Baculovirus surface display: construction and screening of a eukaryotic epitope library.

The baculovirus expression system was utilized to serve as a tool for ligand selection, demonstrating the applicability of the system to the generation and screening of eukaryotic expression libraries. The HIV-1-gp41 epitope 'ELDKWA', specific for the neutralizing human mAb 2F5, was inserted into the antigenic site B of influenza virus hemagglutinin and expressed on the surface of baculovirus infected insect cells. In order to improve the antigenicity of the epitope within the hemagglutinin, and therefore enhance the specific binding of 2F5, we inserted three additional, random amino acids adjacent to the epitope. This pool of hemagglutinin genes was directly cloned into the baculovirus Ac-omega. To identify distinct proteins displayed on the cellular surface, we developed a screening protocol to select for specific binding capacity of individual viral clones. Using fluorescence activated cell sorting (FACS) we isolated a baculovirus clone displaying the epitope with markedly increased binding capacity out of a pool of 8000 variants in only one sorting step. Binding properties of the identified ligand were examined by FACS performing a competition assay.     

Developmental and wound-, cold-, desiccation-, ultraviolet-B-stress-induced modulations in the expression of the petunia zinc finger transcription factor gene ZPT2-2

The ZPT2-2 gene belongs to the EPF gene family in petunia (Petunia hybrida), which encodes proteins with TFIIIA-type zinc-finger DNA-binding motifs. To elucidate a possible function for ZPT2-2, we analyzed its pattern of expression in relation to different developmental and physiological stress signals. The activity of the ZPT2-2 promoter was analyzed using a firefly luciferase (LUC) reporter gene, allowing for continuous measurements of transgene activity in planta. We show that ZPT2-2::LUC is active in all plant tissues, but is strongly modulated in cotyledons upon germination, in leaves in response to desiccation, cold treatment, wounding, or ultraviolet-B light, and in petal tissue in response to pollination of the stigma. Analysis of mRNA levels indicated that the modulations in ZPT2-2::LUC expression reflect modulations in endogenous ZPT2-2 gene expression. The change in ZPT2-2::LUC activity by cold treatment, wounding, desiccation, and ultraviolet-B light suggest that the phytohormones ethylene and jasmonic acid are involved in regulating the expression of ZPT2-2. Although up-regulation of expression of ZPT2-2 can be blocked by inhibitors of ethylene perception, expression in plants is not induced by exogenously applied ethylene. The application of jasmonic acid does result in an up-regulation of gene activity and, thus, ZPT2-2 may play a role in the realization of the jasmonic acid hormonal responses in petunia.     

Characterization of a new degradable polymer scaffold for regeneration of the dermis: In vitro and in vivo human studies

Full thickness skin wounds in humans heal with scars, but without regeneration of the dermis. A degradable poly(urethane urea) scaffold (PUUR), Artelon® is already used to reinforce soft tissues in orthopaedics, and for treatment of osteoarthritis of the hand, wrist and foot. In this paper we have done in vitro experiments followed by in vivo studies to find out whether the PUUR is biocompatible and usable as a template for dermal regeneration. Human dermal fibroblasts were cultured on discs of PUUR, with different macrostructures (fibrous and porous). They adhered to and migrated into the scaffolds, and produced collagen. The porous scaffold was judged more suitable for clinical applications and 4 mm Ø, 2 mm-thick discs of porous scaffold (12% w/w or 9% w/w polymer solution) were inserted intradermally in four healthy human volunteers. The implants were well tolerated and increasing ingrowth of fibroblasts was seen over time in all subjects. The fibroblasts stained immunohistochemically for procollagen and von Willebrand factor, indicating neocollagenesis and angiogenesis within the scaffolds. The PUUR scaffold may be a suitable material to use as a template for dermal regeneration.Key words: dermal regeneration, tissue engineering, polymer scaffold, wound healing, in vitro, in vivo, guided tissue regeneration, human, burns     

Rational Vector Design for Efficient Non-viral Gene Delivery: Challenges Facing the Use of Plasmid DNA

Although non-viral gene delivery is a very straightforward technology, there are currently no FDA-approved gene medicinal products available. Therefore, improving potency, safety, and efficiency of current plasmid DNA vectors will be a major task for the near future. This article will provide an overview on factors influencing production yield and quality as well as safety issues that emerge from the vector design itself. Special focus will be on generating bacterial pDNA vectors by circumventing the use of antibiotic resistance genes, to generate safer gene medicinal products as well as smaller, more efficient DNA vectors.     

Baculovirus display strategies: Emerging tools for eukaryotic libraries and gene delivery.

