Korea Research Institute of Bioscience and Biotechnology
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Heterologous activation of the Hevea PEP16 promoter in the rubber-producing laticiferous tissues of Taraxacum kok-saghyz
No full textHevea brasiliensis, the most abundant rubber crop, is used widely for the commercial production of natural rubber. To reduce the risk of a shortage in the supply of natural rubber that may arise from a single major rubber crop, rubber dandelion (Taraxacum kok-saghyz) has been developed as an alternative rubber-producing crop by using a transgenic approach. However, it is necessary to identify a suitable promoter for the transfer of rubber biosynthesis-related genes to the species. In this study, the promoter region of H. brasiliensis PEP16, which was isolated as a potentially important component in rubber biosynthesis, was sequenced and a pPEP16::GUS fusion construct was introduced into T. kok-saghyz. Histological and fluorometric studies using transgenic T. kok-saghyz plants indicated that the HbPEP16 promoter was highly activated in a laticiferous tissue-specific manner under normal growth conditions and that promoter activation was tightly regulated by various hormones and external signals. These findings suggested that the HbPEP16 promoter may be a useful molecular tool for the manipulation of gene expression in the laticiferous tissues of T. kok-saghyz.
Bakuchicin attenuates atopic skin inflammation
No full textPsoralea corylifolia is a medicinal herb that provides advantageous pharmacological effects against vitiligo and skin rash. Former studies have shown that bakuchicin, a furanocoumarin compound from the fruits of P. corylifolia, has therapeutic effects against inflammation, and infection. This study aimed to define the pharmacological effects of bakuchicin on inflammatory responses and lichenification, the major symptoms of atopic dermatitis (AD). To induce AD-like skin inflammation, we exposed the ears of female BALB/c mice to 2, 4-dinitrochlorobenzene (DNCB) and Dermatophagoides farinae (house dust mite) extract (DFE) for 4 weeks. Intragastric administration of bakuchicin attenuated the symptoms of AD-like skin inflammation, as evident by reductions in ear thickness, erythema, and keratosis. Bakuchicin also reversed increases in auricular epidermal and dermal layer thicknesses, and attenuated eosinophil and mast cell infiltration in AD-induced mice. It also suppressed Th2 gene expression as well as that of pro-inflammatory cytokines and chemokines, such as interleukin (IL)-4, IL-13, IL-31, IL-1β, IL-6, CXCL-1, and CCL-17 in the ear tissue. The levels of total and DFE-specific immunoglobulin (Ig)E, and IgG2a in the mice sera were reduced by the bakuchicin. To investigate the effect of bakuchicin on keratinocytes, experiments were performed using HaCaT cells, the representative cell type used in skin disease studies. Tumor necrosis factor-α and interferon-γ were used to activate keratinocytes. Bakuchicin suppressed Th2 gene expression and that of pro-inflammatory cytokines and chemokines; it also suppressed STAT-1 phosphorylation and the nuclear translocation of NF-κB in activated keratinocytes. These results suggest that bakuchicin attenuated AD symptoms, thus suggesting it as a potential therapeutic agent for the treatment of AD.
CPPF, a novel microtubule targeting anticancer agent, inhibits the growth of a wide variety of cancers
No full textIn the past, several microtubule targeting agents (MTAs) have been developed into successful anticancer drugs. However, the usage of these drugs has been limited by the acquisition of drug resistance in many cancers. Therefore, there is a constant demand for the development of new therapeutic drugs. Here we report the discovery of 5-5 (3-cchlorophenyl)-N-(3-pyridinyl)-2-furamide (CPPF), a novel microtubule targeting anticancer agent. Using both 2D and 3D culture systems, we showed that CPPF was able to suppress the proliferation of diverse cancer cell lines. In addition, CPPF was able to inhibit the growth of multidrug-resistant cell lines that are resistant to other MTAs, such as paclitaxel and colchicine. Our results showed that CPPF inhibited growth by depolymerizing microtubules leading to mitotic arrest and apoptosis. We also confirmed CPPF anticancer effects in vivo using both a mouse xenograft and a two-step skin cancer mouse model. Using established zebrafish models, we showed that CPPF has low toxicity in vivo. Overall, our study proves that CPPF has the potential to become a successful anticancer chemotherapeutic drug.
