RNA interference and its potential applications to chronic HBV treatment: results of a Phase I safety and tolerability study.

Background: RNA interference (RNAi) provides an attractive tool to modulate biological systems, and ultimately, to treat human diseases. We describe early results from a Phase Ib, first-in-human safety and tolerability study of an RNAi-based therapy, NUC B1000, among patients with mild to moderate chronic HBV.

Methods: Three subjects received a single 5 mg DNA dose of NUC B1000 as part of a planned dose escalation study.

The effect of the structure of small cationic peptides on the characteristics of peptide-DNA complexes.

A series of transcriptional activator (TAT)-protein transduction domains (PTDs) modified with hydrophobic amino acids were used as model cationic amphiphilic peptides to study the effect of hydrophobicity on interaction of such peptides with plasmid DNA. The peptide-DNA complexes were analyzed by dynamic light scattering and gel electrophoresis to determine their size and electrokinetic properties at various +/- charge ratios. Peptides in solution were found to have a tendency to aggregate and the hydrodynamic size of the aggregates depends on the structure of peptide.

A spectrophotometric method to quantify linear DNA.

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.

DNA vaccines--challenges in delivery.

DNA vaccines are typically comprised of plasmid DNA molecules that encode an antigen(s) derived from a pathogen or tumor cell. Following introduction into a vaccine, cells take up the DNA, where expression and immune presentation of the encoded antigen(s) takes place. DNA can be introduced by viral or bacterial vectors or through uptake of 'naked' or complexed DNA.

DNA vaccines encoding interleukin-8 and RANTES enhance antigen-specific Th1-type CD4(+) T-cell-mediated protective immunity against herpes simplex virus type 2 in vivo.

Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes.

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine.

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro.

LFA-3 plasmid DNA enhances Ag-specific humoral- and cellular-mediated protective immunity against herpes simplex virus-2 in vivo: involvement of CD4+ T cells in protection.

Adhesion molecules are important for cell trafficking and delivery of secondary signals for stimulation of T cells and antigen-presenting cells (APCs) in a variety of immune and inflammatory responses. Adhesion molecules lymphocyte function-associated antigen (LFA)-1 and CD2 on T cells recognize intercellular adhesion molecule (ICAM)-1 and LFA-3 on APCs, respectively. Recent studies have suggested that these molecules might play a regulatory role in antigen-specific immune responses.

Mechanistic studies of Escherichia coli adenosine-5'-phosphosulfate kinase.

Adenosine-5'-phosphosulfate kinase (APS kinase) catalyzes the formation of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the major form of activated sulfate in biological systems. The enzyme from Escherichia coli has complex kinetic behavior, including substrate inhibition by APS and formation of a phosphorylated enzyme (E-P) as a reaction intermediate. The presence of a phosphorylated enzyme potentially enables the steady-state kinetic mechanism to change from sequential to ping-pong as the APS concentration decreases.

Chain reaction cloning: a one-step method for directional ligation of multiple DNA fragments.

A novel DNA assembly method, chain reaction cloning (CRC), is described. CRC enables the ordered assembly of multiple DNA fragments in a single step. The power of the technique was demonstrated by the directed in vitro assembly of a plasmid comprised of six DNA fragments from a pool of 12 available fragments.

Preclinical safety of DNA vaccines : a method to analyze the distribution of plasmid DNA in animal models.

DNA or genetic vaccines are currently being evaluated for safety and efficacy in human clinical trials in the areas of infectious disease and cancer. Since DNA vaccines induce antibodies and cytotoxic T lymphocytes (CTLs), they are currently being evaluated in humans for both prevention and therapy of HSV-2, HIV-1, and HBV infections, for prevention of influenza and malaria, and therapy of cutaneous T-cell lymphoma (CTCL) and colorectal cancer.

Induction of HSV-gD2 specific CD4(+) cells in Peyer's patches and mucosal antibody responses in mice following DNA immunization by both parenteral and mucosal administration.

A DNA vaccine encoding glycoprotein D (gD) of herpes simplex virus type 2 (pHSV-gD2) was injected via parenteral and mucosal routes to determine the optimal route of delivery for immune stimulation. Generation of distal mucosal immunity following parenteral vaccination was also evaluated. While all routes of DNA vaccine administration resulted in systemic cellular and humoral responses, the intra-muscular (i.

The ORF1 of the gentamicin-resistance operon (aac) of Pseudomonas aeruginosa encodes adenosine 5'-phosphosulphate kinase.

The gentamicin-resistance operon of Pseudomonas aeruginosa (aac) contains two cistrons for which only the second gene product has an identified function. The 813bp second cistron (ORF2) encodes a protein that confers gentamicin resistance by catalysis of the transfer of an acetyl group from acetyl Coenzyme A to gentamicin. The first open reading frame (ORF1) encodes a 23.

Isozymes of S-adenosylmethionine synthetase are encoded by tandemly duplicated genes in Escherichia coli.

The sole biosynthetic route to S-adenosylmethionine, the primary biological alkylating agent, is catalysed by S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase). In Escherichia coli and Salmonella typhimurium numerous studies have located a structural gene (metK) for this enzyme at 63 min on the chromosomal map. We have now identified a second structural gene for S-adenosylmethionine synthetase in E.

Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction.

Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction.

Novel Escherichia coli K-12 mutants impaired in S-adenosylmethionine synthesis.

S-Adenosylmethionine (AdoMet) plays a myriad of roles in cellular metabolism. One of the many roles of AdoMet in Escherichia coli and Salmonella typhimurium is as a corepressor of genes encoding enzymes of methionine biosynthesis. To investigate the metabolic effects of large reductions in intracellular AdoMet concentrations in growing cells, we constructed and examined mutants of E.

Adenosine-5'-phosphosulfate kinase from Escherichia coli K12. Purification, characterization, and identification of a phosphorylated enzyme intermediate.

Adenosine-5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransferase), the second enzyme in the pathway of sulfate activation, has been purified (approximately 300-fold) to homogeneity from an Escherichia coli K12 strain, which overproduces the enzyme activity (approximately 100-fold). The purified enzyme has a specific activity of 153 mumol of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) formed/min/mg of protein at 25 degrees C. The enzyme is remarkably efficient with a Vmax/Km(APS) of greater than 10(8) M-1 s-1, indicating that at physiologically low substrate concentrations the reaction is essentially diffusion limited.

Identification of the reactive sulfhydryl groups of S-adenosylmethionine synthetase.

S-Adenosylmethionine synthetase from Escherichia coli is rapidly inactivated by N-ethylmaleimide. In the presence of excess N-ethylmaleimide inactivation follows pseudo first-order kinetics, and loss of enzyme activity correlates with the incorporation of 2 eq of N-[ethyl-2-3H]maleimide/subunit. Preincubation of the enzyme with methionine and the ATP analog adenylylimidodiphosphate reduced the rate of N-ethylmaleimide incorporation more than 30-fold.

Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli.

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (EC 3.5.3.

Transcription of the speC (ornithine decarboxylase) gene of Escherichia coli is repressed by cyclic AMP and its receptor protein.

The speC gene encoding ornithine decarboxylase (ODC) in Escherichia coli is negatively regulated by cAMP and the cAMP receptor protein (CRP). In minicells transformed with the plasmid pODC bearing speC, cAMP supplementation repressed ODC synthesis. In a cell-free protein synthesizing system directed by pODC, cAMP at 10(-5) M repressed ODC synthesis by 90%.

Antagonistic transcriptional regulation of the putrescine biosynthetic enzyme agmatine ureohydrolase by cyclic AMP and agmatine in Escherichia coli.

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (agmatinase; EC 3.5.3.