ATP-binding cassette (ABC) transporter는 다양한 기질을 세포 밖과 세포 안으로 수송하는 대표적인 수송단백질이다. 곤충에서 ABC transporter는 살충제에 대한 저항성을 발달시키는 중요한 역할을 한다. 현재까지 모델곤충인 초파리를 대상으로 ABC transporter의 살충제 교차저항성에 관한 연구는 많이 수행되어오지 않았다. 본 연구에서는 ABC transporter에 속하는 Mdr49A 유전자가 여섯 종류의 살충제에 보이는 교차저항성 기작을 형질전환 초파리를 이용하여 구명하였다. 초파리 91-R과 91-C 계통은 공통된 조상으로부터 유래되었으며 91-R은 60년 이상 DDT에 노출되었지만 91-C는 어떠한 살충제에도 노출되지 않고 유지되어 왔다. 91-R 계통의 MDR49A 단백질에서 유래된 3개의 아미노산 돌연변이를 형질전환 초파리에 과발현 시켰을 때 carbofuran에 대해서 2.0~6.7배 그리고 permethrin에 대해서 2.5~10.5배의 교차저항성을 나타 낸 반면 다른 약제, abamectin, imidacloprid, methoxychlor, prothiofos에 대해서는 어떠한 교차저항성도 나타내지 않았다. 이상의 결과는 Mdr49A 유전자의 과발현과 더불어 3개의 아미노산 돌연변이는 두 개 약제, carbofuran과 permethrin에 대해 교차저항성 기능을 한다고 제시하고 있다

Insect cuticle or exoskeleton is a complex extracellular matrix formed primarily from structural polysaccharide chitin and protein, and it plays a critical role in protecting them from various environmental stresses and pathogenic infection. Despite of limited composition, insect cuticle has remarkably diverse mechanical properties, ranging from soft and flexible to hard and rigid. My research has been focusing on functional importance of the genes involved in chitin metabolism and cuticle tanning (sclerotization and pigmentation) to comprehensively understand the genetic, enzymatic as well as molecular mechanism underlying differentiation, development and formation of insect cuticular extracellular matrices.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is a component of innate immunity. However, the differentiation between cuticular melanization and innate immunity is not well defined. Here, we demonstrate that the proPO-activating system regulates cuticular melanization in the silkworm pupae using a different mechanism. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, proPOs, and immulectin, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism mediated by the priming of the proPO system.

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is an ectoparasitic pest that feeds on humans as well as other mammals. We investigate that point mutations on the voltage-sensitive sodium channel are associated with the resistance to pyrethroids. Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel (VSSC) α-subunit gene have been identified in deltamethrin-resistant bed bugs. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly. V419L, located in the IS6 transmembrane segment, is a novel mutation. To establish a population-based genotyping method as a molecular resistance monitoring tool, a quantitative sequencing (QS) protocol was developed. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.993). In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to pyrethroids. A pyrethroid-resistant strain showed > 9375- and 6990-fold resistance to deltamethrin and λ-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB method should greatly facilitate the detection and monitoring of pyrethroid resistant bed bugs in the field.

This study examined the anti-osteoclastogenic effects of baicalin on receptor activator of NF-kB ligand (RANKL)- induced RAW264.7 cells. Baicalin is a flavonoid that is produced by Scutellaria baicalensis and is known to have multiple biological properties, including antibacterial, anti- inflammatory and analgesic effects. The effects of baicalin on osteoclasts were examined by measuring 1) cell via- bility; 2) the formation of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells; 3) RANK/RANKL signa- ling pathways and 4) mRNA levels of osteoclast-associated genes. Baicalin inhibited the formation of RANKL-stimu- lated TRAP (+) multinucleated cells and also suppressed the RANKL-stimulated activation of p-38, ERK, cSrc and AKT signaling. Baicalin also inhibited the RANKL-stimu- lated degradation of IĸB in RAW264.7 cells. In addition, the RANKL-stimulated induction of NFATc1 transcription factors was found to be abrogated by this flavonoid. Baica- lin was further found to decrease the mRNA expression of osteoclast-associated genes, including carbonic anhydrase II, TRAP and cathepsin K in the RAW264.7 cells. Our data thus demonstrate that baicalin inhibits osteoclastogenesis by inhibiting the RANKL-induced activation of signaling molecules and transcription factors in osteoclast precursors.

Low level light therapy (LLLT) is known to accelerate the process of wound healing, bone and cartilage formation, and to decrease tissue necrosis and inflammation. It also can be applicable to acute and chronic injury or degenerative disease. Despite forty- plus years of accumulating exprerience of the clinical efficacy of LLLT, their molecular biologic evidence is not fully elucidated. The aim of this review is to explore the role of the ROS system and its molecular biologic mechanism in related with inflammatory response, anti-apoptosis and angiogenesis during LLLT. We suggest that activation of ROS system explains many (if not most) of the observed response of cells to LLLT in vitro, and is likely to play a pivotal role in the response of animals and patients to LLLT for both experimental and clinical indications and diseases.

