Background: Brain-derived neurotrophic factor (BDNF) and its receptor, neurotrophic tyrosine receptor kinase-2 (NTRK2), are well known for their roles in the central nervous and animal reproductive systems. Several studies have observed the extensive expression of BDNF and NTRK2 in non-neuronal tissues, especially reproductive organs. However, most of these studies focused on ovarian development and regulation; thus, scientific research on BDNF and NTRK2 in males is required to determine their roles in the male reproductive system. Therefore, this study aimed to investigate BDNF and NTRK2 expression in bovine testes. Methods: Testes were collected from six Hanwoo bulls (6-8 months old). Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to investigate the mRNA expression of BDNF and NTRK2 in the testes. Western blot analysis was performed to verify the cross-reactivity of BDNF and NTRK2 antibodies with bovine testicular tissues. Immunohistochemistry was conducted to determine BDNF and NTRK2 protein expression in the testes. Results: RT-PCR analysis revealed BDNF and NTRK2 mRNA expression in bovine testes. In Western blotting, BDNF and NTRK2 protein bands were observed at 32 and 45 kDa, respectively. Immunofluorescence demonstrated BDNF expression in the nuclei of spermatogonia and Sertoli cells as well as in the cytoplasm of Leydig cells. NTRK2 was exclusively expressed in Sertoli cells. These results suggest that BDNF plays a potential role in spermatogenesis via BDNF and NTRK2 signaling in bovine testes, a finding supported by previous results in different animal species. Conclusions: The expression patterns of BDNF and NTRK2 indicate their functional importance in the bovine reproductive system.

It is well known that the JNK pathway regulates AMP production against pathogenic infection in both vertebrates and invertebrates. Tenebrio molitor hep (Tmhep) is an homolog of MAP kinase kinase in mammals. Here, we investigate the immunological function of Tmhep in responses in microbial infection using RNA interference technology. The results showed that silencing of Tmhep increased the larval mortality against microbial challenge, as well as reduced AMP production compared to the control group (dsEGFP-treated group). Conclusively, Tmhep plays an critical role in antimicrobial defense in T. molitor larvae.

Behavioral modulation by genetic changes garners a special attention nowadays as an effective means of revealing genetic function on the one hand and broadening the scope of in situ monitoring on the other hand. The cGMP-dependent protein kinase was treated to the western flower thrips, Frankliniella occidentalis. Automatic recognition techniques and computational methods were utilized to investigate behavioral changes across photo- and scoto-phases. Movement behaviors are objectively expressed according to parameter extraction and data structure visualization in different light phases. By comapring with the individuals without treatment, activities of treated thrips were changed including decrease in circadian rhythm. Usefulness of automatic monitoring of insect movement in different genetic strains is further discussed for providing useful information on monitoring and diagnosing natural and unntatural genetic disturbances.

겨울과 같은 환경에서 곤충은 생존과 번성을 위해 생리학적, 생화학적 및 행동적 메커니즘을 이용하고 있다. 대부분의 곤충은 생리학적 적응가운데 급속내한성(Rapid cold hardiness, RCH) 유기를 통해 기온이 급격히 낮아 지는 외부 환경에 대해 빠르게 적응하고 저온조건에서 생존율을 높인다. 열대거세미나방의 경우 행동적 메커니 즘을 통해 따뜻한 곳을 찾아 장거리 비행을 하며, 생존에 유리한 환경으로 이동한다. 본 연구에서는 열대거세미나 방의 생리적 월동능력과 RCH 능력에 관해 조사하였다. 그 결과, RCH에 의해 혈중 글리세롤의 농도가 증가와 체내빙결점이 하강하는 것을 확인할 수 있었다. 또한, RCH(-10℃, 1h)에 노출된 2령 유충기를 대상으로 4령과 5령 유충기에 단기저온(5℃, 30min)에 노출 시 글리세롤 생합성에 관여하는 유전자(glycerol kinase 1, 2)의 발현이 RCH에 노출되지 않은 대조구와 비교하여 빠르게 발현되었다. 이는, 열대거세미나방의 유전자 수준에서 저온에 대한 단기기억이 존재하는 것을 제시한다.

The striped fruit fly (SFF), Zeugodacus scutellata, is an agricultural pest species with a strong and rapid reproductive ability that can cause significant harm. To control the population of these kind of pests, the sterile insect technique (SIT) is being used as one of the effective methods. SIT involves the introduction of sexually transmitted factors that reduce the reproductive capacity of males. This study shows that knocking down the testis-specific serine/threonine protein kinase 1 (Zs-Tssk1) gene alters male fertility and male-initiated types of communication. Since Zs-Tssk1 influences the physiology of the testes, spermatogenesis is also affected, which in turn alters the lifespan of Zs-Tssk1 knock down group in comparison with the control. Based on these results, Zs-Tssk1 may be crucial in reproductive function, and its down-regulation may be helpful in controlling SFF through SIT.

