Membrane interaction and activity of the glycolipid transfer protein

In this study we have addressed the ability of the glycolipid transfer protein (GLTP) to transfer anthrylvinyl-galactosylceramide at different pH and sodium chloride concentrations, and the ability of three different mutants to transfer the fluorescently labeled galactosylceramide between donor and acceptor model membranes. We constructed single tryptophan mutants with site-directed mutagenesis where two of the three tryptophan (W) of wild-type human GLTP were substituted with phenylalanine (F) and named W85 GLTP (W96F and W142F), W96 GLTP (W85F and W142F) and W142 GLTP (W85F and W96F) accordingly. Wild-type GLTP and W96 GLTP were both able to transfer anthrylvinyl-galactosylceramide, but the two variants W85 GLTP and W142 GLTP did not show any glycolipid transfer activity, indicating that the tryptophan in position 96 is crucial for transfer activity. Tryptophan fluorescence emission showed a blue shift of the maximal emission wavelength upon interaction of glycolipid containing vesicle with wild-type GLTP and W96 GLTP, while no blue shift was recorded for the protein variants W85 GLTP and W142 GLTP. The quantum yield of tryptophan emission was highest for the W96 GLTP protein whereas W85 GLTP, W142 GLTP and wild-type GLTP showed a lower and almost similar quantum yield. The lifetime and anisotropy decay of the different tryptophan mutants also changed upon binding to vesicles containing galactosylceramide. Again wild-type GLTP and W96 GLTP showed similar behavior in the presence of vesicles containing glycolipids. Taken together, our data show that the W96 is involved not only in the activity of the protein but also in the interaction between the protein and glycolipid containing membranes.     

Adenovirus type 5 intrinsic adsorption rates measured by surface plasmon resonance

Intrinsic adsorption rates of whole adenovirus type 5 (Ad5) onto a diethylaminoethyl (DEAE) anion exchange surface are measured for the first time by surface plasmon resonance (SPR). Fitting SPR sensorgrams to a two-compartment mass transport reaction model distinguishes intrinsic adsorption rates from slow diffusive Ad5 mass transport. Ad5 is a widely used viral vector for gene therapy that binds electrostatically to surfaces of cells and synthetics such as membranes, chromatographic resins, and glass. Increasing NaCl concentration from 4.8 to 14.4mM shifts binding of whole Ad5 from diffusion control to a regime where both sorption and diffusion affect binding. Intrinsic adsorption rates for Ad5-DEAE interaction are 16 times faster than intrinsic adsorption rates for Ad5 fiber knob interacting with soluble extracellular domain of coxsackievirus adenovirus receptors (s-CAR).     

A platform of high-efficiency nonviral gene transfer in mouse osteoblast cells in vitro

We have previously established mouse genetic models and identified the genetic components of quantitative trait loci (QTL) on mouse chromosomes that contribute to phenotypes such as bone size, bone density, and bone's anabolic response to mechanical loading. However, these regions contain dozens of unknown genes that are needed for functional testing. In this study, we provided a protocol of nucleoporation with high efficiency by using a commercial nucleofection buffer and Gene Pulser to deliver a test gene into bone cells for functional studies. We cloned an osteoblast differentiation-specific geneosterix (Osx) from a mouse bone cDNA library into a pHGCX expression vector and used nucleoporation to deliver pHGCX/Flag-Osx into the nuclei of MC3T3-E1 cells. We then examined the transfection efficiency transgene expression, and function. Our results have demonstrated that nucleoporation can deliver a transgene into MC3T3-E1 osteoblast cells with approx 94% transfection efficiency, and express a functional Flag-Osx fusion protein capable of inducing cell differentiation as measured by an incease in alkaline phosphatase (ALP) activity. Therefore, this experimental system provides a rapid, safe, and efficient cell-based model of high-throughput phenotypic screening to identify candidate genes from physically mapped regions that are important for osteoblast differentiation.     

Heterogeneous nuclear transfer embryos reconstructed by bovine oocytes and camel (Camelus bactrianus) skin fibroblasts and their subsequent development

Summary This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and the bovine oocytes as recipient cytoplasts to investigate the reprogramming of camel somatic cell nuclei in bovine oocyte cytoplasm and the developmental potential of the reconstructed embryos. Serum-starved skin fibroblast cells, obtained from adult camel, were electrically fused into enucleated bovine metaphase II (MII) oocytes that were matured in vitro. The fused eggs were activated by Inomycin with 2 mM/ml 6-dimethylaminopurine. The activated reconstructed embryos were cocultured with bovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum for 168 h. Results showed that 53% of the injected oocytes were successfully fused, 34% of the fused eggs underwent the first egg cleavage, and 100% of them developed to four- or 16-cell embryo stages. The first completed cleavage of xenonuclear transfer camel embryos occurred between 22 and 48 h following activation. This study demonstrated that the reconstructed embryos underwent the first embryonic division and that the reprogramming of camel fibroblast nuclei can be initiated in enucleated bovine MII oocytes.     

