Continued discovery of ligands for G protein-coupled receptors

G protein-coupled receptors are under intense scrutiny as potential targets of drug research, which stems mostly from the sheer size and diversity of this receptor family as well as the recognized high levels of specificity and sensitivity attainable by drugs targeting these receptors. The continued discovery of genes encoding G protein-coupled receptors has provided an extensive reserve of potential therapeutic targets. However, testing experimental therapeutic agents at these receptors requires a high degree of receptor characterization, beginning with the identity of an endogenous ligand. Often, low levels of sequence identity of a newly identified receptor to previously characterized receptors preclude the prompt identification of a ligand. In such cases, innovative techniques commonly referred to as reverse pharmacology have been employed to ascertain the ligand's identity for these "orphan" receptors. To date over 30 endogenous ligands, both novel and previously known, have been paired with orphan G protein-coupled receptors. Here, we briefly summarize the recent identification of neuropeptides W and B and carboxylic acid anions for their respective receptors GPR7, GPR8 and GPR40, GPR41, GPR43.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Effects of elevated carbon dioxide and ozone on the phytochemistry of aspen and performance of an herbivore

The purpose of this study was to assess the independent and interactive effects of CO(2), O(3), and plant genotype on the foliar quality of a deciduous tree and the performance of a herbivorous insect. Two trembling aspen (Populus tremuloides Michaux) genotypes differing in response to CO(2) and O(3) were grown at the Aspen FACE (Free Air CO(2) Enrichment) site located in northern Wisconsin, USA. Trees were exposed to one of four atmospheric treatments: ambient air (control), elevated carbon dioxide (+CO(2); 560 microl/l), elevated ozone (+O(3); ambient x1.5), and elevated CO(2)+O(3). We measured the effects of CO(2) and O(3) on aspen phytochemistry and on performance of forest tent caterpillar (Malacosoma disstria Hübner) larvae. CO(2) and O(3) treatments influenced foliar quality for both genotypes, with the most notable effects being that elevated CO(2) reduced nitrogen and increased tremulacin levels, whereas elevated O(3) increased early season nitrogen and reduced tremulacin levels, relative to controls. With respect to insects, the +CO(2) treatment had little or no effect on larval performance. Larval performance improved in the +O(3) treatment, but this response was negated by the addition of elevated CO(2) (i.e., +CO(2)+O(3) treatment). We conclude that tent caterpillars will have the greatest impact on aspen under current CO(2) and high O(3) levels, due to increases in insect performance and decreases in tree growth, whereas tent caterpillars will have the least impact on aspen under high CO(2) and low O(3) levels, due to moderate changes in insect performance and increases in tree growth.     

Identification of an allele of CLA1 associated with variegation in Arabidopsis thaliana

We have identified a mutant of Arabidopsis thaliana ( lvr111 ) that exhibits a variegated phenotype, reduced isoprenoid pigmentation, and dwarfism in comparison with wild-type plants. Segregation analysis indicated that this phenotype was caused by a single, semi-dominant mutation and PCR-based marker mapping placed the mutation near position 56 on the RI map of chromosome IV. The lvr111 lesion was identified by genomic PCR and sequence analysis as a missense mutation (D306N) in the CLA1 gene (AT4g15560) and complementation analysis confirmed the allelic relationship between lvr111 and CLA1 . CLA1 encodes 1-deoxy- d -xylulose 5-phosphate synthase, which catalyses the first step of the non-mevalonate isoprenoid biosynthetic pathway. These observations demonstrate that, unlike the albinism caused by severe alleles of CLA1 , weaker alleles are associated with leaf variegation.     

Fine needle aspiration biopsy of gastric duplication cysts with endoscopic ultrasound guidance

OBJECTIVE: To evaluate the ability of endoscopic ultrasound (EUS)-guided fine needle aspiration biopsy (FNAB) to diagnose gastric duplication cysts. STUDY DESIGN: FNAB reports from the Department of Pathology, Saint Louis University Hospital, were retrospectively searched for reports of EUS-guided FNABs of the stomach. These reports were then reviewed to find instances in which gastric duplication cysts were diagnosed. The charts of patients diagnosed with gastric duplication cysts were reviewed. RESULTS: Two patients were identified. The first was a 35-year-old, Caucasian male with an asymptomatic submucosal (versus extrinsic) gastric mass discovered during computed tomography of the abdomen. The second was a 44-year-old, Caucasian male with a history of treated low grade B cell lymphoma of mucosa-associated lymphoid tissue (MALToma) who was found to have an asymptomatic gastric wall abnormality by EUS when undergoing follow-up for the MALToma. Respiratory-type epithelial cells were present in each of these gastric duplication cysts. At this writing, both patients were being followed clinically and with imaging. CONCLUSION: Gastric duplication cysts, particularly those that have respiratory-type epithelium, can be diagnosed by EUS-guided FNAB. The diagnosis of gastric duplication cysts by EUS-guided FNAB can preclude surgery, with its associated morbidity.     

