Synthesis and Biological Activity of Sugar-Fluorinated 2′,3′-dideoxy-4′-thioribofuranosyl Nucleosides

Abstract

The efficient DAST fluorination of deoxy-4′-thiopyrimidine nucleosides is reported. The cytidine analogue 3b was marginally effective against HIV.     

Overexpression of Neurospora crassa OR74A glutamate decarboxylase in Escherichia coli for efficient GABA production

This study investigated the effect of glutamate decarboxylase from Neurospora crassa OR74A on GABA production in Escherichia coli. GABA is one of the inhibitory neurotransmitters in the mammalian central nervous system, and can be used as a precursor of promising biopolymer Nylon 4. E. coli that overexpressed N. crassa glutamate decarboxylase was cultured at various pH levels and temperatures to determine optimum conditions for GABA production. When the recombinant E. coli strain was cultured at 30°C and pH 3, a final GABA concentration of 5.26 g/L was obtained from 10 g/L of monosodium glutamate (MSG), corresponding to a GABA yield of 86.23%.     

Change of arthropod abundance in burned forests: Different patterns according to functional guilds

Forest fires are one of the most frequent and important causes of forest disturbances, the occurrence of which is globally increasing due to the effects of climate change. This study aimed to determine the impacts of fire and human activity on arthropod communities in affected forests. Twelve study sites in three burned areas were selected for this study. Intensities of disturbance in the study sites were characterized as follows: Disturbance Degree (DD) 0 (no fire), DD 1 (surface fire), DD 2 (crown fire), and DD 3 (crown fire followed by reforestation). Arthropods were collected using pitfall traps. Fourteen arthropod taxa (families, orders or classes), which are relatively homogeneous in their feeding habits and abundant, were analyzed. Depth of litter layer was selected as an environmental indicator for disturbance intensity, as it decreases linearly as the degree of disturbance increased. Changes of arthropod abundance in response to disturbance differed among functional guilds. As disturbance intensity increased, the abundance of detritivores decreased, but the abundance of herbivores increased. However, the abundance of predators varied between taxa. Formicidae and Araneae increased in disturbed sites, whereas Carabidae and Staphylinidae did not change. The abundance of Thysanura and Diptera was highly correlated with disturbance intensity, and may be suitable as a bioindicator for forest disturbance. Arthropod communities were more heterogeneous in forests of intermediate disturbance.     

Changes of butterfly communities after forest fire

Fires change the diversity and composition of insects in forest ecosystems. In the present study, we examined the change of butterfly communities after a fire including the increase of butterfly richness, grassland species, and generalist species, and more changed communities. Butterflies were surveyed for 5 years after the big Uljin fire in 2007. During each year, butterflies were counted monthly by the line transect method from April to October at two sites (burned vs. unburned, ~ 1.5 km routes). Specialist grassland species decreased in the year of the fire but generalist species did not increase significantly. Butterfly richness did not change but butterfly diversity decreased due to a sudden increase of a species, Polygonia c-aureum. The butterfly community in the year of the fire was different from those in later years, showing temporary change of community in the year of the fire. Species composition was significantly different between burned and unburned sites, but this phenomenon cannot be interpreted as an influence of fire due to highly variable species composition of local butterfly assemblages and the non-repetitive sampling site of the present study.     

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At low dosage rates, it was more effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.     

Development and characterization of a new Bombyx mori cell line for protein expression

A Bombyx mori continuous cell line, designated DZNU-Bm-17, was established from larval ovaries. The cells were initially grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum and 3% heat inactivated B. mori hemolymph at 25 ± 1 °C and later adapted gradually to TNM-FH medium. Partially adhered refractive cells were the predominant cell type in the culture. The cells took about 1055 days to complete 100 passages in TNM-FH medium. The population doubling time of the cell line was about 30–34 h at 25 ± 1 °C. The cell population was largely diploid, but a few triploids and tetraploids were also observed. DNA profiles using simple sequence repeat loci established the differences between the DZNU-Bm-1, Bm-5, DZNU-Bm-12, DZNU-Bm-17, and BmN cell lines. The cell line was susceptible to budded virus of B. mori nucleopolyhedrovirus (BmNPV), and 85–92% of the cells harbored BmNPV with an average of 15 occlusion bodies/infected cell. The cells expressed the luciferase and green fluorescent proteins using the BmNPV bacmid vector. We suggest the usefulness of the DZNU-Bm-17 cell line for BmNPV-based baculoviral expression studies.     

Evaluation of a novel pneumatic bioreactor system for culture of recombinant Chinese hamster ovary cells

Single use culture systems are a tool in research and biotechnology manufacturing processes and are employed in mammalian cell-based manufacturing processes. Recently, we characterized a novel bioreactor system developed by PBS Biotech. The Pneumatic Bioreactor System? (PBS) employs the Air-wheel?, which is a mixing device similar in structure to a water wheel but is driven by the buoyant force of gas bubbles. In this study, we investigated the physical properties of the PBS system, with which we performed biological tests. In 2 L PBS, the mixing times ranged from 6 (30 rpm, 0.175 vvm) to 15 sec (10 rpm, 0.025 vvm). The k_La value reached upto 7.66/h at 0.5 vvm, even without a microsparger, though this condition is not applicable for cell cultures. Also, when a 10 L PBS equipped with a microsparger was evaluated, a k_La value of upto approximately 20/h was obtained particularly in mild cell culture conditions. We performed cultivation of Chinese hamster ovary (CHO) cells in 2 and 10 L PBS prototypes. Results from the PBS were compared with those from an Erlenmeyer flask and conventional stirred tank type bioreactor (STR). The maximum cell density of 10.6 × 106 cells/mL obtained fromthe 2 L PBSwas about 2 times higher than that from the Erlenmeyer flask (5.6 × 10⁶ cells/mL) andwas similar to the STR (9.7 × 10⁶ cells/mL) when the CHO-S cells were cultured. These results support the general suitability of the PBS system using pneumatic mixing for suspension cell cultivation as a novel single-use bioreactor system.