Recombinant baculoviruses have been extensively used as vectors for abundant expression of a large variety of foreign proteins in insect cell cultures. The appeal of the system lies essentially in easy cloning techniques and virus propagation combined with the eukaryotic post-translational modification machinery of the insect cell. Recently, a novel molecular biology tool was established by the development of baculovirus surface display, using different strategies for presentation of foreign peptides and proteins on the surface of budded virions. This eukaryotic display system enables presentation of large complex proteins on the surface of baculovirus particles and has thereby become a versatile system in molecular biology. Surface display strategies play an important role, as they may be used to enhance the efficiency and specificity of viral binding and entry to mammalian cells. In addition, baculovirus surface display vectors have been engineered to contain mammalian promoter elements designed for gene delivery both in vitro and in vivo. Moreover, baculovirus capsid display has recently been developed; this holds promise for intracellular targeting of the viral capsid and subsequent cytosolic delivery of desired protein moieties. Finally, the viruses can accommodate large insertions of foreign DNA and replicate only in insect cells. Together, these are attributes that are very likely to make them important tools in functional genomics and proteomics.     

SIMPROT: Using an empirically determined indel distribution in simulations of protein evolution

Background  

General protein evolution models help determine the baseline expectations for the evolution of sequences, and they have been extensively useful in sequence analysis and for the computer simulation of artificial sequence data sets.     

The effects of salbutamol on epithelial ion channels depend on the etiology of acute respiratory distress syndrome but not the route of administration

Introduction

We investigated the effects of intravenous and intratracheal administration of salbutamol on lung morphology and function, expression of ion channels, aquaporin, and markers of inflammation, apoptosis, and alveolar epithelial/endothelial cell damage in experimental pulmonary (p) and extrapulmonary (exp) mild acute respiratory distress syndrome (ARDS).

Methods

In this prospective randomized controlled experimental study, 56 male Wistar rats were randomly assigned to mild ARDS induced by either intratracheal (n = 28, ARDSp) or intraperitoneal (n = 28, ARDSexp) administration of E. coli lipopolysaccharide. Four animals with no lung injury served as controls (NI). After 24 hours, animals were anesthetized, mechanically ventilated in pressure-controlled mode with low tidal volume (6 mL/kg), and randomly assigned to receive salbutamol (SALB) or saline 0.9% (CTRL), intravenously (i.v., 10 μg/kg/h) or intratracheally (bolus, 25 μg). Salbutamol doses were targeted at an increase of ≈ 20% in heart rate. Hemodynamics, lung mechanics, and arterial blood gases were measured before and after (at 30 and 60 min) salbutamol administration. At the end of the experiment, lungs were extracted for analysis of lung histology and molecular biology analysis. Values are expressed as mean ± standard deviation, and fold changes relative to NI, CTRL vs. SALB.

Results

The gene expression of ion channels and aquaporin was increased in mild ARDSp, but not ARDSexp. In ARDSp, intravenous salbutamol resulted in higher gene expression of alveolar epithelial sodium channel (0.20 ± 0.07 vs. 0.68 ± 0.24, p < 0.001), aquaporin-1 (0.44 ± 0.09 vs. 0.96 ± 0.12, p < 0.001) aquaporin-3 (0.31 ± 0.12 vs. 0.93 ± 0.20, p < 0.001), and Na-K-ATPase-α (0.39 ± 0.08 vs. 0.92 ± 0.12, p < 0.001), whereas intratracheal salbutamol increased the gene expression of aquaporin-1 (0.46 ± 0.11 vs. 0.92 ± 0.06, p < 0.001) and Na-K-ATPase-α (0.32 ± 0.07 vs. 0.58 ± 0.15, p < 0.001). In ARDSexp, the gene expression of ion channels and aquaporin was not influenced by salbutamol. Morphological and functional variables and edema formation were not affected by salbutamol in any of the ARDS groups, regardless of the route of administration.

Conclusion

Salbutamol administration increased the expression of alveolar epithelial ion channels and aquaporin in mild ARDSp, but not ARDSexp, with no effects on lung morphology and function or edema formation. These results may contribute to explain the negative effects of β2-agonists on clinical outcome in ARDS.     

Enhancement of solubility in <Emphasis Type="Italic">Escherichia coli</Emphasis> and purification of an aminotransferase from <Emphasis Type="Italic">Sphingopyxis</Emphasis> sp. MTA144 for deamination of hydrolyzed fumonisin B<Subscript>1</Subscript>

Background  

Fumonisin B₁ is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B₁, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B₁. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity.