Crotonols A and B, two rare tigliane diterpenoid derivatives against K562 cells from Croton tiglium.

Crotonols A and B (1 and 2), two tigliane diterpenoids featuring a rare C-7/C-14 cyclized and novel 5/7/7-fused carbon skeleton, along with the known tigliane wallichiioid A, were isolated from the leaves of Croton tiglium. Their structures were determined through spectroscopic methods, X-ray crystallography and ECD analysis. To the best of our knowledge, crotonol B (2) represents the first example of 13,14-seco-tigliane diterpenoids. Crotonols A and B displayed strong cytotoxic activities against the K562 cell line with IC50 values of 0.20 and 0.21 µM, respectively. Furthermore, crotonol A promoted the apoptosis of K562 cells through the cleavage of PARP and the accumulation of bax as well as the degradation of bcl-2.

Induction of heme oxygenase I (HMOX1) by HPP-4382: a novel modulator of Bach1 activity.

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

4-1BB ligand as an effective multifunctional immunomodulator and antigen delivery vehicle for the development of therapeutic cancer vaccines.

Therapeutic subunit vaccines based on tumor-associated antigens (TAA) represent an attractive approach for the treatment of cancer. However, poor immunogenicity of TAAs requires potent adjuvants for therapeutic efficacy. We recently proposed the tumor necrosis factor family costimulatory ligands as potential adjuvants for therapeutic vaccines and, hence, generated a soluble form of 4-1BBL chimeric with streptavidin (SA-4-1BBL) that has pleiotropic effects on cells of innate, adaptive, and regulatory immunity. We herein tested whether these effects can translate into effective cancer immunotherapy when SA-4-1BBL was also used as a vehicle to deliver TAAs in vivo to dendritic cells (DCs) constitutively expressing the 4-1BB receptor. SA-4-1BBL was internalized by DCs upon receptor binding and immunization with biotinylated antigens conjugated to SA-4-1BBL resulted in increased antigen uptake and cross-presentation by DCs, leading to the generation of effective T-cell immune responses. Conjugate vaccines containing human papillomavirus 16 E7 oncoprotein or survivin as a self-TAA had potent therapeutic efficacy against TC-1 cervical and 3LL lung carcinoma tumors, respectively. Therapeutic efficacy of the vaccines was associated with increased CD4(+) T and CD8(+) T-cell effector and memory responses and higher intratumoral CD8(+) T effector/CD4(+)CD25(+)Foxp3(+) T regulatory cell ratio. Thus, potent pleiotropic immune functions of SA-4-1BBL combined with its ability to serve as a vehicle to increase the delivery of antigens to DCs in vivo endow this molecule with the potential to serve as an effective immunomodulatory component of therapeutic vaccines against cancer and chronic infections.

Coordinated changes in mRNA turnover, translation, and RNA processing bodies in bronchial epithelial cells following inflammatory stimulation.

Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover.

In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon-containing unstable beta-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.

Plasmid host-range: restrictions to F replication in Pseudomonas.

Host-range, a fundamental property of a bacterial plasmid, is primarily determined by the plasmid replication system. To investigate the basis of the restricted host-range of the well-studied F-plasmid of Escherichia coli, we characterized in vitro the interactions of the host DnaA initiation protein and DnaB helicase from Pseudomonas aeruginosa and Pseudomonas putida with the replication origin, oriS, and initiation protein, RepE, of the RepFIA replicon. The results presented here show that a pre-priming complex can form at the F-origin with the replication proteins from the non-native hosts in the presence of RepE. However, RepE cannot form a stable complex with DnaB of P. aeruginosa or P. putida but does stably interact with E. coli DnaB. This unstable association may affect the ability of F to replicate in Pseudomonas. In addition, replication studies in vivo suggest that inefficient expression of the RepE initiation protein from its native promoter in Pseudomonas is a factor in restricting its host-range. This, however, is not the only barrier to F replication, as mini-F derivatives with an alternative promoter for RepE expression do not replicate in P. putida and are not stably maintained in P. aeruginosa.

Trafficking of ODV-E66 is mediated via a sorting motif and other viral proteins: facilitated trafficking to the inner nuclear membrane.

The N-terminal 33 aa of the envelope protein ODV-E66 are sufficient to traffic fusion proteins to intranuclear membranes and the ODV envelope during infection with Autographa californica nucleopolyhedrovirus. This sequence has two distinct features: (i) an extremely hydrophobic sequence of 18 aa and (ii) positively charged amino acids close to the C-terminal end of the hydrophobic sequence. In the absence of infection, this sequence is sufficient to promote protein accumulation at the inner nuclear membrane. Covalent cross-linking results show that the lysines of the motif are proximal to FP25K and/or BV/ODV-E26 during transit from the endoplasmic reticulum to the nuclear envelope. We propose that the 33 aa comprise a signature for sorting proteins to the inner nuclear membrane (sorting motif) and that, unlike other resident proteins of the inner nuclear membrane, ODV-E66 and sortingmotif fusions do not randomly diffuse from their site of insertion at the endoplasmic reticulum to the nuclear envelope and viral-induced intranuclear membranes. Rather, during infection, trafficking is mediated by protein-protein interactions.

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin.

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b.

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli.

Targeting of DNA molecules to specific subcellular positions is essential for efficient segregation, but the mechanisms underlying these processes are poorly understood. In Escherichia coli, several plasmids belonging to different incompatibility groups (F, P1 and RK2) localize preferentially near the midcell and quartercell positions. Here we compare the relative positions of these three plasmids using fluorescence in situ hybridization. When plasmids F and P1 were localized simultaneously using differentially labeled probes, the majority of foci (approximately 75%) were well separated from each other. Similar results were found when we compared the subcellular localization of F with RK2, and RK2 with P1: regardless of the number of foci per cell or growth conditions, most of the foci (70-80%) were not in close proximity to one another. We also localized RK2 in Pseudomonas aeruginosa and Vibrio cholerae, and found that plasmid RK2 localization is conserved across bacterial species. Our results suggest that each plasmid has its own unique subcellular address, implying a mechanism for the stable co-existence of plasmids in which subcellular targeting plays a major role.

A 50-kb plasmid rich in mobile gene sequences isolated from a marine micrococcus.

A 50,709-bp cryptic plasmid isolated from a marine Micrococcus has been sequenced and found to contain a number of putative mobile genetic elements. The coding regions for 11 putative transposases comprise approximately 17% of the total plasmid sequence. The majority of these transposases are located within a 13-kb cluster which includes a 1553-bp direct repeat consisting of a duplicated pair of transposase genes. The remaining putative ORFs showed similarity to a variety of proteins, the most notable being spider silk.