Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris

Pyranose dehydrogenase (PDH) is a fungal flavin-dependent sugar oxidoreductase that is highly interesting for applications in organic synthesis or electrochemistry. The low expression levels of the filamentous fungus Agaricus meleagris as well as the demand for engineered PDH make heterologous expression necessary. Recently, Aspergillus species were described to efficiently secrete recombinant PDH. Here, we evaluate recombinant protein production with expression hosts more suitable for genetic engineering. Expression in Escherichia coli resulted in no soluble or active PDH. Heterologous expression in the methylotrophic yeast Pichia pastoris was investigated using two different signal sequences as well as a codon-optimized sequence. A 96-well plate activity screening for transformants of all constructs was established and the best expressing clone was used for large-scale production in 50-L scale, which gave a volumetric yield of 223 mg L−1 PDH or 1,330 U L−1 d−1 in space–time yield. Purification yielded 13.4 g of pure enzyme representing 95.8% of the initial activity. The hyperglycosylated recombinant enzyme had a 20% lower specific activity than the native enzyme; however, the kinetic properties were essentially identical. This study demonstrates the successful expression of PDH in the eukaryotic host organism P. pastoris paving the way for protein engineering. Additionally, the feasibility of large-scale production of the enzyme with this expression system together with a simplified purification scheme for easy high-yield purification is shown

The effect of low-level laser irradiation (In-Ga-Al-AsP - 660 nm) on melanoma in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) <it>in vitro </it>and the <it>in vivo </it>development of melanoma in mice after laser irradiation.</p> <p>Methods</p> <p>We performed a controlled <it>in vitro </it>study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The <it>in vivo </it>mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm², irradiance 2.5 W/cm²and irradiation times of 60s (dose 150 J/cm²) and 420s (dose 1050 J/cm²) respectively.</p> <p>Results</p> <p>There were no statistically significant differences between the <it>in vitro </it>groups, except for an increase in the hypodiploid melanoma cells (8.48 ± 1.40% and 4.26 ± 0.60%) at 72 h post-irradiation. This cancer-protective effect was not reproduced in the <it>in vivo </it>experiment where outcome measures for the 150 J/cm²dose group were not significantly different from controls. For the 1050 J/cm²dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups.</p> <p>Conclusion</p> <p>LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm²) and high dose (1050 J/cm²) significantly increases melanoma tumor growth <it>in vivo</it>.</p

Long-term land-cover/use change in a traditional farming landscape in Romania inferred from pollen data, historical maps and satellite images

Traditional farming landscapes in the temperate zone that have persisted for millennia can be exceptionally species-rich and are therefore key conservation targets. In contrast to Europe’s West, Eastern Europe harbours widespread traditional farming landscapes, but drastic socio-economic and political changes in the twentieth century are likely to have impacted these landscapes profoundly. We reconstructed long-term land-use/cover and biodiversity changes over the last 150 years in a traditional farming landscape of outstanding species diversity in Transylvania. We used the Regional Estimates of Vegetation Abundance from Large Sites model applied to a pollen record from the Transylvanian Plain and a suite of historical and satellite-based maps. We documented widespread changes in the extent and location of grassland and cropland, a loss of wood pastures as well as a gradual increase in forest extent. Land management in the socialist period (1947–1989) led to grassland expansion, but grassland diversity decreased due to intensive production. Land-use intensity has declined since the collapse of socialism in 1989, resulting in widespread cropland abandonment and conversion to grassland. However, these trends may be temporary due to both ongoing woody encroachment as well as grassland management intensification in productive areas. Remarkably, only 8% of all grasslands existed throughout the entire time period (1860–2010), highlighting the importance of land-use history when identifying target areas for conservation, given that old-growth grasslands are most valuable in terms of biodiversity. Combining datasets from different disciplines can yield important additional insights into dynamic landscape and biodiversity changes, informing conservation actions to maintain these species-rich landscapes in the longer term

Disruption of Lateral Efferent Pathways: Functional Changes in Auditory Evoked Responses

The functional consequences of selectively lesioning the lateral olivocochlear efferent system in guinea pigs were studied. The lateral superior olive (LSO) contains the cell bodies of lateral olivocochlear neurons. Melittin, a cytotoxic chemical, was injected into the brain stem using stereotaxic coordinates and near-field evoked potentials to target the LSO. Brain stem histology revealed discrete damage to the LSO following the injections. Functional consequences of this damage were reflected in depressed amplitude of the compound action potential of the eighth nerve (CAP) following the lesion. Threshold sensitivity and N1 latencies were relatively unchanged. Onset adaptation of the cubic distortion product otoacoustic emission (DPOAE) was evident, suggesting a reasonably intact medial efferent system. The present results provide the first report of functional changes induced by isolated manipulation of the lateral efferent pathway. They also confirm the suggestion that changes in single-unit auditory nerve activity after cutting the olivocochlear bundle are probably a consequence of disrupting the more lateral of the two olivocochlear efferent pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41379/1/10162_2002_Article_3018.pd