Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed

Ectodermal Influx and Cell Hypertrophy Provide Early Growth for All Murine Mammary Rudiments, and Are Differentially Regulated among Them by Gli3

Mammary gland development starts in utero with one or several pairs of mammary rudiments (MRs) budding from the surface ectodermal component of the mammalian embryonic skin. Mice develop five pairs, numbered MR1 to MR5 from pectoral to inguinal position. We have previously shown that Gli3Xt-J/Xt-J mutant embryos, which lack the transcription factor Gli3, do not form MR3 and MR5. We show here that two days after the MRs emerge, Gli3Xt-J/Xt-J MR1 is 20% smaller, and Gli3Xt-J/Xt-J MR2 and MR4 are 50% smaller than their wild type (wt) counterparts. Moreover, while wt MRs sink into the underlying dermis, Gli3Xt-J/Xt-J MR4 and MR2 protrude outwardly, to different extents. To understand why each of these five pairs of functionally identical organs has its own, distinct response to the absence of Gli3, we determined which cellular mechanisms regulate growth of the individual MRs, and whether and how Gli3 regulates these mechanisms. We found a 5.5 to 10.7-fold lower cell proliferation rate in wt MRs compared to their adjacent surface ectoderm, indicating that MRs do not emerge or grow via locally enhanced cell proliferation. Cell-tracing experiments showed that surface ectodermal cells are recruited toward the positions where MRs emerge, and contribute to MR growth during at least two days. During the second day of MR development, peripheral cells within the MRs undergo hypertrophy, which also contributes to MR growth. Limited apoptotic cell death counterbalances MR growth. The relative contribution of each of these processes varies among the five MRs. Furthermore, each of these processes is impaired in the absence of Gli3, but to different extents in each MR. This differential involvement of Gli3 explains the variation in phenotype among Gli3Xt-J/Xt-J MRs, and may help to understand the variation in numbers and positions of mammary glands among mammals

Diamine ligand release from the cisplatin analog [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) in cell culture medium

The stability of the five-membered chelate ring of the cisplatin analog [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) (I) was investigated under typical cell culture conditions (IMEM-Richter's medium with 10% fetal calf serum, 37 Deg). The platinum compd. was radiolabeled with tritium in the meta position of the arom. ring by an acid-catalyzed tritium-exchange reaction, and a reversed-phase HPLC assay with radiochem. detection was developed to monitor for the presence of the free diamine ligand in the cell culture medium. A gradual increase in radioactivity attributed to the free diamine was found in medium contg. the dichloroplatinum(II) complex (ca. 25% after 24 h), indicating that the diamine ligand was being released from the metal atom. When 1 mM glutathione (GSH) was included in the incubation medium, the amt. of free diamine nearly doubled after 24 h, while the amt. of radioactivity attributed to serum protein-platinum adducts decreased relative to incubations without GSH. On the other hand, the omission of serum form the incubations resulted in a dramatic decrease in the amt. of radioactivity eluting under the diamine peak, while the concns. of the 2 methionine-Pt adducts, which formed in a 1:1 ratio, rose. Through the use of liq. secondary ion mass spectroscopy, the 2 methionine-Pt adducts were identified as monomethionine metabolites of the title compd., whereby the 2 chloride ligands were replaced by the amino acid. These compds. are probably diastereomers since the sulfur of methionine can coordinate to platinum with equal probability either cis or trans to the R-configured benzylamine carbon. On the basis of the chem. shifts of the MeS groups in the 250-MHz 1H NMR, it is concluded that a S,N-five-membered chelate ring is present in these methionine-Pt adducts

The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor-4

<p><b>Copyright information:</b></p><p>Taken from "The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor"</p><p>http://www.biomedcentral.com/1472-6807/7/66</p><p>BMC Structural Biology 2007;7():66-66.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2099433.</p><p></p> (B) Representative calcium traces elicited upon stimulation with different concentrations of neohesperidin dihydrochalcone (NHDC) in HEK293T-G16Gust44 cells cotransfected with DNA for hTAS1R2/hTAS1R3. (C,D) Calcium responses of cells cotransfected with DNA for different rat and human subunits or chimeras upon stimulation with 1 mM NHDC (C), or 30 mM D-Tryptophan (D). R, rat receptor subunit; H human receptor subunit; HR, receptor chimera comprising the N-terminal extra cellular domain of the human receptor subunit fused to the corresponding heptahelical domain of the rat; RH, comprising the N-terminal extracellular domain of the rat receptor subunit fused to the corresponding heptahelical domain of the human receptor

The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor-1

<p><b>Copyright information:</b></p><p>Taken from "The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor"</p><p>http://www.biomedcentral.com/1472-6807/7/66</p><p>BMC Structural Biology 2007;7():66-66.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2099433.</p><p></p> different lactisole concentrations. No lactisole present (filled circles, solid line), 50 μM lactisole (filled triangles up, dashed line), and 100 μM lactisole (filled squares, dash-dotted line). (E) ECvalues for acesulfame K, aspartame, NHDC, and cyclamate in the absence or presence of 50 μM and 100 μM lactisole

The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor-3

<p><b>Copyright information:</b></p><p>Taken from "The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor"</p><p>http://www.biomedcentral.com/1472-6807/7/66</p><p>BMC Structural Biology 2007;7():66-66.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2099433.</p><p></p>tisole in HEK293T-G16Gust44 cells cotransfected with DNA for wild type hTAS1R2/hTAS1R3 (filled circle, solid line), C801I(open diamond, short dashed line), Y699L(open circle, dash-dot-dotted line), Y699F(filled triangle up, medium dashed line), or W775A(filled hexagon, dash-dotted line) and hTAS1R2 DNA. (B) Concentration-dependent responses of HEK293T-G16Gust44 cells to cyclamate cotransfected with DNA for hTAS1R3 (filled circle, solid line), S726A(open triangle up, medium dashed line), C801I(open diamond, short dashed line), or W775A(filled hexagon, dash-dotted line) and hTAS1R2

The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor-0

<p><b>Copyright information:</b></p><p>Taken from "The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor"</p><p>http://www.biomedcentral.com/1472-6807/7/66</p><p>BMC Structural Biology 2007;7():66-66.</p><p>Published online 12 Oct 2007</p><p>PMCID:PMC2099433.</p><p></p>ked by ICL and ECL, respectively. Amino acids that influence allosteric modulator activity in hCaSR, hmGlu2, rmGlu1 and 5 are marked in grey. Positions that alter the response of the sweet receptor to lactisole, cyclamate, or NHDC are shown in red. Asterisks indicate residues involved in receptor activation by NHDC (orange) or cyclamate (blue). Green asteriks denote residues mediating sensitivity of the receptor to lactisole