Our previous study demonstrated that Coprisin, a peptide from Copris tripartitus infected with bacterial pathogens, has an antibacterial activity. We assessed in this study whether Coprisin caused cellular toxicity in various mammalian cell lines. Coprisin selectively caused a marked drop of cell viability in Jurkat T cells, U937 cells and AML-2 cells belonging to the human leukemia cells but not in Caki cells and Hela cells. Fragmentation of DNA, a maker of apoptosis, was also confirmed in theleukemia cell lines but not in other cells. The Coprisin-induced apoptosis in leukemia cells was mediated by AIF (apoptosis inducing factor), a caspase -independent pathway.

Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, post-mating behaviors are triggered by sex peptide (SP), a key modulatory substance produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors by acting on the sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the central nervous system (CNS). Interestingly, however, SPR is also expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. Based on these observations, we speculated that SPR may have additional ligands that are only distantly related to SP, if at all. If so, then this also raises questions on the evolution of SP-SPR signaling. To begin to address these questions, we set out to identify additional ligands for SPR. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes and Aplysia SPRs in vitro, yet are unable to trigger post-mating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for post-mating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors. Therefore, we propose that SPR has a different ancestral function, with a role in post-mating behavior arising only recently in Drosophila evolution, concomitant with the emergence of its novel SP ligand.

COPRISIN is an antibiotic substance extracted from Copris tripartitus. This study is intended to identify various cell biological stimuli that COPRISIN, widely known as an antibacterial substance, has on human cells and to identify its molecule mechanism. A variety of human cell lines were divided into epithelial cells including kidney cells or womb cells, and immunocyte including T cells or macrophages and, after their being cultivated and maintained, cell biological changes of the respective cells according to COPRISIN treatment were compared. As a result, it was confirmed that, different from other experiment cells, COPRISIN specifically caused cell kill in T cells and macrophages. That is, fragmentation of DNA, typical characteristics observed in the process of apoptosis, was confirmed in the nucleus of cells dying owing to COPRISIN treatment. An Apoptosis process is one dependent upon activity of caspase family protein, it was proved that COPRISIN medium cell kill process was one through a caspase-independent route such as AIF. Though it was found out that transcription of TNF-α and extracellular TNF-α secretion increased in blood cells stimulated by COPRISIN, it was also confirmed that TNF-α was a major medium factor in a COPRISIN induced cell kill process from the fact that a cell kill process by COPRISIN was not inhibited at all with TNF-α inhibiting antibody treatment. Above results revealed that COPRISIN, different from other tissue origin cells including kidney cells, can specifically induce apoptosis in immunocyte, which is caused by a caspase-independent cell signal transmission route.

A glucose-regulated protein 78 (grp78) gene, which is belongs to a heat shock cognate 70 (hsc70) subfamily, was cloned from Indian meal moth, Plodia interpunctella. Its full length cDNA was 2679 bp and contains a 1980 bp open reading frame. The translated amino acid sequence consists of 660 residues with a calculated molecular mass of 72,975 Da and an isoelectronic point (pI) of 5.27. It contains several highly conserved functional motifs of the Hsp70 family and, particularly, C-terminal motif of KDEL that is characteristic for endoplasmic reticulum (ER) hsc70. Its deduced amino acid sequence shows a high identity (83-94%) with Hsc70s of other insects and grouped with Hsc70-3 among 5 Hsc70 members of Drosophila melanogaster. During development the grp78 transcript level was high in egg, feeding larval and adult stages but low in molting and wandering larval and pupal stages. Particularly its level was higher in the gut than integument and fat body of fifth instars. Furthermore its level was greatly decreased when fifth instar larvae were starved for 48 hrs but recovered at 3-6 hrs after re-fed diet. Our data suggests that grp78 is a member of hsc70 gene that belongs to ER and may have a role for energy metabolism at cellular level.

Identification of spermatogonial stem cell-specific surface molecules is important in understanding the molecular mechanisms underlying the maintenance and differentiation of these cells. We have found that spermatogonia from busulfan treated mice expressed an autoantigen that distinguishes between undifferentiated and differentiated spermatogonia. Four to six weeks after busulfan treatment, germ cells located in the basal compartment of seminiferous epithelium show isotype-specific IgG deposits that form due to autoimmunity. Before busulfan treatment, the level of testicular IgG was very low but IgG levels began to increase after week 4 and peaked at week 6. When cells from the busulfan treated testis were analyzed using laser scanning cytomeoy (LSC), the frequency of cells positive for IgG deposits, 6-integrin, and 1-integrin were 16.53.8%, 11.82.6%, and 9.0 1.4%, respectively. Immunofluorescent staining suggested that most, if not all of the cells with IgG-deposits isolated from a laminin-coated dish, were also positive for a spermatogonial stem cell marker \ulcorner6-integrins as well as for a germ cell-specific marker TRA 98. We determined serum and intratesticular IgG levels and the soundness of seminiferous tubule basement membrane from busulfan treated mice using electron microscopy, in order to study the mechanism responsible for IgG deposits in spermatogonia. We found that the basement membranes of seminiferous tubules from busulfan treated mice were severely impaired when compared to those of normal adult, neonates and w/wv mice. Furthermore, new blood cells were observed in the surface of the damaged basement membrane along the seminiferous tubules. These results suggest that the IgG in spermatogonial stem cells accumulates from circulating blood through the impaired basement membranes induced by busulfan treatment. Taken together, our study suggests that IgG can be used as a new marker for undifferentiated spermatogonia cells.