Phosphorylation-specific status of RNAi triggers in pharmacokinetic and biodistribution analyses.

The RNA interference (RNAi)-based therapeutic ARC-520 for chronic hepatitis B virus (HBV) infection consists of a melittin-derived peptide conjugated to N-acetylgalactosamine for hepatocyte targeting and endosomal escape, and cholesterol-conjugated RNAi triggers, which together result in HBV gene silencing. To characterize the kinetics of RNAi trigger delivery and 5΄-phosphorylation of guide strands correlating with gene knockdown, we employed a peptide-nucleic acid (PNA) hybridization assay. A fluorescent sense strand PNA probe binding to RNAi duplex guide strands was coupled with anion exchange high performance liquid chromatography to quantitate guide strands and metabolites.

Dose response in rodents and nonhuman primates after hydrodynamic limb vein delivery of naked plasmid DNA.

The efficacy of gene therapy mediated by plasmid DNA (pDNA) depends on the selection of suitable vectors and doses. Using hydrodynamic limb vein (HLV) injection to deliver naked pDNA to skeletal muscles of the limbs, we evaluated key parameters that affect expression in muscle from genes encoded in pDNA. Short-term and long-term promoter comparisons demonstrated that kinetics of expression differed between cytomegalovirus (CMV), muscle creatine kinase, and desmin promoters, but all gave stable expression from 2 to 49 weeks after delivery to mouse muscle.

Myofiber Damage Evaluation by Evans Blue Dye Injection.

Evans blue dye (EBD) can be used in live mice to study muscle pathology or injury, including exercise-induced muscle damage. EBD is excluded from intact cell membranes but leaks into cells, including muscle fibers, when the cell membrane is ruptured. EBD can be visualized by its autofluorescence under a fluorescence microscope.

Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.

Various plasmids were delivered into rodent limb muscles by hydrodynamic limb vein (HLV) injection of naked plasmid DNA (pDNA). Some of the pDNA preparations caused significant muscle necrosis and associated muscle regeneration 3 to 4 days after the injection whereas others caused no muscle damage. Occurrence of muscle damage was independent of plasmid sequence, size, and encoded genes.

Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.

Evans blue dye (EBD) is used to mark damaged and permeable muscle fibers in mouse models of muscular dystrophy and as an endpoint in therapeutic trials. We counted EBD-positive muscle fibers and extracted EBD from muscles sampled throughout the hindlimbs in young adult and old mdx mice to determine if the natural variability in morphology would allow measurement of a functional improvement in one limb compared to the contralateral limb. Following one bout of rotarod or treadmill exercise that greatly increased serum creatine kinase levels, the number of EBD(+) muscle fibers in 12-19-month-old mdx mice increased 3-fold, EBD in the muscles increased, and, importantly, contralateral pairs of muscles contained similar amounts of EBD.

Functional efficacy of dystrophin expression from plasmids delivered to mdx mice by hydrodynamic limb vein injection.

In these studies we delivered by hydrodynamic limb vein (HLV) injection plasmid DNA (pDNA) expressing the full-length mouse dystrophin gene to skeletal muscles throughout the hind limbs of the mdx mouse model for Duchenne muscular dystrophy (DMD). We evaluated the levels and stability of dystrophin expression and measured the resulting muscle protection, using Evans blue dye (EBD) to mark the damaged myofibers. Plasmid delivery was as efficient in the dystrophic mice as in wild-type mice and equally efficient in young adult and old mice, as long as the dose of pDNA was adjusted for the target muscle weight.

K12-biotinylated histone H4 marks heterochromatin in human lymphoblastoma cells.

Covalent modifications of histones play crucial roles in chromatin structure and genomic stability. Recently, we reported a novel modification of histones: biotinylation of lysine residues. Here we provide evidence that K12-biotinylated histone H4 (K12Bio H4) maps specifically to both heterochromatin (alpha satellite repeats in pericentromeric regions) and transcriptionally repressed chromatin (gamma-G globin and interleukin-2) in human lymphoblastoma cells.

Biotin supplementation decreases the expression of the SERCA3 gene (ATP2A3) in Jurkat cells, thus, triggering unfolded protein response.

Protein folding in the endoplasmic reticulum (ER) depends on Ca(2+); uptake of Ca(2+) into the ER is mediated by sarco/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3). The 5'-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors.

K4, K9 and K18 in human histone H3 are targets for biotinylation by biotinidase.

Histones are modified post-translationally, e.g. by methylation of lysine and arginine residues, and by phosphorylation of serine residues.

The expression of genes encoding ribosomal subunits and eukaryotic translation initiation factor 5A depends on biotin and bisnorbiotin in HepG2 cells.

Biotin affects gene expression at both the transcriptional and the posttranscriptional level; biotin metabolites might have biotin-like activities with regard to gene expression. Here, human hepatocarcinoma (HepG2) cells were used (i) to identify clusters of biotin-dependent genes, (ii) to determine whether the naturally occurring metabolite bisnorbiotin affects gene expression and (iii) to determine whether biotin and bisnorbiotin affect the expression of genes coding for ribosomal subunits and translation initiation factors. HepG2 cells were cultured in media containing deficient (0.

