimisation procedure to select a small subset of molecules that gives the best match with experimental data. These experimental data consist of Molecular Weight (MW), elemental analysis and NMR spectroscopy, including both 1 H and 13 C data. First, we validate the method by testing a number of single model compounds. Then we use a real asphaltene data set available in the literature. Different values of the MW were used as input parameter. We tested two specific values of the MW in detail, representing the peri-condensed and archipelago structure respectively: MW= 750 and MW = 4190. For both MWs, we generated 10 sets of 5000 samples each.The samples were then optimized with respect to the experimental objective function. Then we calculate the value of the objective function as an average over all the simulation runs. It turns out that the value of the objective function is significantly smaller for MW=750 than for MW=4190. This means that the lower Molecular Weight of 750 provides the best match with the experimental data. As an example, one of the optimised QMR asphaltene structures generated was then used as input in Molecular Dynamics (MD) simulations to study the formation of nano-aggregates.

Simulations of mixed cationic/anionic wormlike micellar systems have been carried out for a wide range of compositions, including pure anionic and cationic systems. It was found that the wormlike micelle formed by only cationic surfactant molecules is unstable and transforms to a set of small spherical micelles. Adding anionic surfactants with a short hydrophobic chain (only eight carbon atoms) results in stable wormlike micelles. The 34/66 cationic/anionic worm is stable and symmetrical, while the 50/50 mixture yields a flattened worm, indicating a phase transition to the lamellar phase. All these observations are in excellent agreement with the experimental results of Raghavan et al. (Langmuir 2002, 18, 3797), and they provide a molecular mechanism for their observations. The addition of octyltrimethylammonium chloride increases the radius of the worm due to the bigger hydrophobic part. Meanwhile, the length of the worms decreases with the concentration of cationic surfactant and reaches a minimum for the 50/50 mixture. The latter system is of special interest due to a zero surface charge density. The worm with the electrostatically neutral surface was used to investigate intermicellar interactions. The molecular dynamics (MD) simulations show that the merging process requires a substantial activation energy even in the case of reduced electrostatic repulsion.

ABSTRACT:A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.The superfamily of cytochrome(s) P450 (P450) plays a key role in hepatic and extrahepatic drug metabolism, and qualitative and quantitative analyses of the P450 isozyme expression in a particular organ are critical in predicting a metabolic fate of a particular drug or in examination of the potential drug-drug interaction. The number of sequenced and named different P450 isozymes surpassed 3100 (dnelson.utmem.edu/CytochromeP450.html), and the degree of sequence homology, particularly among P450s belonging to the same subfamily, is high (Nelson et al., 1996). None of the existing research approaches to the analysis of individual P450 forms, which include specific P450 inhibitors (Halpert et al., 1994;Kobayashi et al., 2003) or substrates (Kobayashi et al., 2002;Stresser et al., 2002), antibodybased identification (Gelboin et al., 1999;Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999;Zhang et al., 1999), is in a position to provide reliable quantitative and qualitative information on the individual P450 composition in a given type of microsomes. First, only a minority of known P450 isozymes is fully characterized by substrate specificity, and since they exhibit a broad, often overlapping substrate specificity, there is no known substrate or inhibitor that is absolutely specific for an individual P450 isozyme. Second, the high degree of sequence homology among members of the P450 superfamily confounds high specificity of antibody-based analysis, particularly among members of the same subfamily. Third, the application of a quantitative mRNA analysis for the evaluation of P450 isozyme expression...

