Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Targeted inhibition of the EGFR pathways enhances Zn‐BC‐AM PDT‐induced apoptosis in well‐differentiated nasopharyngeal carcinoma cells

Epidermal growth factor receptor (EGFR), a receptor often expressed in nasopharyngeal carcinoma (NPC) cells, is one of the recently identified molecular targets in cancer treatment. In the present study, the effects of combined treatment of Zn‐BC‐AM PDT with an EGFR inhibitor AG1478 were investigated. Well‐differentiated NPC HK‐1 cells were subjected to PDT with 1 µM of Zn‐BC‐AM and were irradiated at a light dose of 1 J/cm² in the presence or absence of EGFR inhibitor AG1478. Specific protein kinase inhibitors of downstream EGFR targets were also used in the investigation. EGFR, Akt, and ERK were found constitutively activated in HK‐1 cells and the activities could be inhibited by the EGFR inhibitor AG1478. A sub‐lethal concentration of AG1478 was found to further enhance the irreversible cell damage induced by Zn‐BC‐AM PDT in HK‐1 cells. Pre‐incubation of the cells with specific inhibitors of EGFR (AG1478), PI3k/Akt (LY294002), or MEK/ERK (PD98059) before light irradiation were found to enhance Zn‐BC‐AM PDT‐induced formation of apoptotic cells. The efficacy of Zn‐BC‐AM PDT can be increased through the inhibition of EGFR/PI3K/Akt and EGFR/MEK/ERK signaling pathways in NPC cells. Combination therapy with Zn‐BC‐AM PDT and EGFR inhibitors may further be developed for the treatment of advanced NPC. J. Cell. Biochem. 108: 1356–1363, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of sinomenine on gene expression of the IL-1 beta-activated human synovial sarcoma

Sinomenine is an alkaloid with pharmacological effects of anti-inflammation, anti-angiogenesis, anti-arthritis and immunosuppression. This study aimed to investigate the effect of sinomenine on gene expression of human synovial sarcoma cells (Hs701.T) activated by IL-1 beta. The proliferative effect of sinomenine was examined in the presence or absence of IL-1 beta by the [3H]-thymidine incorporation and MTT assay, respectively. Using DNA microarray technology and RT-PCR, the activating action of IL-1 beta and modulatory effect of sinomenine on Hs701.T were simultaneously determined. Results showed that IL-1 beta could stimulate the proliferation and gene expression of Hs701.T cells. Sinomenine could significantly inhibit proliferation of IL-1 beta-activated Hs701.T cells and suppress expression of 17 genes including IL-6, PlGF, Daxx, and HSP27. These genes were found to be important in tumor progression through the mediation of inflammation, cell adhesion, proliferation, apoptosis and angiogenesis. In conclusion, our study provides supplementary information for the further studies on the pharmacological effects of sinomenine acting on synovial sarcoma.     

The Effect of pH and Heat Pre-Treatments on the Physicochemical and Emulsifying Properties of β-lactoglobulin

The overall goal of this research was to investigate structure-function mechanisms associated the emulsifying properties β-lactoglobulin (β-LG). Specifically the physicochemical (i.e., surface charge and hydrophobicity, size and interfacial tension) and emulsifying (i.e., emulsification activity (EAI) and stability indices (ESI)) properties of β-LG were investigated in response to changes in pH (3.0, 5.0 and 7.0) and heat pre-treatment conditions (25, 55 and 85 °C). Hydrophobicity was found to be greatest at pH 5.0/85 °C, whereas at all conditions it was significantly lower. Surface charge on β-LG was found to be neutral at?~?pH 3.9, regardless of conditions. Aggregate size was also found to be highest at pH 5.0/85 °C (avg. hydrodynamic radii of ~714 nm), corresponding to a reduced net surface charge and high hydrophobicity. Little size dependence of aggregates was observed at pH 3.0 regardless to the temperature pre-treatments (radii ~120 nm). In contrast, at pH 7.0 slight temperature dependence was apparent, where treatments at 25, 55 and 85 °C led to radii of 412.8, 307.2 and 232.3 nm, respectively. Overall, the addition of β-LG to a canola oil–water system resulted in a decline in interfacial tension from ~28 mN/m to ~18 mN/m, however the effect of pH/temperature conditions was minimal. EAI was found to be highest when β-LG solutions displayed high surface charge combined with moderate hydrophobicity. In contrast, ESI was higher under conditions where β-LG solutions remained in a native (25 °C) or fully denatured state (85 °C) versus one in where partially unravelling may be occurring (55 °C).     

The Melanogenesis-Inhibitory Effect and the Percutaneous Formulation of Ginsenoside Rb1

Ginsenoside Rb1 (Rb1) is the most predominant ginsenoside isolated from the roots of ginseng (Panax ginseng C. A. Meyer). This compound is active in various human biological pathways that are involved in human collagen synthesis and inhibition of cell apoptosis. In this study, the skin-whitening effects of Rb1 were investigated in B16 melanoma cells. Our results showed that Rb1 inhibited melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 cells in a dose-dependent manner, which collectively indicated that Rb1 may have skin-whitening effects and may be formulated into skin-whitening products for skin care. Accordingly, a ginsenoside collagen transdermal patch was developed as a vehicle to topically deliver Rb1 into pig skin. The percutaneous permeation, retention within skin, and release in vitro of Rb1 from seven transdermal patch formulas were studied. It was determined that the best formula for ginsenoside collagen transdermal patch is made of protein collagen hydrolysate powder (PCHP) 2.0% (w/w), methyl cellulose (MC) 0.5% (w/w), polyethyleneglycol 6000 (PEG6000) 0.5% (w/w), ginsenoside 0.036% (w/w), azone 0.4% (v/w), menthol 0.20% (w/w), and water.     

Remotely Monitoring Change in Vegetation Cover on the Montebello Islands,Western Australia,in Response to Introduced Rodent Eradication

The Montebello archipelago consists of 218 islands; 80 km from the north-west coast of Western Australia. Before 1912 the islands had a diverse terrestrial fauna. By 1952 several species were locally extinct. Between 1996 and 2011 rodents and cats were eradicated, and 5 mammal and 2 bird species were translocated to the islands. Monitoring of the broader terrestrial ecosystem over time has been limited. We used 20 dry-season Landsat images from 1988 to 2013 and estimation of green fraction cover in nadir photographs taken at 27 sites within the Montebello islands and six sites on Thevenard Island to assess change in vegetation density over time. Analysis of data averaged across the 26-year period suggests that 719 ha out of 2169 ha have increased in vegetation cover by up to 32%, 955 ha have remained stable and 0.6 ha have declined in vegetation cover. Over 492 ha (22%) had no vegetation cover at any time during the period analysed. Chronological clustering analysis identified two breakpoints in the average vegetation cover data occurring in 1997 and 2003, near the beginning and end of the rodent eradication activities. On many islands vegetation cover was declining prior to 1996 but increased after rodents were eradicated from the islands. Data for North West and Trimouille islands were analysed independently because of the potential confounding effect of native fauna being introduced to these islands. Mala (Lagorchestes hirsutus) and Shark Bay mice (Pseudomys fieldi) both appear to suppress native plant recruitment but not to the same degree as introduced rodents. Future research should assess whether the increase in vegetation cover on the Montebello islands is due to an increase in native or introduced plants.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.