Measurements of the dielectric properties of peripheral blood mononuclear cells and trophoblast cells using AC electrokinetic techniques

The separation of trophoblast cells from the maternal circulation could provide a valuable diagnostic tool for prenatal diagnosis of genetic abnormalities. This has been attempted using antibody methods, but due to non-specificity of the antibodies, maternal cell contamination remains a problem. We have investigated the potential of dielectrophoretic separation methods as a means of isolating trophoblast cells from mixed peripheral blood mononuclear cells. To determine the potential of this method the dielectric properties of trophoblast cells and mixed peripheral blood mononuclear cells were measured using dielectrophoretic crossover and single cell electrorotation methods. Both dielectrophoretic crossover data and electrorotation data gave an average specific membrane capacitance of the peripheral blood mononuclear cells of 11.5 mF m(-2). Trophoblast cells prepared using three different methods had a higher average specific membrane capacitance in the range 13-18 mF m(-2). The differences in capacitance between the cell types could be exploited as the basis of an AC electrokinetic-based system for the separation of trophoblast cells from peripheral blood mononuclear cells.     

A cell array biosensor for environmental toxicity analysis

In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.     

Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles

This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40-200 nm as determined using photon correlation spectroscopy (PCS) and 40-80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding beta-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect and Lipofectin were also used. In contrast to chitosan particles, the duration of expression for both SuperFect and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect and Lipofectin however, were observed to engender marked cytotoxicity. Poly-D,L-lactide (PLA) nanoparticles (40-80 nm) and poly-L-lactide (PLLA) lamellae (2-6 microm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.     

Enhancement of the anti-herpetic effect of trichosanthin by acyclovir and interferon

Trichosanthin (TCS) is a type I ribosome-inactivating protein that has a wide range of pharmacological activities. The present study investigated the effectiveness of TCS on herpes simplex virus (HSV-1). The anti-viral activity and toxicity of TCS on Vero cells were measured. Results showed that the ED(50), TD(50) and the therapeutic indices were 38.5, 416.5 and 10.9 microg/ml, respectively. Anti-viral activity of TCS was substantially potentiated when it was used in conjunction with other anti-viral agents. The ED(50) of TCS was reduced 125-fold by acyclovir at a concentration of 0.001 microg/ml, which was practically devoid of significant anti-viral activity. Similarly, the ED(50) of TCS was reduced 100-fold by interferon-alpha2a at a concentration of 100 IU/ml. In conclusion, TCS is effective against HSV-1 and other anti-viral agents such as acyclovir or interferon can potentiate its action substantially.     

Metabolic engineering of noviose: heterologous expression of novWUS and generation of a new hybrid antibiotic, noviosylated 10-deoxymethynolide/narbonolide, from Streptomyces venezuelae YJ003-OTBP1

NovW, novU and novS genes have been characterized as dTDP-4-keto-6-deoxy-D-glucose 3-epimerase, C-5 methyltransferase and dTDP-glucose 4-ketoreductase, respectively involved in noviose biosynthetic pathway. We have cloned and expressed the Streptomyces spheroids novWUS genes in S. venezuelae YJ003-OTBP1. This established the function of novWUS and, at the same time, it also proved that the noviosyl derivative of 10-deoxymethynolide(2)/narbonolide(4) obtained from S. venezuelae YJ003-OTBP1 is a novel hybrid antibiotic.     

Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice.

The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP-1-like cis-elements in the beta-globin locus control region. Three members, p45-Nf-e2, Nrf-1 and Nrf-2 have been identified in mammals. Expression of p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide range of tissues. To determine the function of Nrf-1, targeted disruption of the Nrf-1 gene was carried out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive erythropoiesis and die in utero.     

Mode of action investigation for the antibacterial cationic anthraquinone analogs

Reported previously by our group, we have developed a novel class of antibacterial cationic anthraquinone analogs with superb potency (MIC <1μg/mL) against Gram positive (G+) pathogens including Methicillin-resistant Staphylococcus aureus (MRSA). However, most of these compounds only manifest modest antibacterial activity against Gram negative (G-) bacteria. Further investigation on the antibacterial mode of action using fluorogenic dyes reveals that these compounds exert two different modes of action that account for the difference in their antibacterial profile. It was found that most of the compounds exert their antibacterial activity by disrupting the redox processes of bacteria. At high concentration, these compounds can also act as membrane disrupting agents. This information can help to design new therapeutics against various bacteria.     

DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.     

Heat Shock Stress Induces Cleavage and Activation of PAK2 in Apoptotic Cells

Heat shock induces a stress response in mammalian cells and can also lead to apoptotic cell death. Here we report that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be drastically activated in several cell types by heat shock. Immunoblot analysis revealed that this 36-kDa MBP kinase can be recognized by an antibody against the C-terminal region of a family of p21^Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as tools, we further demonstrated that heat shock can induce cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment in mouse Balb/c 3T3 and human Hep 3B cells. The kinetic profile of appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in these cells induced by heat shock. In addition, the heat shock-induced cleavage and activation of PAK2 was found to be closely associated with both DNA fragmentation and activation of an ICE/CED-3 family cysteine protease termed caspase-3 in heat shock-treated Hep 3B cells. Moreover, blockage of the activation of caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors of caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could substantially diminish the extent of heat shock-induced cleavage/activation of PAK2. Overall, our results point out that PAK2 is cleaved and activated during the heat shock-induced apoptotic cell death process and suggest that caspase-3 is involved in this process.     

Modelling a Wolbachia Invasion Using a Slow–Fast Dispersal Reaction–Diffusion Approach

This paper uses a reaction–diffusion approach to examine the dynamics in the spread of a Wolbachia infection within a population of mosquitoes in a homogeneous environment. The formulated model builds upon an earlier model by Skalski and Gilliam (Am. Nat. 161(3):441–458, 2003), which incorporates a slow and fast dispersal mode. This generates a faster wavespeed than previous reaction–diffusion approaches, which have been found to produce wavespeeds that are unrealistically slow when compared with direct observations. In addition, the model incorporates cytoplasmic incompatibility between male and female mosquitoes, which creates a strong Allee effect in the dynamics. In previous studies, linearised wavespeeds have been found to be inaccurate when a strong Allee effect is underpinning the dynamics. We provide a means to approximate the wavespeed generated by the model and show that it is in close agreement with numerical simulations. Wavespeeds are approximated for both Aedes aegypti and Drosophila simulans mosquitoes at different temperatures. These wavespeeds indicate that as the temperature decreases within the optimal temperature range for mosquito survival, the speed of a Wolbachia invasion increases for Aedes aegypti populations and decreases for Drosophila simulans populations.