Search

The genetic complexity, clinical variability, and inaccessibility of affected tissue in neurodegenerative and neuropsychiatric disorders have largely prevented the development of effective disease-modifying therapeutics. A precision medicine approach that integrates genomics, deep clinical phenotyping, and patient stem cell models may facilitate identification of underlying biological drivers and targeted drug development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

FlyFactorSurvey (http://pgfe.umassmed.edu/TFDBS/) is a database of DNA binding specificities for Drosophila transcription factors (TFs) primarily determined using the bacterial one-hybrid system. The database provides community access to over 400 recognition motifs and position weight matrices for over 200 TFs, including many unpublished motifs. Search tools and flat file downloads are provided to retrieve binding site information (as sequences, matrices and sequence logos) for individual TFs, groups of TFs or for all TFs with characterized binding specificities. Linked analysis tools allow users to identify motifs within our database that share similarity to a query matrix or to view the distribution of occurrences of an individual motif throughout the Drosophila genome. Together, this database and its associated tools provide computational and experimental biologists with resources to predict interactions between Drosophila TFs and target cis-regulatory sequences.

Background: Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) or ChIP followed by genome tiling array analysis (ChIP-chip) have become standard technologies for genome-wide identification of DNA-binding protein target sites. A number of algorithms have been developed in parallel that allow identification of binding sites from ChIP-seq or ChIP-chip datasets and subsequent visualization in the University of California Santa Cruz (UCSC) Genome Browser as custom annotation tracks. However, summarizing these tracks can be a daunting task, particularly if there are a large number of binding sites or the binding sites are distributed widely across the genome., Results: We have developed ChIPpeakAnno as a Bioconductor package within the statistical programming environment R to facilitate batch annotation of enriched peaks identified from ChIP-seq, ChIP-chip, cap analysis of gene expression (CAGE) or any experiments resulting in a large number of enriched genomic regions. The binding sites annotated with ChIPpeakAnno can be viewed easily as a table, a pie chart or plotted in histogram form, i.e., the distribution of distances to the nearest genes for each set of peaks. In addition, we have implemented functionalities for determining the significance of overlap between replicates or binding sites among transcription factors within a complex, and for drawing Venn diagrams to visualize the extent of the overlap between replicates. Furthermore, the package includes functionalities to retrieve sequences flanking putative binding sites for PCR amplification, cloning, or motif discovery, and to identify Gene Ontology (GO) terms associated with adjacent genes., Conclusions: ChIPpeakAnno enables batch annotation of the binding sites identified from ChIP-seq, ChIP-chip, CAGE or any technology that results in a large number of enriched genomic regions within the statistical programming environment R. Allowing users to pass their own annotation data such as a different Chromatin immunoprecipitation (ChIP) preparation and a dataset from literature, or existing annotation packages, such as GenomicFeatures and BSgenome, provides flexibility. Tight integration to the biomaRt package enables up-to-date annotation retrieval from the BioMart database.

The ordered expression of genes after growth factor stimulation in G(1) supportsthe onset of DNA replication. To characterize regulatory events during S-phase when cell cycle progression has become growth factor independent, we have profiled the expression of over 7,000 human genes using GeneChip DNA microarray analysis. HeLa cells were synchronized at the beginning of S-phase by thymidine/aphidicolin block, and RNA populations were analyzed throughout the S and G(2) phases. Expression of genes involved in DNA replication is maximal during early S-phase, whereas histone mRNAs peak at mid S-phase. Genes related to cell proliferation, including those encoding cyclins, oncoproteins, growth factors, proteins involved in signal transduction, and DNA repair proteins, follow distinct temporal patterns of expression that are functionally linked to initiation of DNA replication and progression through S-phase. The timing of expression for many genes in tumor-derived HeLa cells is highly conserved when compared with normal cells. In contrast, a number of genes show growth phenotype-related expression patterns that may directly reflect loss of stringent growth control in tumor cells. Our data reveal there is a core subset of cell growth-related genes that is fundamental to cycling cells irrespective of cell growth phenotype.

