Renal cell carcinoma (RCC) accounts for 11,000 deaths per year in the United States. When detected early, generally serendipitously by imaging conducted for other reasons, long term survival is generally excellent. When detected with symptoms, prognosis is poor. Under these circumstances, a screening biomarker has the potential for substantial public health benefit. The purpose of this study was to evaluate the utility of urine metabolomics analysis for metabolomic profiling, identification of biomarkers, and ultimately for devising a urine screening test for RCC. Fifty urine samples were obtained from RCC and control patients from two institutions, and in a separate study, urine samples were taken from 13 normal individuals. Hydrophilic interaction chromatography-mass spectrometry was performed to identify small molecule metabolites present in each sample. Cluster analysis, principal components analysis, linear discriminant analysis, differential analysis, and variance component analysis were used to analyze the data. Previous work is extended to confirm the effectiveness of urine metabolomics analysis using a larger and more diverse patient cohort. It is now shown that the utility of this technique is dependent on the site of urine collection and that there exist substantial sources of variation of the urinary metabolomic profile, although group variation is sufficient to yield viable biomarkers. Surprisingly there is a small degree of variation in the urinary metabolomic profile in normal patients due to time since the last meal, and there is little difference in the urinary metabolomic profile in a cohort of pre-and postnephrectomy (partial or radical) renal cell carcinoma patients, suggesting that metabolic changes associated with RCC persist after removal of the primary tumor. After further investigations relating to the discovery and identity of individual biomarkers and attenuation of residual sources of variation, our work shows that urine metabolomics analysis has potential to lead to a diagnostic assay for RCC. The study of all endogenously produced metabolites, known as metabolomics (or metabonomics), is the youngest of the omics sciences. It is becoming increasingly clear that, of all of the omics techniques, metabolomics has the greatest potential for biomarker discovery because this technique defines the signature of the actual processes that are occurring within the body rather than examining compounds (such as untranscribed DNA or pre-or post-translationally modified proteins) that may be superfluous to these processes (1). In addition, there is a relatively small number of metabolites to examine (with the notable exception of plants, which produce a plethora of secondary metabolites) as compared with genes, transcripts, and proteins in their respective omics fields, and therefore the data germane to metabolomics are more easily handled and analyzed. Proponents of metabolomics provide convincing justification that this technique offers more immediate translational benefit than the other omics fi...

The human sinoatrial node (SAN) efficiently maintains heart rhythm even under adverse conditions. However, the specific mechanisms involved in the human SAN’s ability to prevent rhythm failure, also referred to as its robustness, are unknown. Challenges exist because the three-dimensional (3D) intramural structure of the human SAN differs from well-studied animal models, and clinical electrode recordings are limited to only surface atrial activation. Hence, to innovate the translational study of human SAN structural and functional robustness, we integrated intramural optical mapping, 3D histology reconstruction, and molecular mapping of the ex vivo human heart. When challenged with adenosine or atrial pacing, redundant intranodal pacemakers within the human SAN maintained automaticity and delivered electrical impulses to the atria through sinoatrial conduction pathways (SACPs), thereby ensuring a fail-safe mechanism for robust maintenance of sinus rhythm. During adenosine perturbation, the primary central SAN pacemaker was suppressed, whereas previously inactive superior or inferior intra-nodal pacemakers took over automaticity maintenance. Sinus rhythm was also rescued by activation of another SACP when the preferential SACP was suppressed, suggesting two independent fail-safe mechanisms for automaticity and conduction. The fail-safe mechanism in response to adenosine challenge is orchestrated by heterogeneous differences in adenosine A1 receptors and downstream GIRK4 channel protein expressions across the SAN complex. Only failure of all pacemakers and/or SACPs resulted in SAN arrest or conduction block. Our results unmasked reserve mechanisms that protect the human SAN pacemaker and conduction complex from rhythm failure, which may contribute to treatment of SAN arrhythmias.

