Ablation of Doublecortin-Like Kinase 1 in the Colonic Epithelium Exacerbates Dextran Sulfate Sodium-Induced Colitis

Doublecortin-like kinase 1 (Dclk1), a microtubule-associated kinase, marks the fifth lineage of intestinal epithelial cells called tuft cells that function as tumor stem cells in Apc mutant models of colon cancer. In order to determine the role of Dclk1 in dextran sulfate sodium (DSS) induced colonic inflammation both intestinal epithelial specific Dclk1 deficient (Villin^Cre;Dclk1^f/f) and control (Dclk1^f/f) mice were fed 3% DSS in drinking water for 9 days, allowed to recover for 2 days, and killed. The clinical and histological features of DSS-induced colitis were scored and immunohistochemical, gene expression, pro-inflammatory cytokines/chemokines, and immunoblotting analyses were used to examine epithelial barrier integrity, inflammation, and stem and tuft cell features. In DSS-induced colitis, Villin^Cre;Dclk1^f/f mice demonstrated exacerbated injury including higher clinical colitis scores, increased epithelial barrier permeability, higher levels of pro-inflammatory cytokines and chemokines, decreased levels of Lgr5, and dysregulated Wnt/b-Catenin pathway genes. These results suggest that Dclk1 plays an important role in regulating colonic inflammatory response and colonic epithelial integrity.     

Actin networks regulate the cell membrane permeability during electroporation

Transient physical disruption of cell membranes by electric pulses (or electroporation) has significance in biomedical and biological applications requiring the delivery of exogenous (bio)molecules to living cells. We demonstrate that actin networks regulate the cell membrane permeability during electroporation. Disruption of actin networks increases the uptake of membrane-impermeable molecules such as propidium iodide during electroporation. Our experiments at different temperatures ranging from 11 °C to 37 °C show that molecular uptake during electroporation increases with temperature. Furthermore, by examining the temperature-dependent kinetics of propidium iodide uptake, we infer that the activation energy barrier of electroporation is lowered when the actin networks are disrupted. Our numerical calculations of transmembrane voltage show that the reduced activation energy barrier for the cells with disrupted actin is not a consequence of the changes in transmembrane voltage associated with changes in the cell shape due to the disruption of actin, indicating that this could be due to changes in membrane mechanical properties. Our results suggest that the current theoretical models of electroporation should be advanced further by including the contributions of the cytoskeletal networks on the cell membrane permeability during the delivery of exogenous materials.     

Bacoside A downregulates matrix metalloproteinases 2 and 9 in DEN‐induced hepatocellular carcinoma

Cancer metastasis is a complex multi‐step process, responsible for a majority of cancer‐related deaths by affecting the critical organs and causing complications in therapies. Hepatocellular carcinoma is a multi‐factorial disease and is the third most common cause of cancer related mortality worldwide. Clinical and experimental studies have shown that MMP‐2 and MMP‐9 are involved in tumor invasion and metastases and their elevated expression has been associated with poor prognosis. Our recent studies showed a strong anti‐oxidant and hepatoprotective effects of bacoside A (BA) against carcinogen. Nevertheless the effect of BA on the activities and expression of MMP‐2 and MMP‐9 during hepatocellular carcinoma is not yet recognized. Therefore, the present study was designed to assess the same. Results of gelatin zymography study showed that BA co‐treatment significantly decreased the activities of MMP‐2 and MMP‐9, which is increased during hepatocellular carcinoma. Further immunoblot analysis showed decreased expression of MMP‐2 and MMP‐9 in rats co‐treated with BA compared to DEN‐induced hepatocellular carcinoma. Our results reveal that BA exerts its anti‐metastatic effect against DEN‐induced hepatocellular carcinoma by inhibiting the activities and expressions of MMP‐2 and MMP‐9. Copyright © 2010 John Wiley & Sons, Ltd.     

Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0.13 and 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, respectively. Through 1-photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.     

Properties of film from splendid squid (Loligo formosana) skin gelatin with various extraction temperatures

Properties of film from splendid squid (Loligo formosana) skin gelatin extracted at different temperatures (50-80°C) were investigated. Tensile strength (TS) and elongation at break (EAB) of films decreased, but water vapour permeability (WVP) increased (P<0.05) as the extraction temperature increased. Increase in transparency value with coincidental decrease in lightness was observed with increasing extraction temperatures. Electrophoretic study revealed that degradation of gelatin became more pronounced with increasing extraction temperatures. As a consequence, their corresponding films had the lower mechanical properties. FTIR spectra of obtained gelatin films revealed the significant loss of molecular order of the triple helix. Thermogravimetric analysis indicated that F80 exhibited the higher heat susceptibility and weight loss. Loosen structure was observed in film prepared from gelatin with increasing extraction temperatures. Thus, the temperature used for gelatin extraction from splendid squid skin directly affected the properties of corresponding films.     

Quantitative proteomics of heavy metal stress responses in Sydney rock oysters

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 μg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.     

Synthesis, antimicrobial and antioxidant evaluation of quinolines and bis(indolyl)methanes

An improved and practical synthesis of substituted quinolines and bis(indolyl)methanes was achieved under microwave condition using Zn(OTf)(2) as catalyst. The synthesized compounds have been screened for antimicrobial and antioxidant activities.     

Optimization of labile esters for esterase-assisted accumulation of nitroxides into cells: a model for in vivo EPR imaging

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.     

Properties and Characteristics of Multi-Layered Films from Tilapia Skin Gelatin and Poly(Lactic Acid)

Multi-layered films based on tilapia skin gelatin and poly(lactic acid) (PLA) were characterized, in comparison with the control gelatin and PLA films. Three different layers of multi-layered films (PLA/Gelatin/PLA) were visualized by scanning electron microscopic (SEM) analysis. The synergetic effect of lamination was evidenced by the increased mechanical properties (P < 0.05). Multi-layered films had higher water vapor barrier property and water resistance, compared to control gelatin film (P < 0.05). Gelatin films showed increased lightness (L*) with coincidental decrease in total color difference (?E*) in the presence of PLA layers (P < 0.05). Transparency and solubility of films decreased with increasing ratio of PLA (P < 0.05). In addition, multi-layered films showed the enhanced hydrophobicity and thermal stability as evidenced by increased water contact angle and degradation temperature, respectively. Thus, PLA/Gelatin/PLA multi-layered film with improved water vapour barrier property could serve as bio-degradable packaging material for wider applications.