Pooled analysis of loss of heterozygosity in breast cancer: a genome scan provides comparative evidence for multiple tumor suppressors and identifies novel candidate regions

Somatic loss of heterozygosity (LOH) has been widely reported in breast cancer as a means of identifying putative tumor-suppressor genes. However, individual studies have rarely spanned more than a single chromosome, and the varying criteria used to declare LOH complicate efforts to formally differentiate regions of consistent versus sporadic (random) loss. We report here the compilation of an extensive database from 151 published LOH studies of breast cancer, with summary data from >15,000 tumors and primary allelotypes from >4,300 tumors. Allelic loss was evaluated at 1,168 marker loci, with large variation in the density of informative observations across the genome. Using studies in which primary allelotype information was available, we employed a likelihood-based approach with a formal chromosomal instability and selection model. The approach seeks direct evidence for preferential loss at each locus compared with nearby loci, accounts for heterogeneity across studies, and enables the direct comparison of candidate regions across the genome. Striking preferential loss was observed (in descending order of significance) in specific regions of chromosomes 7q, 16q, 13q, 17p, 8p, 21q, 3p, 18q, 2q, and 19p, as well as other regions, in many cases coinciding with previously identified candidate genes or known fragile sites. Many of these observations were not possible from any single LOH study, and our results suggest that many previously reported LOH results are not systematic or reproducible. Our approach provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.     

Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.     

Do capuchin monkeys, Cebus apella, know what conspecifics do and do not see?

Capuchin monkeys were tested in five experiments in which two individuals competed over food. When given a choice between retrieving a piece of food that was visible or hidden from the dominant, subordinate animals preferred to retrieve hidden food. This preference is consistent with the hypotheses that either (1) the subordinate knew what the dominant could and could not see or (2) the subordinate was monitoring the behaviour of the dominant and avoiding the piece of food that it approached. To test between these alternatives, we released subordinates with a slight head start forcing them to make their choice (between a piece of food hidden or visible to the dominant) before the dominant entered the area. Unlike chimpanzees, Pan troglodytes, subordinates that were given a head start did not preferentially approach hidden pieces of food first. Therefore, our experiments provide little support for the hypothesis that capuchin monkeys are sensitive to what another individual does or does not see. We compare our results with those obtained with chimpanzees in the same paradigm and discuss the evolution of primate social cognition. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.      

Purification and characterization of T cell protein tyrosine phosphatase reveals significant functional homology to protein tyrosine phosphatase-1B

We have developed a protocol for rapid purification of T cell protein tyrosine phosphatase (TCPTP) and the structurally related protein tyrosine phosphatase-1B (PTP-1B) from bacterial cells. The pH profile for TCPTP was bell-shaped with an optimum of 5.5. The catalytic domain and full-length versions of TCPTP bound a potent inhibitor with affinities similar to those of PTP-1B. The K(m) values for the catalytic domains of TCPTP and PTP-1B increased with increasing ionic strength, whereas the k(cat) values remained unchanged. Arrhenius plots revealed that TCPTP and PTP-1B possess similar activation energies of 25.3+/-1.2 and 18.4+/-3.0 kJ/mol, respectively. Increasing solvent microviscosity (up to 40% (w/v) sucrose) did not affect k(cat)/K(m) of either enzyme. However, high sucrose concentrations protected both enzymes from thermal inactivation. These studies show that, although they share a 72% amino acid sequence identity within their catalytic domains, TCPTP and PTP-1B are functionally very similar in vitro.     

Oxidative damage to ferritin by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a alpha-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1-10mM ALA produced dose-dependent decreases in tryptophan fluorescence (11-35% after 5h), and a partial depletion of protein thiols (18% after 24h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.