Carboxylesterase catalyzed 18O-labeling of carboxylic acid and its potential application in LC-MS/MS based quantification of drug metabolites

LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel ¹⁸O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H₂¹⁸O in the presence of the enzyme, generating singly- and doubly-¹⁸O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds – indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery.     

Melatonin Receptors on Ovine Pars Tuberalis: Characterization and Autoradiographicai Localization

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand [¹²⁵l]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction.     

Which constructs can predict emotional exhaustion in a working population? A study into its determinants

The main objective of this study was to examine the psychosocial stress model developed by Taylor and Aspinwall with emotional exhaustion as the outcome variable. Respondents, 409 men and 346 women, who had a paid job for at least 20 hours per week, completed questionnaires concerning demographic variables, personality, temperament, work pressure, workload, perceived social support, appraisal, coping, and emotional exhaustion. Structural equation analyses provided only partial support for the validity of the model. First, on theoretical and statistical grounds, one more path linking external resources to social support was added. Second, contrary to expectations, coping styles did not predict emotional exhaustion. To conclude, when coping is measured retrospectively, it does not add to our understanding of emotional exhaustion. It is suggested that future studies should be longitudinal and include objective measures of stressors and psychosocial health outcomes in addition to self‐reports. Copyright © 2007 John Wiley & Sons, Ltd.     

Aza-peptides. III. Experimental structural analysis of aza-alanme and aza-asparagine-containing peptides

To determine the structural perturbations induced by the CαH→Nα exchange in aza-peptides, we have examined by H NMR and IR spectroscopy various derivatives of the aza-analogues of alanine, aspartic acid and asparagine in different organic solvents with increasing polarity. Their general formulas are: R'-AzXaa-NR²R³, R'-Pro-AzXaa-NR²R³ and R-AzXaa-Pro-NR²R³ (where AzXaa denotes the aza-analogue of the amino acid residue Xaa = Ala, Asp, Asn; R = Boc, Z; R², R³= H, Me, iPr). The aza-analogue of an amino acid residue appears to be a strong p-turn-inducing motif, and the AzAsn carboxamide side-chain is capable of interacting, as a proton donor, with the preceding peptide carbonyl group.     

Precise and accurate measurement of proton T1 in human brain in vivo: Validation and preliminary clinical application

Precise and accurate inversion-recovery (PAIR) magnetic resonance (MR) measurements of T1 were obtained in eight brain regions and cerebrospinal fluid of 26 healthy volunteers. Accuracy of the technique was assessed by measuring T1 in small fluid volumes with the PAIR technique and with two independent spectroscopic techniques. The mean difference between T1 measured with PAIR and with the two spectroscopic techniques was 3.1% ± 1.3. The precision (reproducibility) of measurements with the PAIR technique was excellent. The coefficient of variation (CV) across 16 measurements in a head phantom was 2.0%, compared with a CV of 2.7% across 45 separate measurements in a single subject. The within-subject CV was 1.8% ± 0.6 in white matter and 1.4% ± 1.0 in basal ganglia. The between-subject CV in 26 healthy volunteers was 3.6% ± 0.6 in white matter and 4.1% ± 1.9 in basal ganglia. Comparison between a patient with an active recurrent brain tumor and an agematched patient with an inactive brain tumor showed that T1 was significantly elevated throughout the brain of the active-tumor patient, especially in white matter tracts, even though no tumor or edema was detected in the white matter on standard MR images. Comparisons between five brain tumor patients and four healthy volunteers of similar age showed that T1 was significantly and substantially elevated throughout the white matter tracts and in the caudate nucleus, putamen, and thalamus. These results are consistent with the hypothesis that white matter tracts are selectively vulnerable to edema and that T1 increases in white matter are a sensitive indicator of patient status or tumor aggressiveness.     

Time after excision and temperature alter ex vivo tissue relaxation time measurements

Previously unreported effects of tissue storage were recently observed in the authors' experimental magnetic resonance (MR) studies. To evaluate the effect of elapsed time after excision and storage temperature on tissue relaxation time measurements, tissue samples from the liver, pancreas, kidney, testis, spleen, and brain were obtained in rats. T1 and T2 were first measured within 5 minutes of excision, and between subsequent measurements, tubes were kept in a water bath at 40°C, at room temperature (28°C), or in an ice bath (4°C). Cellular and organellar integrity was assessed with electron microscopy and correlated with the MR findings. At 40°C (20-MHz spectrometer), the T1 of liver decreased from 280 msec ± 8 to 212 msec ± 10 during the first 60 minutes; the T1 of pancreas decreased from 276 msec ± 3 to 208 msec ± 2. Other tissues showed less than a 5% decrease in T1. T2 changes were smaller than T1 changes in all tissues. Electron microscopy of pancreatic acinar cells showed postmortem changes in mitochondria evolving over the first 60 minutes after death. Manganese loading experiments implicated mitochondrial manganese stores in the observed enhanced postmortem decrease in T1. This study calls into question reported relaxation time data for liver and pancreas. MR studies of excised tissues must account for time and temperature to prevent systematic experimental errors.     

重油和沥青中饱和碳的定量估计

建立了较完整的估计重油和沥青中饱和碳浓度数方法，其中包括环烷桥头碳、环烷甲基取代碳、环烷烷基（≥Ｃ２）取代碳等。基于理论分析，建立了估计环间的桥链和各种平均结构参数的方法，包括平均芳环环核数和环烷环环核数、芳环和环烷环烷工取代度、单元片上芳环和环烷环数。从实验数据出发，提出了烷基链长分布的公式。     

轻稀土与联吡啶和菲罗啉三元配合物的合成与表征

合成了稀土（Ｌｎ）与２，２－联吡啶（Ｌ１）、１，１０－菲罗啉（Ｌ２）的三元固态配合物。通过元素分析、ＩＲ谱、ＴＧＡ谱和摩尔电导等对该系列配合物进行了表征，实测结果与通式ＬｎＬ１Ｌ２Ｃｌ３·３Ｈ２Ｏ符合较好。抗菌试验表明，该系列配合物有较强的广谱抗菌作用。     

Identification of functional GRP-preferring bombesin receptors on human melanoma cells

Bombesin was originally isolated from amphibian skin, wherease its mammalian counterpart, gastrin-releasing peptide (GRP), was first identified in the nervous system of the gastrointestinal tract. Whether GRP is present in the human skin is not known. Bombesin-like peptides are also known to modulate growth. We therefore investigated whether human melanoma cell lines express functional GRP-preferring bombesin receptors and whether they alter growth or other specific cellular functions of these tumour cells. GRP receptor mRNA was found in HBL, D-10, Me-28 and A375-6 cell lines, but only A375-6 cells express a large number of high-affinity binding sites for [¹²⁵I]-[Tyr⁴] bombesin ( K _d 0.31 ± 0.04 nmol L⁻¹, 3880 ± 429 binding sites per cell). Bombesin dose-dependently increased cytosolic calcium, but did not alter interleukin (IL) 1β-induced reduction of cell viability or IL-6 secretion, both A375-6-specific cell functions. Growth of A375-6 cells was not altered by bombesin or the specific GRP receptor antagonist BIM26226 as measured by [³H]-thymidine incorporation or methylene blue assay, whereas insulin alone or in combination with other potential growth factors dose-dependently stimulated growth of these cells. The newly characterized GRP-preferring bombesin receptors on highly malignant human melanoma cells could initiate studies of growth effects on solid tumours or in vivo scanning using radiolabelled tracers.