HepG2 cells develop signs of riboflavin deficiency within 4 days of culture in riboflavin-deficient medium.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential coenzymes in redox reactions. For example, FAD is a coenzyme for both glutathione reductase and enzymes that mediate the oxidative folding of secretory proteins. Here we investigated short-term effects of moderately riboflavin-deficient culture medium on flavin-related responses in HepG2 hepatocarcinoma cells.

Biological functions of biotinylated histones.

Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase.

High-throughput immunoblotting identifies biotin-dependent signaling proteins in HepG2 hepatocarcinoma cells.

Biotin affects the abundance of mRNA coding for approximately 10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.

Biotin deficiency stimulates survival pathways in human lymphoma cells exposed to antineoplastic drugs.

Cells may respond to nutrient deficiency or death signals with nuclear translocation of the transcription factor nuclear factor kappaB (NF-kappaB), which activates transcription of anti-apoptotic genes. Here we tested the hypothesis that biotin deficiency stimulates NF-kappaB-dependent survival pathways in human lymphoma cells, enhancing resistance to antineoplastic agents. Lymphoma (Jurkat) cells were cultured in biotin-deficient (0.

Roles for nutrients in epigenetic events.

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing.

Biotin supplementation increases expression of the cytochrome P450 1B1 gene in Jurkat cells, increasing the occurrence of single-stranded DNA breaks.

DNA microarray studies provided evidence that biotin supplementation increases the abundance of mRNA encoding cytochrome P(450) 1B1 (CYP1B1) in human lymphocytes. CYP1B1 hydroxylates procarcinogens, generating electrophilic mutagens. Here, we sought to identify the signaling pathways that increase the expression of CYP1B1 in biotin-supplemented human T (Jurkat) cells and to determine whether activation of the CYP1B1 gene is associated with increased occurrence of single-stranded DNA breaks.

Biotin supply affects rates of cell proliferation, biotinylation of carboxylases and histones, and expression of the gene encoding the sodium-dependent multivitamin transporter in JAr choriocarcinoma cells.

Background: Placental transfer of nutrients and secretion of hormones is essential for normal fetal development.

Aim Of The Study: To determine whether biotin supply affects biotin homeostasis, proliferation rates, and progesterone secretion in placenta cells.

Methods: JAr choriocarcinoma cells were cultured in media containing deficient (25 pmol/L), physiological (250 pmol/L), or pharmacological concentrations (10,000 pmol/L) of biotin for three weeks; markers for biotin homeostasis, proliferation, and hormone secretion were quantified.

The nuclear abundance of transcription factors Sp1 and Sp3 depends on biotin in Jurkat cells.

Biotin affects gene expression in mammals; however, the signaling pathways leading to biotin-dependent transcriptional activation and inactivation of genes are largely unknown. Members of the Sp/Krüppel-like factor family of transcription factors (e.g.

Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells.

Here the hypothesis was tested that monocarboxylate transporters (MCT) mediate biotin transport in human lymphoid cells. Uptake of [(3)H]biotin was measured in human lymphoid cells [peripheral blood mononuclear cells (PBMC) and Jurkat cells] under conditions known to affect MCT-mediated transport. When biotin uptake into PBMC was measured in the presence of excess concentrations of competitors (MCT substrates) and MCT inhibitors, transport rates decreased significantly to <75 and <67%, respectively, of controls.

Interleukin-2 receptor-gamma -dependent endocytosis depends on biotin in Jurkat cells.

Biotin has been credited with having beneficial effects on immune function despite observations that biotin supplementation causes decreased secretion of interleukin-2. Here this paradox was addressed by determining whether receptor-dependent internalization of interleukin-2 by immune cells depends on biotin. Theoretically, this would be consistent with both decreased net secretion of interleukin-2 by biotin-supplemented cells (causing increased endocytosis) and beneficial effects of biotin on immune function (causing increased receptor signaling).

Synthesis of a rabbit polyclonal antibody to the human sodium-dependent multivitamin transporter.

In mammalian cells, biotin is covalently attached to carboxylases and histones and is required for cell proliferation and function. Cellular uptake of biotin (as well as pantothenic acid and lipoic acid) is mediated by the sodium-dependent multivitamin transporter, SMVT. Studies of cellular biotin homeostasis have been hampered by the lack of an antibody to SMVT.

Exposure to UV light causes increased biotinylation of histones in Jurkat cells.

Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage.

Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities.

Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.

Biotinidase catalyzes debiotinylation of histones.

Background: Posttranslational modifications of histones play important roles in processes such as regulation of gene expression and DNA repair. Recently, evidence has been provided that histones in human cells are modified by covalent attachment of biotin. AIM OF THE STUDY To determine whether the reverse process (debiotinylation of histones) occurs in biological samples and whether debiotinylation is an enzyme-mediated process; and to characterize the enzyme that mediates debiotinylation of histones.

Biotin supply affects expression of biotin transporters, biotinylation of carboxylases and metabolism of interleukin-2 in Jurkat cells.

Biotin supply may affect transcription of genes and biotinylation of proteins in cells. In this study, Jurkat cells were used to model effects of biotin supply on biotin homeostasis and interleukin-2 metabolism in immune cells. Cells were cultured in media containing deficient (25 pmol/L), physiologic (250 pmol/L), or pharmacologic concentrations (10,000 pmol/L) of biotin for 4 wk.