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

Crystal structure of NirD,the small subunit of the nitrite reductase NirbD from Mycobacterium tuberculosis at 2.0 Å resolution

NirD is part of the nitrite reductase complex NirBD that catalyses the reduction of nitrite to NH₃ in nitrate assimilation and anaerobic respiration. The crystal structure analysis of NirD from Mycobacterium tuberculosis shows a double β‐sandwich fold. NirD is related in three‐dimensional structure and sequence to the Rieske proteins; however, it does not contain any Fe–S cluster or other cofactors that might be involved in electron transfer. A cysteine residue at the protein surface, conserved in NirD homologues lacking the iron–sulfur cluster might be important for the interaction with NirB and possibly stabilize one of the Fe–S centers in this subunit. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Studying the thermal effects of a clinically-extracted vascular tissue during cryo-freezing

Cryosurgery is known to be a suitable treatment for unresectable tumors by employing extremely low temperature to induce cryo-lesion. However, large blood vessels in the tissue may induce insufficient freezing at the targeted area. In this work, we have developed a cryo-freezing model specifically dedicated to tumors with a complex blood vessel network taken from CT-scanned images. The model was validated with in-vitro experimental data. Adopting an appropriate mesh size, the simulated results achieved an excellent agreement with the experimental data at a maximum error of 3.4%. The validated model was applied to study an optimal cryotherapy in the treatment of a human liver. The movement of 265 K isotherm and the thermal influence of blood flow were investigated. Key results indicated that large neighboring vessels could significantly influence the shape of the ice fronts, but they exerted less impact on the development of the lethal temperature boundary. The freezing duration, distance to the vessel, blood flow rate and blood vessel size were observed to be key parameters in determining the optimal cryoablation. A case study further demonstrated that by precisely controlling the cryo-freezing process, up to 44.6% of the unintended tissue freezing could be achieved. Pragmatically, this work provides surgeons with essential information on how to precisely tune key parameters to promote greater surgical success.     

Structure-based phylogeny of polyene macrolide antibiotic glycosyltransferases

Antibiotic glycosyltransferases (AGts) attach unusual deoxy-sugars to aglycons so antibiotics can exert function. It has been reported that polyene macrolide (PEM) AGts have different evolutionary origin when compared with other polyketide AGts, and our previous analysis have suggested that they could be results of horizontal gene transfer (HGT) from eukaryotes. In this paper, we compared the structures of PEM AGts with structures of eukaryotes and other AGts, and then built models of the representative PEM AGts and GT-1 glycosyltransferases. We also constructed the Neighbor-Joining (NJ) trees based on the normalized Root Mean Square (RMS) distance, the Bayesian tree guided by structural alignments, and carried out analysis on several key conserved residues in PEM AGts. The NJ tree showed a close relationship between PEM AGts and eukaryotic glycosyltransferases, and Bayesian tree further supported their affinity with UDP-glucuronosyltransferases (UGTs). Analysis on key conserved residues showed that PEM AGts may have similar interaction mechanism such as in the formation of hydrogen bonds as eukaryotic glycosyltransferases. Using structure-based phylogenetic approaches, this study further supported that PEM AGts were the result of HGT between prokaryotes and eukaryotes.     

Spermine, a molecular switch regulating EGFR, integrin β3, Src, and FAK scaffolding

Intracellular polyamine levels are highly regulated by the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting reaction in polyamine biosynthesis, producing putrescine, which is subsequently converted to spermidine and spermine. We have shown that polyamines regulate proliferation, migration, and apoptosis in intestinal epithelial cells. Polyamines regulate key signaling events at the level of the EGFR and Src. However, the precise mechanism of action of polyamines is unknown. In the present study, we demonstrate that ODC localizes in lamellipodia and in adhesion plaques during cell spreading. Spermine regulates EGF-induced migration by modulating the interaction of the EGFR with Src. The EGFR interacted with integrin β3, Src, and focal adhesion kinase (FAK). Active Src (pY418-Src) localized with FAK during spreading and migration. Spermine prevented EGF-induced binding of the EGFR with integrin β3, Src, and FAK. Activation of Src and FAK was necessary for EGF-induced migration in HEK293 cells. EGFR-mediated Src activation in live HEK293 cells using a FRET based Src reporter showed that polyamine depletion significantly increased Src kinase activity. In vitro binding studies showed that spermine directly binds Src, and preferentially interacts with the SH2 domain of Src. The physical interaction between Src and the EGFR was severely attenuated by spermine. Therefore, spermine acts as a molecular switch in regulating EGFR-Src coupling both physically and functionally. Upon activation of the EGFR, integrin β3, FAK and Src are recruited to EGFR leading to the trans-activation of both the EGFR and Src and to the Src-mediated phosphorylation of FAK. The activation of FAK induced Rho-GTPases and subsequently migration. This is the first study to define mechanistically how polyamines modulate Src function at the molecular level.     

Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.