The kinetics of calcium movements in the isolated perfused rat liver were examined using compartmental analysis of the efflux profiles of 45Ca2+ from 45Ca(2+)-equilibrated livers under a variety of calcium concentrations and hormonal treatments. From the 45Ca2+ efflux profiles, we determined that a three compartment model was appropriate to describe the movements of calcium in the liver on the time scale of the experiments. Hormonal treatment with the alpha-adrenergic agonist, phenylephrine, or the vasoactive peptide, vasopressin, during the efflux period lowered significantly the rate of transfer of Ca2+ between the internal compartments at all of the calcium concentrations employed. Also, phenylephrine treatment leads to increased transfer of Ca2+ into the liver from the perfusate. The temporal characteristics of the phenylephrine and vasopressin sensitive Ca2+ pools were examined by pulsing livers, loaded for variable periods of time with 45Ca2+, with the two hormones during the efflux of 45Ca2+ to measure the kinetics of Ca2+ exchange in the hormone-sensitive pools. Results from these experiments indicate that the rate of unstimulated Ca2+ efflux, k2, for the phenylephrine and vasopressin sensitive Ca2+ pools, modeled as a one compartment system, are the same, 0.074 and 0.078 min-1 for phenylephrine and vasopressin respectively, corresponding to half times for turnover of the pool(s) of 9.3 and 8.9 min, respectively.

The methods described here represent a flexible set of procedures for investigating the metabolism of the branched-chain alpha-keto acids and other substances in perfused organs, notably the rat heart and liver. These procedures have been used to investigate many aspects of the metabolism of the branched-chain alpha-keto acids not discussed here, such as the effects on branched-chain alpha-keto acid metabolism by exposure to alpha-adrenergic agents, by inhibition of the monocarboxylate translocator, and by the coinfusion of other metabolites.

Platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)) is a potent lipid mediator which stimulates hepatic glycogenolysis, causes hepatic vasoconstriction, and stimulates the production of cyclooxygenase-derived metabolites of arachidonic acid, primarily prostaglandin (PG) D2 in the perfused liver. Following infusion of platelet-activating factor (1 nM) in the perfused rat liver the production of PGD2, measured in the effluent perfusate, increased 4-fold after only 2 min. Infusion of the cyclooxygenase inhibitor, ibuprofen (50 microM), abolished the stimulated production of PGD2 and thromboxane B2 in response to AGEPC without significantly affecting the hepatic glycogenolytic or vasoconstrictive responses to AGEPC. Contrary to previous reports, these observations do not support the suggestion that cyclooxygenase-derived metabolites mediate directly either the glycogenolytic or the vasoactive effects of AGEPC in the perfused rat liver.

The properties of ornithine decarboxylase from Phycomyces blakesleeanus were examined. Enzyme from mycelial cultures was extracted and purified approximately 70-fold. The apparent molecular weight is 96K. The Michaelis constants with respect to ornithine and pyridoxal 5'-phosphate are 90 and 0.37 microM, respectively. Putrescine is a potent competitive inhibitor with a Ki of 75 microM. Exposure of ornithine decarboxylase to sulfhydryl-modifying reagents resulted in a rapid inhibition of activity. In vivo addition of putrescine produced characteristic decreases in cellular ornithine decarboxylase activity. Light stimulation of dark-adapted mycelial cultures also decreased cellular ornithine decarboxylase activity.

