Genomic locations of glucokinase genes in diverse mammalian genomes.

1<p>Bases spanning the N-terminus to C-terminus of the encoded protein.</p>2<p>Y, indicates that all 11 exons (2 tissue specific and 9 shared) could be predicted from the genomic sequence. N, indicates that at least one exon could not be predicted.</p>3<p>None indicates that a gene was not annotated in the genome assembly.</p>4<p>A liver specific 1^st exon was not found in the genomic sequence, but there is no gap in this sequence.</p

Repetitive DNA content of introns within mammalian glucokinase genes.

1<p>Sequence between the beta-cell and liver specific 1^st exons.</p>2<p>Sequence between liver-specific 1^st exon and exon 2.</p>3<p>Combined sequences of introns 2 through 9.</p

Alignment of glucokinase gene sequences from diverse mammals.

<p>A genomic sequence alignment was generated by MultiPipMaker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824-Schwartz1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824-Schwartz2" target="_blank">[25]</a>. The sequence is numbered (in kilobases, k) from the 5′ end of the liver-specific transcript, with 5′ flanking sequence numbered backwards. Exons are represented as tall boxes, and are numbered from the 5′ end of the transcripts. The arrow, labeled GCK, represents the liver-specific glucokinase transcript. Beta refers to the 1^st exon of the beta-cell-specific GCK transcript, which is spliced to join exon 2. Tissue-specific first exons are labeled as 1B, for the pancreatic beta-cell-specific 1^st exon, and 1L, for the liver-cell-specific 1^st exon. Filled tall boxes are coding exon sequences, while shaded boxes are untranslated sequences. The percentage sequence identity (if above 50%) of the mouse, cow, horse, hyrax, and Tasmanian devil GCK genomic sequences to the human genomic sequence is shown for each species below the human genomic region schematic. Repetitive DNA elements, and sequence shown high GC content are also identified using the symbols shown in box at the lower right.</p

Transcriptional activity of deletions of the human glucokinase 5′ flanking sequence.

<p>A. Schematic illustrations of the human glucokinase promoter deletion constructs used to test promoter activity are shown on the left. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824.s001" target="_blank">Fig. S1</a> for the locations of genomic fragments within the gene. Reporter gene activity in two human cell lines, HepG2 and L-02 is shown on the right. Asterisk (*) indicates reporter gene activity that is significantly greater than that from pGL2 basic. Data are the Mean ± SD of three independent experiments. B and C. Responsiveness of the human glucokinase promoter constructs to insulin and fetal calf serum (serum) in HepG2 (B) and L-02 (C) cells. Basal refers to culture without insulin or serum. Constructs from part A were incubated in the presence of absence of 100 nM insulin or 10% fetal calf serum for 20 hours. Data are the Mean ± SD of three independent experiments. Asterisk (*) indicates conditions that have significantly greater activity than the basal level for that construct (P<0.05).</p

Analysis of the transcriptional activity of fragments of intron 1 of the human GCK gene.

<p>A. Activity of large fragments spanning most of intron 1. B Activates of subfragments of intronic fragment Int-3 (I-3). Schematics on the left indicate the relative positions of fragments of intron 1 inserted downstream of a luciferase reporter gene in the pGK-1049Luc reporter plasmid. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824.s001" target="_blank">Fig. S1</a> for the locations of genomic fragments within the gene. Reporter gene activities of the constructs in human L-02 and HepG2 cells are shown on the right. Data are the Mean ± SD of three independent experiments. Asterisk (*) indicates constructs that have significantly different activity than the −1049 construct (P<0.05).</p

Intron 1 sequences regulate expression of the human glucokinase promoter.

<p>A. Schematic illustrations of reporter gene constructs with 1049 or 3815 bases of liver-specific glucokinase 5′ flanking sequence with or without intron 1 are shown on the left. Solid boxes are the luciferase-coding regions. Thin lines represent glucokinase 5′ flanking sequences (1049 or 3815 bases) while the broken lines represent intron 1 sequences. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045824#pone.0045824.s001" target="_blank">Fig. S1</a> for the locations of genomic fragments within the gene. To the right are the luciferase reporter activities detected in two human liver cell lines (L-02 and HepG2), a hamster pancreatic beta-cell line (HIT) and a human intestinal cell line (HCT8). Data are the Mean ± S.D. of three independent experiments. Asterisk (*) indicates reporter gene activity that is significantly greater than that from pGL2 basic. Significant differences in the activity of constructs that differed due to the presence or absence of intronic sequences are indicated by the pound symbol (#), with the comparison shown by the lines (P<0.05). B and C, Responsiveness of the human glucokinase reporter constructs to insulin and fetal calf serum (serum) in HepG2 (B) and L-02 (C) cells. Basal refers to culture without insulin or serum. Constructs from part A were incubated in the presence of absence of 100 nM insulin or 10% fetal calf serum for 20 hours. Data are the Mean ± SD of three independent experiments. Asterisk (*) indicates conditions that have significantly greater activity than the basal level for that construct (P<0.05).</p

Potential transcription factor binding sites near the human glucokinase liver-specific 1^st exon.

1<p>Location of the binding site relative to liver-specific mRNA start site.</p>2<p>Name of protein that potentially binds to the human glucokinase genomic sequence.</p

Design, Synthesis, and Evaluation of Tetrahydropyrrolo[1,2‑<i>c</i>]pyrimidines as Capsid Assembly Inhibitors for HBV Treatment

The discovery of novel tetrahydropyrrolo­[1,2-<i>c</i>]­pyrimidines derivatives from <b>Bay41_4109</b> as hepatitis B virus (HBV) inhibitors is herein reported. The structure–activity relationship optimization led to one highly efficacious compound <b>28a</b> (IC₅₀ = 10 nM) with good PK profiles and the favorite L/P ratio. The hydrodynamic injection model in mice clearly demonstrated the efficacy of <b>28a</b> against HBV replication