ABSTRACT:The main targets of our investigation were cytochrome P450 isozymes (P450), the key enzymes of the hepatic drug-metabolizing system. Current research approaches to the identification of individual P450 forms include specific P450 inhibitors or substrates, antibody-based identification, and mRNA-based expression profiling. All of these approaches suffer from one common disadvantage-they all are indirect methods. On the other hand, current developments in mass spectrometry provide a direct and reliable approach to protein identification with sensitivity in the femtomole or low picomole range. In this study we have used high-accuracy, matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-based peptide mapping to perform direct identification of distinct P450 isozymes in various rat and rabbit liver microsomes. For the first time, the P450 isozyme composition of clofibrate-induced rat and phenobarbital-induced rabbit liver microsomes was determined by peptide mass fingerprinting (PMF). Application of MALDI TOF-based PMF allows differential identification of such highly homologous P450s as CYP2B1 and CYP2B2. We have found that CYP2A10 previously reported only in rabbit olfactory and respiratory nasal mucosa is present in phenobarbital (PB)-induced rabbit liver microsomes. Two other rabbit P450s, earlier identified only by screening a cDNA library, were found to be present in PB-induced rabbit liver microsomes. In summary, direct identification of P450s by proteomic technique offers advantages over other methods with regard to identification of distinct P450 isozymes and should become a standard approach for characterizing microsomes.Cytochrome P450 isozymes (P450) are the key enzymes of the hepatic drug-metabolizing system. Eukaryotic P450s are membrane proteins that are expressed in varying amounts, and many forms differ very little in their amino acid sequence and catalytic properties. Currently the number of sequenced and named distinct P450s exceeds 1925 (dnelson. utmem.edu/CytochromeP450.html). Since individual P450 isozymes exhibit a broad, often overlapping substrate specificity, knowledge of the P450 composition in a particular type of microsomes is critical in predicting drug/substrate interactions and formation of reactive intermediates. Current research approaches to the identification of individual P450 forms include: specific P450 inhibitors or substrates (Halpert et al., 1994;Kobayashi et al., 2002), antibody-based identification (Shou et al., 2000), and mRNA-based analysis (Chow et al., 1999). However, only a limited number of almost 2000 P450s is characterized in terms of substrate specificity and has available antibodies, not to mention the fact that, to the best of our knowledge, there is no known substrate or inhibitor that is absolutely specific for only one P450 isozyme. As for antibody-based techniques, the main problem is a very high degree of sequence homology characteristic for P450 isozymes. For example, neither polyclonal nor monoclonal antibodies can distinguish C...

We describe results of the ongoing development of a computer algorithm to optimize asphaltene molecular weight (MW) by systematically deriving quantitative molecular representations (QMRs) for asphaltenes and a comparison to experimental data. The QMR method consists of the generation and optimization of molecular structures based on experimental data, including elemental analysis, nuclear magnetic resonance, and MW. In this case, we use the MW as a variable in the problem, keeping all other experimental variables constant, and systematically investigate the effect of an increasing MW on the quality of the model optimization, as measured by the penalty or objective function. In this fashion, we find an optimal value for the MW using the QMR algorithm. We observed a significant increase in the objective function when optimizing sets of increasing target MW. We then analyzed the individual contributions of the different properties of the optimized sets toward the objective function and tested a number of possible alterations to the sampling parameters in an attempt to reduce the value of the objective function. It was found that constructing a single set of molecules, prior to optimization, from smaller subsets that are generated using a variety of sampling parameters could yield a great reduction in the value of the objective function following optimization. The resulting asphaltene molecular structures may be used as input for molecular dynamics simulations.

G protein-coupled trace amine-associated receptor 1 (TAAR1) is expressed in several brain regions and modulates dopaminergic activity partially by affecting D2 dopamine receptor function. In vitro, the nonselective dopamine agonist apomorphine can activate mouse and rat TAAR1. The aim of the present study was to evaluate whether apomorphine activity at the rodent TAAR1 observed in in vitro studies contributes to its behavioral manifestation in mice. For this purpose, we compared the behavioral effects of a wide range of apomorphine doses in wild type (WT) and TAAR1 knockout (TAAR1-KO) mice. Apomorphine-induced locomotor responses (0.01-4.0 mg/kg) were tested in locomotor activity boxes, and stereotypic behavior at 5 mg/kg was tested by ethological methods. A gnawing test was used to analyze the effects of the highest dose of apomorphine (10 mg/kg). No statistically significant differences were observed between TAAR1-KO and WT mice following inhibitory pre-synaptic low doses of apomorphine. At higher doses (2.0-5.0 mg/kg), apomorphine-induced climbing behavior was significantly reduced in TAAR1 mutants relative to WT controls. Moreover, the lack of TAAR1 receptors decreased certain types of stereotypies (as reflected in by measures of the global stereotypy score, licking but not sniffing or gnawing) that were induced by high doses of apomorphine. These data indicate that apomorphine activity at TAAR1 contributes to some behavioral manifestations, particularly climbing, in rodents following high doses of this drug. The contribution of TAAR1 to apomorphine-induced climbing in rodents should be considered when apomorphine is used as a screening tool in the search for potential antipsychotics.

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