Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 +/- 2.6 ng/ml at day 9. Maximal NGF production was 8.46 +/- 2.1 pg/10(3) cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p 相似文献   

Age-related decline in carriage of ampicillin-resistant Escherichia coli in young calves

The presence of ampicillin-resistant Escherichia coli (Amp(r) E. coli) in the fecal flora of calves was monitored on a monthly basis in seven cohorts of calves. Calves were rapidly colonized by Amp(r) E. coli, with peak prevalence in cohort calves observed in the 4 months after the calves were born. The prevalence of calves yielding Amp(r) E. coli in cohorts consistently declined to low levels with increasing age of the calves (P < 0.001).     

Integrative bioinformatics for functional genome annotation: trawling for G protein-coupled receptors

G protein-coupled receptors (GPCR) are amongst the best studied and most functionally diverse types of cell-surface protein. The importance of GPCRs as mediates or cell function and organismal developmental underlies their involvement in key physiological roles and their prominence as targets for pharmacological therapeutics. In this review, we highlight the requirement for integrated protocols which underline the different perspectives offered by different sequence analysis methods. BLAST and FastA offer broad brush strokes. Motif-based search methods add the fine detail. Structural modelling offers another perspective which allows us to elucidate the physicochemical properties that underlie ligand binding. Together, these different views provide a more informative and a more detailed picture of GPCR structure and function. Many GPCRs remain orphan receptors with no identified ligand, yet as computer-driven functional genomics starts to elaborate their functions, a new understanding of their roles in cell and developmental biology will follow.     

Computer simulations improve university instructional laboratories

Laboratory classes are commonplace and essential in biology departments but can sometimes be cumbersome, unreliable, and a drain on time and resources. As university intakes increase, pressure on budgets and staff time can often lead to reduction in practical class provision. Frequently, the ability to use laboratory equipment, mix solutions, and manipulate test animals are essential learning outcomes, and "wet" laboratory classes are thus appropriate. In others, however, interpretation and manipulation of the data are the primary learning outcomes, and here, computer-based simulations can provide a cheaper, easier, and less time- and labor-intensive alternative. We report the evaluation of two computer-based simulations of practical exercises: the first in chromosome analysis, the second in bioinformatics. Simulations can provide significant time savings to students (by a factor of four in our first case study) without affecting learning, as measured by performance in assessment. Moreover, under certain circumstances, performance can be improved by the use of simulations (by 7% in our second case study). We concluded that the introduction of these simulations can significantly enhance student learning where consideration of the learning outcomes indicates that it might be appropriate. In addition, they can offer significant benefits to teaching staff.     

Evidence of glutathione transferase complexing and signaling in the model nematode Caenorhabditis elegans using a pull-down proteomic assay

Phage display techniques using random peptide interactions have supported the role of mammalian glutathione transferase (GST) as part of a signalling pathway for both oxidative stress and an apoptosis pathway. Little is known about the interaction of nonmammalian GST with other proteins. GSTs have been implicated in the development of chronic nematode infections by neutralising cytotoxic products arising from host immune initiated reactive oxygen species (ROS) assault. In this study we attached one of the key GSTs expressed in the model nematode Caenorhabditis elegans to an affinity support matrix and directly identified major interacting proteins by two-dimensional electrophoresis and peptide mass fingerprinting before and following oxidative stress. Nematode GST does not appear to be a stand-alone enzyme and interacts with many types of proteins in both normal and ROS stress conditions. Pull-down proteomic presents a flexible, label free, rapid and economical assay without specialised ligand fishing equipment to identify protein binding partners.     

Fish as Bioreactors: Transgene Expression of Human Coagulation Factor VII in Fish Embryos

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.     

Generation of marker-free transgenic maize by regular two-border <Emphasis Type="Italic">Agrobacterium</Emphasis> transformation vectors

By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome. This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation. In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders. These results also provide evidence that both the right and left borders can initiate and terminate T-strands. Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions.     

BPROMPT: A consensus server for membrane protein prediction

Protein structure prediction is a cornerstone of bioinformatics research. Membrane proteins require their own prediction methods due to their intrinsically different composition. A variety of tools exist for topology prediction of membrane proteins, many of them available on the Internet. The server described in this paper, BPROMPT (Bayesian PRediction Of Membrane Protein Topology), uses a Bayesian Belief Network to combine the results of other prediction methods, providing a more accurate consensus prediction. Topology predictions with accuracies of 70% for prokaryotes and 53% for eukaryotes were achieved. BPROMPT can be accessed at http://www.jenner.ac.uk/BPROMPT.     

Maternal condition, yolk androgens and offspring performance: a supplemental feeding experiment in the lesser black-backed gull (Larus fuscus)

It has been proposed that the maternal androgens in avian egg yolk enhance offspring fitness by accelerating growth and improving competitive ability. Because egg quality is strongly influenced by maternal condition, we predicted that females in good condition would produce high-quality eggs with relatively high androgen content. We experimentally enhanced maternal condition by supplementary feeding lesser black-backed gulls (Larus fuscus) during egg formation and compared the concentrations of androstenedione (A4), 5alpha-dihydrotestosterone (DHT) and testosterone (T) in their eggs with those in eggs laid by control females. We also measured circulating levels of T in females immediately after laying. Egg androgens could affect offspring performance directly through chick development and/or indirectly through changes in the competitive ability of a chick relative to its siblings. To avoid confounding these two routes, and to separate effects operating through the egg itself with those operating through experimental changes in parental chick rearing capacity, we fostered eggs from both maternal treatment groups singly into the nests of unmanipulated parents. Contrary to expectation, mothers with experimentally enhanced body condition laid eggs with lower levels of androgens, while exhibiting higher circulating T concentrations post-laying. Despite these lower levels of egg androgen, offspring hatched from eggs laid by mothers in good condition did not show reduced growth or survival when reared in the absence of sibling competition. Our results demonstrate that yolk androgen concentrations vary with the body condition of the female at the time of egg formation and that females in good condition reduced the yolk androgen content of their eggs without altering offspring performance.     

Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86

There is great interest in understanding the role of costimulatory molecules in immune activation. In both the influenza and HIV DNA immunization models, several groups have reported that coimmunization of mice with plasmids encoding immunogen and CD86, but not CD80, effectively boosts Ag-specific T cell activation. This difference in immune priming provided an opportunity to examine the functional importance of different regions of the B.7 molecules in immune activation. To examine this issue, we developed a series of chimeric CD80 and CD86 constructs as well as deletion mutants, and examined their immune activating potential in the DNA vaccine model. We demonstrate that the lack of an Ig constant-like region in the CD80 molecule is critically important to the enhanced immune activation observed. CD80 C-domain deletion mutants induce a highly inflammatory Ag-specific cellular response when administered as part of a plasmid vaccine. The data suggest that the constant-like domains, likely through intermolecular interactions, are critically important for immune regulation during costimulation and that engineered CD80/86 molecules represent more potent costimulatory molecules and may improve vaccine adjuvant efficacy.