To investigate whether functional polymorphisms exist in the C-reactive protein (CRP) gene, i.e., ones that contribute directly to differences in baseline CRP among individuals, we sequenced a 1,156-nucleotide-long stretch of the CRP gene promoter in 287 ostensibly healthy people. We identified two single-nucleotide polymorphisms (SNPs), a bi-allelic one at nucleotide -409 (G-->A), and a tri-allelic one at -390 (C-->T-->A), both resident within the hexameric core of transcription factor binding E-box elements. Electrophoretic mobility shift assays confirmed that the SNP within the sequence (-412)CACGTG(-407) (E-box 1) modulates transcription factor binding, and that the one within (-394)CACTTG(-389) (E-box 2) supports transcription factor binding only when the -390 T allele is present. The commonest of four E-box 1/E-box 2 haplotypes (-409G/-390T) identified in the population supported highest promoter activity in luciferase reporter assays, and the rarest one (-409A/-390T) supported the least. Importantly, serum CRP in people with these haplotypes reproduced this rank order, i.e., people with the -409G/-390T haplotype had the highest baseline serum CRP (mean +/- SEM 10.9 +/- 2.25 microg/ml) and people with the -409A/-390T haplotype had the lowest (5.01 +/- 1.56 microg/ml). Furthermore, haplotype-associated differences in baseline CRP were not due to differences in age, sex, or race, and were still apparent in people with no history of smoking. At least two other SNPs in the CRP promoter lie within E-box elements (-198 C-->T, E-box 4, and -861 T-->C, E-box 3), indicating that not only is the quality of E-box sites in CRP a major determinant of baseline CRP level, but also that the number of E-boxes may be important. These data confirm that the CRP promoter does encode functional polymorphisms, which should be considered when baseline CRP is being used as an indicator of clinical outcome. Ultimately, development of genetic tests to screen for CRP expression variants could allow categorization of healthy people into groups at high versus low future risk of inflammatory disease.

␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA C ) is a ligand-gated ion channel with pharmacological properties distinct from the GABA A receptor. To date, only three binding domains in the recombinant 1 GABA C receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the 1 GABA C receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA C receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a ␤-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the 1 GABA C receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA C receptor binding pocket was proposed and discussed in comparison with the GABA A receptor binding pocket. GABA A1 and GABA C receptors are both GABA-gated chloride channels but with very different pharmacological properties. In fact, that is the basis for their classification. Although both types of receptors can be activated by GABA, the GABA A receptor can be specifically antagonized by bicuculline. In contrast, the GABA C receptor is insensitive to bicuculline but can be selectively antagonized by 1,2,5,6-tetrahydropyridine-4-yl)-methylphosphinic acid (1). The functional properties of these two types of GABA receptors are also different. For example, the GABA C receptor has higher GABA sensitivity but slower activation and deactivation kinetics than the GABA A receptor (2). The GABA C receptor also shows almost no desensitization (2), whereas the GABA A receptor exhibits strong desensitization (3). Although the functional differences could arise from differences in the structural architecture of the GABA binding pockets, distinct antagonist profiles of GABA A and GABA C receptors indicate that their agonist/antagonist binding pockets are not the same. Studies over the last 15 years have shaped a relatively complete model for the GABA A receptor (4 -9). In contrast, structural information on the GABA C receptor agonist binding pocket is far from complete. Some candidate binding residues in several potential "binding loops" are still undefined.GABA-gated ion channels belong to the ligand-gated ion channel family, which also includes nicotinic acetylcholine receptors, serotonin-3 receptors, and glycine receptors. The study of nicotinic receptors in the past two decades h...

The evolutionary relationships among arthropod hemocyanins and insect hexamerins were investigated. A multiple sequence alignment of 12 hemocyanin and 31 hexamerin subunits was constructed and used for studying sequence conservation and protein phylogeny. Although hexamerins and hemocyanins belong to a highly divergent protein superfamily and only 18 amino acid positions are identical in all the sequences, the core structures of the three protein domains are well conserved. Under the assumption of maximum parsimony, a phylogenetic tree was obtained that matches perfectly the assumed phylogeny of the insect orders. An interesting common clade of the hymenopteran and coleopteran hexamerins was observed. In most insect orders, several paralogous hexamerin subclasses were identified that diversified after the splitting of the major insect orders. The dipteran arylphorin/LSP-1-like hexamerins were subject to closer examination, demonstrating hexamerin gene amplification and gene loss in the brachyceran Diptera. The hexamerin receptors, which belong to the hexamerin/hemocyanin superfamily, diverged early in insect evolution, before the radiation of the winged insects. After the elimination of some rapidly or slowly evolving sequences, a linearized phylogenetic tree of the hexamerins was constructed under the assumption of a molecular clock. The inferred time scale of hexamerin evolution, which dates back to the Carboniferous, agrees with the available paleontological data and reveals some previously unknown divergence times among and within the insect orders.