Receptor tyrosine kinase c-Kit, a marker found on interstitial cells of Cajal (ICCs), is expressed in Leydig cells, which are testicular interstitial cells. The expression of other ICC markers has not yet been reported. In this study, we investigated the expression of c-Kit and anoctamin 1 (ANO1), another ICC marker, in mouse testes. In addition, the relationship between c-Kit and ANO1 expression and Leydig cell function was investigated. We observed that c-Kit and ANO1 were predominantly expressed in mouse Leydig cells. The mRNA and protein of c-Kit and ANO1 were expressed in TM3, a mouse Leydig cell line. LH induced an increase in intracellular Ca2+ concentration, membrane depolarization, and testosterone secretion, whereas these signals were inhibited in the presence of c-Kit and ANO1 inhibitors. These results show that c-Kit and ANO1 are expressed in Leydig cells and are involved in testosterone secretion. Our findings suggest that Leydig cells may act as ICCs in testosterone secretion.

Salivary glands are exocrine glands that secrete saliva into the oral cavity, and secreted saliva plays essential roles in oral health. Therefore, maintaining the salivary glands in an intact state is required for proper production and secretion of saliva. To investigate a specific signaling pathway that might affect the maintenance of mouse submandibular gland (SMGs), RNA sequencing was performed. In SMGs, downregulated expression patterns of Rho-associated protein kinase (ROCK) signaling pathway-related genes, including Rhoa, Rhob, Rhoc, Rock1, and Rock2, were observed. Gene expression profiling analyses of these genes indicate that the ROCK signaling pathway is a potential signal for SMG maintenance.

Periodontal disease is an inflammatory disease that affects the destruction of the bone supporting the tooth and connective tissues surrounding it. Periodontal ligament fibroblasts (PDLFs) induce overexpression of matrix metalloproteinase (MMP) involved in periodontal diseaseʼs inflammatory destruction. Osteoclasts take part in physiological bone remodeling, but they are also involved in bone destruction in many kinds of bone diseases, including osteoporosis and periodontal disease. This study examined the effect of baicalin on proteolytic enzymesʼ production and secretion of inflammatory cytokines in PDLFs and RAW 264.7 cells under the lipopolysaccharide (LPS)-induced inflammatory conditions. Baicalin inhibited the expression of the protein, MMP-1 and MMP-2, without affecting PDLFs’ cell viability, suggesting its possibility because of the inhibition of phosphorylation activation of mitogen-activated protein kinase’s p38, and the signal transduction process of nuclear factor κB (NFκB)-related protein. Also, baicalin reduced the expression of MMP-8 and MMP-9 in RAW 264.7 cells. This reduction is thought to be due to the inhibition of the signal transduction process of NFκB-related proteins affected by inhibiting p65RelA phosphorylation. Also, baicalin inhibited the secretion of nitric oxide and interleukin-6 induced by LPS in RAW 264.7 cells. These results suggest that baicalin inhibits connective tissue destruction in periodontal disease. The inhibition of periodontal tissue destruction may be a therapeutic strategy for treating inflammatory periodontal-diseased patients.

L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl- 2 family and MAPK/Akt signaling pathways.

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

Toll and IMD pathways play an important role in producing antimicrobial peptides (AMPs) through NF-κB in insects. The functions of IκB kinase (IKK) complex regulating the NF-κB signaling cascade have not yet been investigated in Tenebrio model. Here, we identified TmIKK-β (or TmIrd5) which contains 2,112 bp encoding 703 amino acid residues. Domain analysis shows that TmIKK-β contains one Serine/Threonine protein kinases catalytic domain. Developmental expression patterns indicate that TmIKK- β gene was highly expressed in early pupal (P1) and adult (A5) stages. Tissue specific profiles show that TmIKK-β was highly expressed in the integuments in last instar larvae, and fat body and hemocytes in 5 day-old adults. TmIKK-β1 transcripts were strongly induced at 3 and 12 h-post injection of E. coli, and 3 h-post injection of S. aureus or C. albicans in hemocytes. In gut, TmIKK-β transcripts were slightly induced by E. coli (at 6, 9 and 24 h) and C. albicans (at 24 h), while it was not induced by S. aureus challenge. Moreover, it was highly induced at 6 h-post injection of E. coli and then it was gradually decreased in the fat body. To understand the immunological role of TmIKK-β, gene specific RNAi and mortality assay was performed. Depletion of TmIKK-β mRNA leads to increase microbial susceptibility of larvae against E. coli, S. aureus and C. albicans. In addition, induction patterns of fourteen AMP genes in response to microbial challenge was tissue specifically investigated in TmIKK-β–silenced T. molitor larvae. The results suggest that expression of ten AMP genes out of fourteen genes were drastically decreased by TmIKK-β RNAi in fat body, suggesting that TmIKK-β plays an important role in antimicrobial innate immune responses.

