Construction and characterization of novel chimeric β-glucosidases with Cellvibrio gilvus (CG) and Thermotoga maritima (TM) by overlapping PCR

In order to create novel β-glucosidase constructs, 8 kinds of chimeric β-glucosidases were constructed using overlapping polymerase chain reaction (PCR) based on Cellvibrio gilvus (CG) and Thermotoga maritima (TM) genes. Two kinds of novel chimeric β-glucosidases (No. 6 and No. 8 type) were selected and their properties characterized. SDS-PAGE analysis showed that both constructs had a molecular mass of 80 kDa. The optimum pH of No. 6 chimeric β-glucosidase was found to be 3.0 and 5.0, showing varying maximum activity according to the buffer used. No. 8 chimeric enzyme was found to be optimally active at a pH of 4.5 and the optimum temperature of No.6 and No.8 chimeric β-glucosidases was reported to be 60°C, respectively. The Km values of both novel chimeric enzymes were calculated to be 0.012 mM and 0.0082 mM, respectively and the characteristics of the novel chimeric enzymes were to lie between those of the parental enzymes.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

Jasmonate-dependent plant defense restricts thrips performance and preference

Background  

The western flower thrips (Frankliniella occidentalis [Pergande]) is one of the most important insect herbivores of cultivated plants. However, no pesticide provides complete control of this species, and insecticide resistance has emerged around the world. We previously reported the important role of jasmonate (JA) in the plant's immediate response to thrips feeding by using an Arabidopsis leaf disc system. In this study, as the first step toward practical use of JA in thrips control, we analyzed the effect of JA-regulated Arabidopsis defense at the whole plant level on thrips behavior and life cycle at the population level over an extended period. We also studied the effectiveness of JA-regulated plant defense on thrips damage in Chinese cabbage (Brassica rapa subsp. pekinensis).     

Two Helminthic Cases of Human Mummy Remains from Joseon-Period Graves in Korea

Our previous research on coprolite specimens from the mummies of Joseon Dynasty (1392–1910 CE) has revealed various species of parasite eggs. Herein, we added 2 new helminthic cases of human remains from Joseon-period graves in the Republic of Korea (Korea). The organic materials precipitated on the hip bones of 2 half-mummied cases (Goryeong and Gwangmyeong cases) were collected, rehydrated, and examined by a microscope. In the sample from Goryeong-gun (gun=County), ova of Trichuris trichiura, Clonorchis sinensis, and Metagonimus spp. were detected, and eggs of T. trichiura and A. lumbricoides were found from the sample of Gwangmyeong-si (si=City). By adding this outcome to the existing data pool, we confirm our previous estimates of Joseon-period parasite infection rates. The overall rates of A. lumbricoides, T. trichiura, and C. sinensis decreased dramatically from Joseon to the modern period. In Goryeong mummy specimen, we also found Metagonimus spp. eggs that has rarely been detected in archaeological samples so far.     

Synergistic anticancer effect of docosahexaenoic acid and isoliquiritigenin on human colorectal cancer cells through ROS-mediated regulation of the JNK and cytochrome c release

Molecular Biology Reports - A large body of research has demonstrated a synergistic anticancer effect between docosahexaenoic acid (DHA) and standard chemotherapy regimens against colorectal cancer...     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

Geographical Distribution and Relative Abundance of Vectors of Scrub Typhus in the Republic of Korea

A survey to determine the geographical distribution and relative abundance of potential vectors of scrub typhus was conducted from October to November 2006 at 13 localities throughout the Republic of Korea. Apodemus agrarius accounted for 97.6% (80/82) of all rodents, while only 2 Myodes regulus (2/82) were collected. A total of 10,860 chiggers were collected from A. agrarius belonging to 4 genera and 8 species, while only Walchia fragilis (40) was collected from Myodes regulus. Leptotrombidium pallidum (8,137; 74.9%), a vector of scrub typhus, was the predominant species collected from A. agrarius followed by Leptotrombidium scutellare (2,057, 18.9%), Leptotrombidium palpale (279; 2.7%), Leptotrombidium orientale (232; 2.1%), and Leptotrombidium zetum (79; 0.7%), Neotrombicula tamiyai (58; 0.5%), Euschoengastica koreaensis (16; 0.1%), and Cheladonta ikaoensis (2; < 0.1%). L. pallidum was the predominant chigger collected at collection sites in Gangwon (100%), Gyeonggi (87.2%), Chungnam (100%), Chungbuk (100%), Jeonbuk (73.9%), Jeonnam (77.0%), and Gyeongbuk (66.1%) provinces, whereas L. scutellare was the predominant chigger collected in Gyeongnam province (77.9%) and Jeju Island (62.3%). Data suggest a correlation between chigger population abundance and human cases of scrub typhus in Korea.     

Efficiency Evaluation of Nozawa-Style Black Light Trap for Control of Anopheline Mosquitoes

House-residual spraying and insecticide-treated bed nets have achieved some success in controlling anthropophilic and endophagic vectors. However, these methods have relatively low efficacy in Korea because Anopheles sinensis, the primary malaria vector, is highly zoophilic and exophilic. So, we focused our vector control efforts within livestock enclosures using ultraviolet black light traps as a mechanical control measure. We found that black light traps captured significantly more mosquitoes at 2 and 2.5 m above the ground (P < 0.05). We also evaluated the effectiveness of trap spacing within the livestock enclosure. In general, traps spaced between 4 and 7 m apart captured mosquitoes more efficiently than those spaced closer together (P > 0.05). Based on these findings, we concluded that each black light trap in the livestock enclosures killed 7,586 female mosquitoes per trap per night during the peak mosquito season (July-August). In May-August 2003, additional concurrent field trials were conducted in Ganghwa county. We got 74.9% reduction (P < 0.05) of An. sinensis in human dwellings and 61.5% reduction (P > 0.05) in the livestock enclosures. The black light trap operation in the livestock enclosures proved to be an effective control method and should be incorporated into existing control strategies in developed countries.     

Designing the substrate specificity of d-hydantoinase using a rational approach

Enzymes that exhibit superior catalytic activity, stability and substrate specificity are highly desirable for industrial applications. These goals prompted the designed substrate specificity of Bacillus stearothermophilus d-hydantoinase toward the target substrate hydroxyphenylhydantoin (HPH). Positions crucial to substrate specificity were selected using structural and mechanistic information on the structural loops at the active site. The size and hydrophobicity of the involved amino acids were rationally changed, and the substrate specificities of the designed d-Hyd mutants were investigated. As a result, M63I/F159S exhibited about 200-fold higher specificity for HPH than the wild-type enzyme. Systematic mutational analysis and computational modeling also supported the rationale used in the design.