Bioremediation: A perspective

In this opinion paper, we offer our perspective on our bioremediation research along with the methods to assess its effectiveness as a safe and beneficial technology to remediate selected soil sites. The isolation and characterization of bacterial isolates from chemically contaminated soils, their survival and catabolic activity in contaminated soil, toxicity testing in chemically contaminated soils, molecular‐based methods of detection such as the polymerase chain reaction (PCR) and DNA probing are discussed. By using numerous conventional microbiological, chemical techniques, and molecular based methods, bioremediation can be studied in a comprehensive manner and the technology transferred to the commercial sector.     

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

Ubiquitin‐Activated Interaction Traps (UBAITs) identify E3 ligase binding partners

We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (Ub iquitin‐A ctivated I nteraction T raps) are E3‐ubiquitin fusion proteins and, in an E1‐ and E2‐dependent manner, the C‐terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co‐purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester‐linked lariat intermediate or through an E2 thioester intermediate, and both WT and active‐site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double‐strand break repair. Using the RNF168 UBAIT, we identify H2AZ—a histone protein involved in DNA repair—as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.     

Characterization of humoral responses in mice immunized with plasmid DNAs encoding SARS-CoV spike gene fragments

The immunological characteristics of SARS-CoV spike protein were investigated by administering mice with plasmids encoding various S gene fragments. We showed that the secreting forms of S1, S2 subunits and the N-terminus of S1 subunit (residues 18-495) were capable of eliciting SARS-CoV specific antibodies and the region immediate to N-terminus of matured S1 protein contained an important immunogenic determinant for elicitation of SARS-CoV specific antibodies. In addition, mice immunized with plasmids encoding S1 fragment developed a Th1-mediated antibody isotype switching. Another interesting finding was that mouse antibodies elicited separately by plasmids encoding S1 and S2 subunits cooperatively neutralized SARS-CoV but neither the S1 nor S2 specific antibodies did, suggesting the possible role of both S1 and S2 subunits in host cell docking and entry. These results provide insights into understanding the immunological characteristics of spike protein and the development of subunit vaccines against SARS-CoV.     

Intracellular trafficking of polyamidoamine-poly(ethylene glycol) block copolymers in DNA delivery

The delivery of nucleic acids has the potential to revolutionize medicine by allowing previously untreatable diseases to be clinically addressed. Viral delivery systems have shown immunogenicity and toxicity dangers, but synthetic vectors have lagged in transfection efficiency. Previously, we developed a modular, linear-dendritic block copolymer architecture with high gene transfection efficiency compared to commercial standards. This rationally designed system makes use of a cationic dendritic block to condense the anionic DNA and forms complexes with favorable endosomal escape properties. The linear block provides biocompatibility and protection from serum proteins, and can be functionalized with a targeting ligand. In this work, we quantitate performance of this system with respect to intracellular barriers to gene delivery using both high-throughput and traditional approaches. An image-based, high-throughput assay for endosomal escape is described and applied to the block copolymer system. Nuclear entry is demonstrated to be the most significant barrier to more efficient delivery and will be addressed in future versions of the system.     

Complex predictive eye pursuit in monkey: a model system for cerebellar studies of skilled movement

Smooth pursuit eye movements provide a good model system for cerebellar studies of complex motor control in monkeys. First, the pursuit system exhibits predictive control along complex trajectories and this control improves with training. Second, the flocculus/paraflocculus region of the cerebellum appears to generate this control. Lesions impair pursuit and neural activity patterns are closely related to eye motion during complex pursuit. Importantly, neural responses lead eye motion during predictive pursuit and lag eye motion during non-predictable target motions that require visual control. The idea that flocculus/paraflocculus predictive control is non-visual is also supported by a lack of correlation between neural activity and retinal image motion during pursuit. Third, biologically accurate neural network models of the flocculus/paraflocculus allow the exploration and testing of pursuit mechanisms. Our current model can generate predictive control without visual input in a manner that is compatible with the extensive experimental data available for this cerebellar system. Similar types of non-visual cerebellar control are likely to facilitate the wide range of other skilled movements that are observed.     

The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers: HYBRID MOLECULES COMPOSED OF ACETYLCHOLINESTERASE AND BUTYRYLCHOLINESTERASE DIMERS ARE UP-REGULATED DURING DEVELOPMENT OF CHICKEN BRAIN*

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE_T and BChE_T with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q_N-GPI). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q_N-GPI always consist of AChE_T and BChE_T homodimers. The dimer formation of AChE_T and BChE_T depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal “t-peptides” in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE_T or BChE_T homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.     

Boats, Pathways, and Aquatic Biological Invasions: Estimating Dispersal Potential with Gravity Models

Biological invaders can have dramatic effects on the environment and the economy. To most effectively manage these invaders, we should consider entire pathways, because multiple species are dispersed through the same vectors. In this paper, we use production-constrained gravity models to describe movement of recreational boaters between lakes – potentially the most important pathway of overland dispersal for many aquatic organisms. These models are advantageous because they require relatively easily acquired data, hence are relatively easy to build. We compare linear and non-linear gravity models and show that, despite their simplicity, they are able to capture important characteristics of the recreational boater pathway. To assess our model, we compared observed data based on creel surveys and mailed surveys of recreation boaters to the model output. Specifically, we evaluate four metrics of pathway characteristics: boater traffic to individual lakes, distances traveled to reach these lakes, Great Lakes usage and movement from the Great Lakes to inland waters. These factors will influence the propagule pressure (hence the probability of establishment of invasive populations) and the rate of spread across a landscape. The Great Lakes are of particular importance because they are a major entry point of non-indigenous species from other continents, hence will act as the origin for further spread across states. The non-linear model had the best fit between model output and empirical observations with r² =0.80, r² =0.35, r² =0.57, and r² =0.36 for the four metrics, respectively. For the distances traveled to individual lakes, r² improved from 0.35 to 0.76 after removal of an outlier. Our results suggest that we were able to capture distances traveled to most but not all lakes. Thus, we demonstrate that production-constrained gravity models will be generally useful for modeling invasion pathways between non-contiguous locations.