The oxidative decarboxylation and subsequent production of glucose from alpha-ketobutyrate were studied using perfused livers from fasted rats. The production of 14CO2 from alpha-keto-[1-14C]butyrate increased monotonically while the production of glucose from alpha-ketobutyrate was biphasic as the perfusate concentration of alpha-ketobutyrate was increased. The biphasic gluconeogenic response using alpha-ketobutyrate as the gluconeogenic precursor was similar to that observed with propionate. The decarboxylation of alpha-ketobutyrate was found to be exquisitely sensitive to the effects of the monocarboxylate transport inhibitor, alpha-cyanocinnamate. Infusion of beta-hydroxybutyrate caused a substantial inhibition of alpha-ketobutyrate decarboxylation while dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, did not stimulate the metabolism of alpha-ketobutyrate but was inhibitory. The effects of alpha-ketobutyrate infusion on pyruvate decarboxylation were tested and it was found that at low perfusate pyruvate concentrations (ca. 0.25 mM) increasing alpha-ketobutyrate led to increasing inhibition of pyruvate decarboxylation, while at high perfusate pyruvate concentrations (ca. 2.5 mM) an initial inhibition was apparent which did not increase substantially with increasing alpha-ketobutyrate concentrations. The results obtained indicate that the regulation of alpha-ketobutyrate metabolism by oxidative decarboxylation differs significantly from that of pyruvate. In addition, while the rate of gluconeogenesis using alpha-ketobutyrate as a precursor was remarkably similar to that using propionate as a gluconeogenic precursor, the effects of alpha-ketobutyrate on the oxidative decarboxylation of pyruvate were qualitatively different from the effects of propionate on pyruvate metabolism.

In the perfused rat liver, platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), infusion produces an extensive but transient glycogenolytic response which at low AGEPC concentrations (i.e., 10(-11) M) is markedly dependent upon the perfusate calcium levels. The role of calcium in the glycogenolytic response of the liver to AGEPC was investigated by assessing the effect of AGEPC on various calcium pools in the intact liver. Livers from fed rats were equilibrated with 45Ca2+, and the kinetics of 45Ca2+ efflux were determined in control, AGEPC-stimulated, and phenylephrine-stimulated livers during steady-state washout of 45Ca2+. AGEPC treatment had only a slight if any effect on the pattern of steady-state calcium efflux from the liver, as opposed to major perturbations in the pattern of calcium efflux effected by the alpha-adrenergic agonist phenylephrine. Infusion of short pulses of AGEPC during the washout of 45Ca2+ from labeled livers caused a transient release of 45Ca2+ which was not abolished at low calcium concentrations in the perfusate. Moreover, there occurred no appreciable increase in the total calcium content in the liver perfusate at either high or low concentrations of calcium in the perfusion fluid. Infusion of latex beads, which are removed by the reticuloendothelial cells, caused the release of hepatic 45Ca2+ in a fashion similar to the case with AGEPC. Our findings indicate that AGEPC does not perturb a major pool of calcium within the liver as occurs upon alpha-adrenergic stimulation; it is likely that AGEPC mobilizes calcium from a smaller yet very important pool, very possibly from nonparenchymal cells in the liver.

The regulation of gluconeogenesis from alpha-ketoisovalerate and propionate was investigated in perfused livers from fasted rats. With alpha-ketoisovalerate as the gluconeogenic precursor, infusion of beta-hydroxybutyrate and acetate stimulated the rate of alpha-ketoisovalerate decarboxylation, but inhibited the rate of glucose production. Oleate, on the other hand, inhibited both alpha-ketoisovalerate decarboxylation and glucose production. When propionate was the primary gluconeogenic substrate, oleate, beta-hydroxybutyrate, and acetate infusion did not significantly alter hepatic glucose production. The present studies suggest that gluconeogenesis from alpha-ketoisovalerate is regulated at the level of various dehydrogenases prior to formation of propionyl-CoA, but subsequent to the branched-chain alpha-keto acid dehydrogenase reaction.

Mean transit times for the movement of extracellular and intracellular reference compounds through isolated perfused rat livers were determined during exposure of livers to platelet-activating factor (AGEPC; 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) and the alpha-adrenergic agonist phenylephrine, using the multiple indicator dilution technique. From the outflow profiles of rapid bolus injections of 3H-sucrose and 14C-urea given to the liver, the estimated intracellular volume of distribution of small freely permeant substances, Vi, and the ratio of intracellular to extracellular space, were computed. Exposure of the liver to AGEPC decreased Vi and by 32 and 34%, respectively, from control values, whereas infusion of phenylephrine increased Vi by 16% and by 33%. The results indicate that the hemodynamic effects of AGEPC in perfused rat liver cause the apparent loss of tissue space accessible to small permeant compounds. Phenylephrine, although increasing hepatic vascular resistance, measured at the portal vein, by the same magnitude as AGEPC, led to an increase in the apparent tissue space accessible to this same species.