Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells.

Background: Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context.

A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha.

RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III-promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR.

Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells.

Purpose: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis.

The antithrombotic and anti-inflammatory effects of BCX-3607, a small molecule tissue factor/factor VIIa inhibitor.

Tissue factor (TF) is a transmembrane glycoprotein that binds its zymogen cofactor, Factor VIIa (FVIIa) on the cell surface. Together (TF/FVIIa) they activate Factor X (FX) and Factor IX (FIX) and start the extrinsic pathway of blood coagulation. As such, the TF/FVIIa complex plays an important role in normal physiology as well as in thrombotic diseases such as unstable angina (UA), disseminated intravascular coagulation (DIC), and deep vein thrombosis (DVT).

Down-regulated expression of PPARalpha target genes, reduced fatty acid oxidation and altered fatty acid composition in the liver of mice transgenic for hTNFalpha.

The present study investigated the hepatic regulation of fatty acid metabolism in hTNFalpha transgenic mice. Reduced hepatic mRNA levels and activities of carnitine palmitoyltransferase-II (CPT-II) and mitochondrial HMG-CoA synthase were observed, accompanied by decreased fatty acid oxidation, fatty acyl-CoA oxidase and fatty acid synthase (FAS) activities and down-regulated gene expression of mitochondrial acetyl-CoA carboxylase 2 (ACC2). The mRNA levels of peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARdelta were reduced.

Downregulation of tissue factor by RNA interference in human melanoma LOX-L cells reduces pulmonary metastasis in nude mice.

Tissue factor (TF) is the membrane receptor of the serine protease coagulation factor VIIa (FVIIa). Formation of the TF/FVIIa complex initiates the coagulation cascade. We used short hairpin RNA (shRNA)-mediated RNA interference to knock down TF expression in the human metastatic melanoma cell line LOX-L.

Regulated compartmentalization of the putative DEAD-box helicase MDDX28 within the mitochondria in COS-1 cells.

We recently cloned a putative DEAD-box helicase MDDX28 and found that it was localized to the nuclei and mitochondria of COS-1 cells. The mitochondrial localization of MDDX28 is largely diffuse. We have, however, used immunofluorescence and immunogold cytochemistry to show that the MDDX28 protein is localized in a distinct mitochondrial subcompartment in 5-10% of COS-1 cells.

Knockdown of C-terminal Src kinase by siRNA-mediated RNA interference augments T cell receptor signaling in mature T cells.

C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h.

The epidermal growth factor receptor (EGFR) and proline rich tyrosine kinase 2 (PYK2) are involved in tissue factor dependent factor VIIa signalling in HaCaT cells.

Binding of the coagulation protease factor VIIa to its receptor Tissue Factor (TF) induces intracellular signals in several cell types including HaCaT keratinocytes. TF belongs to the cytokine receptor family, but is most likely not alone in transferring the complete TF/FVIIa signal over the plasma membrane. The protease activated receptor PAR2 is involved in factor VIIa and factor Xa signal transduction.

Coagulation factor VII, R353Q polymorphism, and serum choline-containing phospholipids in males at high risk for coronary heart disease.

Introduction: Elevated levels of coagulation factor VII (FVII) have been associated with increased risk for myocardial infarction (MI). The R353Q polymorphism of the FVII gene has been shown to modify plasma levels of FVII, and has in some studies also been associated with reduced risk for MI.

Objectives: To examine the R353Q polymorphism of the FVII gene and the relation to myocardial infarction (MI), cardiovascular disease (CVD), and diabetes, and furthermore, to elucidate the association between the polymorphism and plasma levels of FVII coagulant activity (FVIIc), FVII antigen (FVIIag), activated FVII (FVIIa), and serum choline-containing phospholipids (PC).

An algorithm for selection of functional siRNA sequences.

Randomly designed siRNA targeting different positions within the same mRNA display widely differing activities. We have performed a statistical analysis of 46 siRNA, identifying various features of the 19bp duplex that correlate significantly with functionality at the 70% knockdown level and verified these results against an independent data set of 34 siRNA recently reported by others. Features that consistently correlated positively with functionality across the two data sets included an asymmetry in the stability of the duplex ends (measured as the A/U differential of the three terminal basepairs at either end of the duplex) and the motifs S1, A6, and W19.

Involution of thymus and lymphoid depletion in mice expressing the hTNF transgene.

Tumour necrosis factor (TNF) is involved in the pathogenesis of several diseases. In mice, human TNF signals only through p55, one of two murine TNF receptors. We here report a study of growth, viability and morphological alterations in transgenic mice expressing a low constitutive and tissue-restricted level of human TNF in vivo.

A novel gene mutation in the 60s loop of human coagulation factor VII - inhibition of interdomain crosstalk.

A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8.

Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus.

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases.

Effects of dietary fat quantity and composition on fasting and postprandial levels of coagulation factor VII and serum choline-containing phospholipids.

Dietary fat influences plasma levels of coagulation factor VII (FVII) and serum phospholipids (PL). It is, however, unknown if the fat-mediated changes in FVII are linked to PL. The present study aimed to investigate the effects of dietary fat on fasting and postprandial levels of activated FVII (FVIIa), FVII coagulant activity (FVIIc), FVII protein (FVIIag) and choline-containing PL (PC).

Two novel mutations in the human coagulation factor VII promoter.

The factor VII genes of five unrelated Finnish female patients, F1-F5, with moderate bleeding tendency, were screened for mutations using single strand conformational polymorphisms and DNA sequencing. Heterozygous shifts were detected in exons 5 and 8 for patient F1, and sequencing confirmed the presence of the silent dimorphism H115H, the polymorphism R353Q and the mutation A294V. The patient F1 was also heterozygous for a novel -59T/G transversion mutation in the Hepatocyte nuclear factor 4-binding site.

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway.

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications.

Transport signals and transcription-dependent nuclear localization of the putative DEAD-box helicase MDDX28.

The human protein MDDX28 is a putative RNA helicase and a nucleocytoplasmic shuttling protein also localized to the mitochondria. Its localization is novel among RNA helicases. We have studied its intracellular targeting signals and show that the first 20 amino acids of MDDX28 are necessary and sufficient for both mitochondrial import and nuclear export of the protein.

Tolerance for mutations and chemical modifications in a siRNA.

Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modifications in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions.

PSKH1, a novel splice factor compartment-associated serine kinase.

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells. Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes. The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood.

Human TNF-alpha in transgenic mice induces differential changes in redox status and glutathione-regulating enzymes.

Tumor necrosis factor a (TNF-alpha) is a pleiotropic cytokine involved in several diseases. Various effects of TNF-alpha are mediated by the induction of a cellular state consistent with oxidative stress. Glutathione (GSH) is a major redox-buffer of eukaryotic cells and is important in the defense against oxidative stress.

Factor VIIa induces tissue factor-dependent up-regulation of interleukin-8 in a human keratinocyte line.

Tissue factor (TF), a transmembrane receptor for the serine protease coagulation factor VII(a) (FVIIa), is the main initiator of the coagulation cascade. Through incompletely elucidated mechanisms, TF serves additional functions in tumor-associated angiogenesis and metastasis. We have studied interleukin-8 (IL-8) as a possible link between TF-FVIIa complex formation and subsequent processes.

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs.