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.     

On community structure in high alpine grasslands

Previously published data on production biology, energy use efficiency, nutrient availability and vulnerability were reinterpreted for the explanation of community processing and structure in high altitude grasslands of the Alps. The final goal of the approach was to present a characterization of these plant communities in generalized terms.The keystone-concept was applied, and the alpine grasslands then defined as plant communities with one or a few keystone species. Associated species show a varying degree of interdependence from these keystones. They form guilds exploiting resources not needed by the latter. As many of these species belong to these groups, and alpine grasslands are commonly species rich, a high functional redundancy is typical for these communities.     

Effects of GABA on acutely isolated neurons from the gustatory zone of the rat nucleus of the solitary tract

Responses of acutely isolated neurons from the rostral nucleus of the solitary tract (rNST) to GABA receptor agonists and antagonists were investigated using whole-cell recording in current clamp mode. The isolated neurons retain their morphology and can be divided into multipolar, elongate and ovoid cell types. Most rNST neurons (97%), including all three cell types, respond to GABA with membrane hyperpolarization and a reduction in input resistance. The GABA(A) receptor agonist muscimol reduces neuronal input resistance in a concentration-dependent manner, whereas the GABA(B) receptor agonist baclofen had no effect on any of the neurons tested. The GABA and muscimol reversal potentials were both found to be -75 mV Both the GABA competitive antagonist picrotoxin and the GABA(A) receptor antagonist bicuculline block the effect of GABA in a concentration-dependent manner. These results suggest that GABA activates all neurons in the rNST and that inhibitory synaptic activity is important in brainstem processing of gustatory and somatosensory information.      

Replacement of receptor cells in the hamster vomeronasal epithelium after nerve transection

Chemoreceptor cells in the vomeronasal and olfactory epithelium are replaced following experimentally induced degeneration. This study analyzes quantitatively the time course and degree of vomeronasal receptor cell replacement. Unilateral transection of the vomeronasal nerves in adult hamster was used to induce a retrograde degeneration of receptor cells in the vomeronasal organ. Histological measurement of both number of receptor cells and epithelial thickness were made for recovery times from 0 to 60 days. After nerve transection, there was a gradual degeneration of receptor cells, the number decreasing to 50% of control by day 2 and 16% by day 6. During days 7-15 maximum receptor cell replacement was observed. Cell number increased rapidly and reached a peak on day 15. At recovery times of 40-60 days, cell number returned to the control level. Epithelial thickness, however, decreased to 60-70% during the degeneration period (days 4-6) and did not return to control levels. After 40-60 days epithelial thickness remained at 70% of control. These results demonstrate that vomeronasal receptor cells are replaced following degeneration, but epithelial thickness does not return to control levels. These findings suggest that the number of replacement cells is not limited by the reduced thickness of the epithelium, and that recovery mechanisms may function to restore an optimum number of receptor cells.      

Beiträge zur Flora der Umgebung von Innsbruck

Ohne Zusammenfassung     

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21^Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21^Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21^Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.