Anti-tumor activity mediated by protein and peptide transduction of HIV viral protein R (Vpr)

Peptides that are capable of traversing the cell membrane, via protein transduction domains (PTDs), are attractive either directly as drugs or indirectly as carriers for the delivery of therapeutic molecules. One such PTD, a HIV-1 Tat derived peptide has successfully delivered a variety of "cargoes" including proteins, peptides and nucleic acids into cells. There also exists other naturally occurring membrane permeable peptides which have potential as PTDs. Specifically, one of the accessory proteins of HIV (viral protein R; i.e., Vpr), which is important in controlling viral pathogenesis, possesses cell transduction domain characteristics. Related to these characteristics, Vpr has also been demonstrated to induce cell cycle arrest and host/target cell apoptosis, suggesting a potential anticancer activity for this protein. In this report we assessed the ability of Vpr protein or peptides, with or without conjugation to a PTD, to mediate anti-cancer activity against several tumor cell lines. Specifically, several Vpr peptides spanning carboxy amino acids 65-83 induced significant (i.e., greater than 50%) in vitro growth inhibition/toxicity of murine B16.F10 melanoma cells. Likewise, in in vitro experiments with other tumor cell lines, conjugation of Vpr to the Tat derived PTD and transfection of this construct into cells enhanced the induction of in vitro apoptosis by this protein when compared to the effects of transfection of cells with unconjugated Vpr. These results underscore the potential for Vpr based reagents as well as PTDs to enhance anti-tumor activity, and warrants further examination of Vpr protein and derived peptides as potential therapeutic agents against progressive cell proliferative diseases such as cancer. ©2009 Landes Bioscience

Metabolic Networks of Sodalis glossinidius: A Systems Biology Approach to Reductive Evolution

Background: Genome reduction is a common evolutionary process affecting bacterial lineages that establish symbiotic or pathogenic associations with eukaryotic hosts. Such associations yield highly reduced genomes with greatly streamlined metabolic abilities shaped by the type of ecological association with the host. Sodalis glossinidius, the secondary endosymbiont of tsetse flies, represents one of the few complete genomes available of a bacterium at the initial stages of this process. In the present study, genome reduction is studied from a systems biology perspective through the reconstruction and functional analysis of genome-scale metabolic networks of S. glossinidius. Results: The functional profile of ancestral and extant metabolic networks sheds light on the evolutionary events underlying transition to a host-dependent lifestyle. Meanwhile, reductive evolution simulations on the extant metabolic network can predict possible future evolution of S. glossinidius in the context of genome reduction. Finally, knockout simulations in different metabolic systems reveal a gradual decrease in network robustness to different mutational events for bacterial endosymbionts at different stages of the symbiotic association. Conclusions: Stoichiometric analysis reveals few gene inactivation events whose effects on the functionality of S. glossinidius metabolic systems are drastic enough to account for the ecological transition from a free-living to hostdependent lifestyle. The decrease in network robustness across different metabolic systems may be associated with th

The regulation of the putrescine biosynthetic gene, speB, encoding agmatine ureohydrolase in Escherichia coli

Agmatine ureohydrolase (EC.3.5.3.11) was purified 3,300 fold from an E.coli strain by ammonium sulphate precipitation, heat treatment, ion-exchange column chromatography, gel permeation column chromatography and chromatofocusing. The active enzyme is probably a dimer of identical subunits with a molecular weight of 38,000, and a pI of 8.3. The Km for agmatine was 1.3 mM, L-arginine was a competitive inhibiter and ornithine inhibited in a mixed manner. In crude extracts the enzyme was associated with another factor, probably a protein, which reduced the pI to 5.5 and decreased the activity of the enzyme. This factor also blocked reaction with antibodies raised against the purified enzyme in rabbits. In a strain known to express both biosynthetic and biodegradative ornithine decarboxylase and arginine decarboxylase, there was evidence for only the biosynthetic form of agmatine ureohydrolase. -- Agmatine ureohydrolase was negatively regulated by cAMP and the cAMP receptor protein, CRP. The specific activity of agmatine ureohydrolase was determined in crude extracts prepared from wild type strains of E.coli, from strains carrying a mutation in the structural gene for adenylcyclase (EC.4.6.1.1) (cya) or from strains carrying mutations in both cya and cAMP receptor protein gene (crp). Cyclic AMP when added to a glucose based medium repressed the specific activity of agmatine ureohydrolase, in contrast, cAMP induced the specific activity of β -galactosidase in both the wild type and the cya mutant, but not in the crp mutant. Addition of 1 mM agmatine to a glucose based medium induced an the specific activity of agmatine ureohydrolase in wild type, ∆cya or ∆cya, ∆crp strains. Chloramphenicol (150 μg/ml) abolished the inducibility of agmatine ureohydrolase by agmatine. In mutants blocked in the steps leading to the biosynthesis of polyamines, the addition of putrescine repressed the specific activities of arginine decarboxylase and ornithine decarboxylase, but did not affect agmatine ureohydrolase activity. The negative regulation of speA, speB and speC (encode arginine decarboxylase, agmatine ureohydrolase and ornithine decarboxylase respectively) by cAMP was shown not to be mediated by the repressive effect of cAMP on glutamine synthetase (EC.6.3.1.2), as cAMP repressed the expression of all three genes in a strain deleted for glnA (encodes glutamine synthetase). -- The speB gene was cloned into the plasmid vector pBR322 at the BamHI site, and was localised by exonuclease digestion of the plasmid pKA5. The extent of digestion of the plasmid was determined and was related to the ability of these religated plasmids to restore agmatine ureohydrolase activity to an speBsup- strain of E.coli. Using a cell-free transcription and translation system the direction of transcription and the approximate location of the promoter was determined. The direction of transcription was also determined by cloning fragments in both directions into the promoterless genes of lacZ (encodes β -galactosidase) and galK {encodes galactokinase). The ability of these hybrid genes to confer enzymatic activities (β -galactosidase or galactokinase) to strains deleted in their respective genes confirmed the location of the speB promoter. -- cAMP:CRP was shown to interact with the promoters of speA and speB using a cell-free transcription and translation system in which cloned copies of the genes served as templates. This was also shown in vivo, by demonstrating that cAMP, inhibited the expression of β -galactosidase and galactokinase in plasmids carrying fusions of the speA and speB promoters to the structural genes of these enzymes. In addition, cAMP supplementation of minicells carrying the plasmid pKA5 (carries speB) caused repression of agmatine ureohydrolase synthesis. Cyclic AMP decreased and agmatine increased the steady state mRNA concentrations of speB, while those of lacZ were increased by cAMP in the ∆cya strain. In contrast, cAMP did not affect the steady state concentrations of lacZ or speB mRNAs in the ∆crp strain