It has been well known that IKK-β, -ε and –γ play a pivotal role in IMD pathway. In this study, TmIKK-ε was identified and their functions in countering pathogenic infections were investigated. We identified TmIKK-ε gene which including 2,196 bp nucleotides (encoding 731 amino acid residues). Domain analysis of TmIKK-ε indicates that there is one Serine/Threonine protein kinases catalytic domain. TmIKK-ε gene was highly expressed in 2 day-old pupal stage and the expression was gradually decreased until 1 day-old adults. Then the expression was slightly increased until 4 day-old adult stage. Tissue specific expression of TmIKK-ε mRNA was high in the gut, integuments and hemocytes in last instar larvae, and fat body, Malpighian tubules and testis in 5-daysold adult. In hemocytes, TmIKK-ε was drastically induced by E. coli injection after 3 h and by S. aureus at 3 and 12 h-post injection. In gut, expression level of TmIKK-ε was high at 6 h-post injection of microbial injection. Expression of TmIKK-ε in fat body was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-ε, gene specific RNAi and mortality assay were performed. TmIKK-ε RNAi caused increased larval mortality against E. coli, not S. aureus and C. albicans. Finally, to investigate the induction patterns of Tenebrio fourteen AMP genes in response TmIKK-ε RNAi, three microorganisms were treated into TmIKK-ε-silenced T. molitor larvae. Nine out of fourteen AMP genes were not induced by microbial challenge in TmIKK-β dsRNA-injected group. Taken together, our results indicate that TmIKK-ε may regulates nine antimicrobial peptide genes in response to microbial challenge in T. molitor fat body.

IKK-γ is an essential protein to form IKK complex which regulate NF-κB. We identified TmIKK-γ (or TmKenny) gene which has 1,521 bp of nucleotides encoding 506 amino acid residues. Domain analysis of TmIKK-γ shows that there are one NF-κB essential modulator (NEMO) domain and a leucine zipper domain. Expression of TmIKK-γ gene was gradually increased from egg to 2-day-old pupal stage, dramatically decreased until 7 day-old pupal stage, and then it was gradually increased. TmIKK-γ transcripts were highly expressed in fat body and hemocytes in late instar larvae and integuments, fat body and Malpighian tubules in 5 day-old adult. TmIKK-γ was drastically induced by E. coli after 3 h challenges and by S. aureus at 3 and 12 h-post injection in hemocytes. TmIKK-γ was not induced by C. albicans although it was significantly induced by E. coli (at 3, 6 and 24 h) and S. aureus (at 9 h) in gut. In fat body, expression of TmIKK-γ was drastically induced by E. coli at 3 and 24 h-post injection while it was not significantly induced by S. aureus and C. albicans. To understand the immunological role of TmIKK-γ, gene specific RNAi and mortality assay was performed. larval mortality against microbial challenge was dramatically increased by TmIKK-γ RNAi. Furthermore, we investigate the tissue specific induction patterns of fourteen AMP genes in response TmIKK-γ dsRNA-treatment. In fat body, ten AMP genes out of fourteen was not significantly induced by microbial challenge in TmIKK-γ dsRNA-treated group. Based on these results, TmIKK-γ might play an important role in antimicrobial innate immune responses in Tenebrio molitor.

Arabidopsis nucleoside diphosphate kinase 2 (AtNDPK2) is an upstream signaling molecule that has been shown to induce stress tolerance in plants. In this study, the AtNDPK2 gene, under the control of a stress-inducible SWPA2 promoter, was introduced into the genome of tall fescue (Festuca arundinacea Schreb.) plants. The induction of the transgene expression mediated by methyl viologen (MV) and NaCl treatments were confirmed by RT-PCR and northern blot analysis, respectively. Under salt stress treatment, the transgenic tall fescue plants (SN) exhibited lower level of H2O2 and lipid peroxidation accumulations than the non-transgenic (NT) plants. The transgenic tall fescue plants also showed higher level of NDPK enzyme activity compared to NT plants. The SN plants were survived at 300 mM NaCl treatment, whereas the NT plants were severely affected. These results indicate that stress-inducible overexpression of AtNDPK2 might efficiently confer the salt stress tolerance in tall fescue plants.

The p21-activated kinases (PAKs) are a family of serine/threonine protein kinases and activated by binding with activated Rho GTPases such as Rac or Cdc42. PAKs regulate actin cytoskeletal remodeling, cell motility, cell survival, and apoptosis. Also, PAKs are involved in several diseases such as cancer, virus infectious diseases, mental retardation, Alzheimer and Parkinson's diseases. Therefore, the substances that are able to inhibit PAK activation can be used as powerful tools and medicines for PAK relative diseases or specific inhibitors for study of PAK signaling pathway. In this study, we investigated and characterized the 5 compounds of 4-benzene-1, 2-naphthoquinone (NQ) family as candidate substances to inhibit the PAK1 activation in vitro and in cells. Binding between p21-binding domain (PBD) of PAK1 and Cdc42 was blocked by 5 NQ-compounds in ELISA assay. Myelin basic protein (MBP) phosphorylation was dramatically reduced by treatment of these compounds in vitro kinase assay for Cdc42-induced or constitutive active PAK1 mutant. Also, phosphorylation at Thr 423 of transfected PAK1 was inhibited by treatment of 5 NQ-compounds in 293T cells, respectively. Finally, NQ-5 inhibited strongly the PAK1 activation by PDGF stimulation and cell motility in PDGF-induced wound migration assay in NIH 3T3 cells. Therefore, these NQ compounds will be good candidates as target molecules to regulate PAK1-related diseases or inhibitors to study PAK1 signaling pathway.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.