Molecular phylogenetics analysis of geoduck (Panopea japonica) apply metallothionein gene
No full textMetallothionein (MT) plays an important role in protecting cells from harmful substances. MT is a protein with less than seven kDa with large amounts of cysteine residues, which have been studied as a phylogenetic biomarker with unique patterns not only in vertebrates but also in Mollusca. Panopea japonica is a giant clam that lives on the coast of the East Sea in Korea. Nevertheless, little research has been done on the genetic resources of P. japonica, and the amount of genetic information registered in the NCBI is very limited. In this study, a metallothionein sequence consisting of 74 amino acids with 222 bp nucleotide was obtained from the transcriptome data by using the GS FLX 454 platform. In Silico analysis of PjMT gene was conducted with ClustalX and MEGAX programs. With the previously reported Mollusca MT data, we verified the PjMT sequence as a potential candidate for molecular marker, and also confirmed that PjMT was clustered in Myoida order.
Phylogenetic analysis of endangered wild animal class II Satsuma myomphala using the argrinine kinase gene
No full textArginine kinase (AK) belongs to the phosphagen kinase (PK) super family group of enzyme that has an essential role in maintaining energy homeostasis by catalyzing the re-phosphorylation of ADP. The AK gene is used for phylogenetic analysis in arthropods, mollusks, and Nematoda, and has been reported to branch in the Phylum unit. Satsuma myomphala has been reported as an indigenous species in Northeast Asia, including Korea and Japan. Particularly in Korea, it was registered as an endangered wildlife class 2 in 2017. Therefore, the preservation of species and securing genetic resources became more important. The genetic resources of S. myomphala registered on the NCBI GenBank have only six nucleotides and two proteins registered. The AK sequence was extracted from the RNAseq data which was obtained from Illumina Hiseq 2500 platfrom. After sequencing, de novo assembly and clustering, 103,774 unigenes were generated. Through annotation step by using the PANM database and BLAST program, we obtained AK sequence of 354 amino acid residues containing a 1,062 bp coding region. The AK sequence was verified using the BLAST program in the NCBI nr database, and phylogenetic analysis with the AK sequences of Gastropoda, Bivalvia, and Cephalopoda registered in NCBI was conducted. A phylogenetic dendrogram was generated using the MEGA program, and we confirmed the phylogenetic relationships. In addition, the 2D and 3D structures of the AK gene were predicted.
Hispidulin alleviates imiquimod-induced psoriasis-like skin inflammation by inhibiting splenic Th1/Th17 cell population and keratinocyte activation
No full textPsoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes accompanied by increased infiltration of immune cells. Previous studies have demonstrated that hispidulin (4′,5,7-trihydroxy-6-methoxyflavone, HPD) has various pharmacological benefits such as anti-fungal, anti-inflammation, and anti-allergic effects. This study investigated the effectiveness of HPD to treat psoriasis using an imiquimod (IMQ)-induced mouse model and activated keratinocytes. IMQ was topically applied to the back skin of mice for six consecutive days, and the mice were orally administered HPD. Based on the histological observation and immunological analysis, oral administration of HPD suppressed psoriatic characteristics including skin thickness, psoriasis area severity index, transepidermal water loss, and neutrophil infiltration. HPD alleviated pathologically increased levels of immunoglobulin G2a, myeloperoxidase, and tumor necrosis factor-α. Splenic Th1 and Th17 cell populations were also reduced by HPD in the murine model. In addition, in activated keratinocytes, HPD inhibited gene expression of Th1- and Th17-associated cytokines and chemokines, and phosphorylation of mitogen-activated protein kinases and nuclear factor-κB. In summary, HPD alleviates psoriasis skin inflammation in vivo and in vitro. Therefore, we suggest that HPD would be a potent therapeutic candidate for the treatment of psoriasis.
Antioxidant compounds for the inhibition of enzymatic browning by polyphenol oxidases in the fruiting body extract of the edible mushroom Hericium erinaceus
No full textMushrooms are attractive resources for novel enzymes and bioactive compounds. Nevertheless, mushrooms spontaneously form brown pigments during food processing as well as extraction procedures for functional compounds. In this study, the dark browning pigment in the extract derived from the edible mushroom Hericium erinaceus was determined to be caused by the oxidation of endogenous polyphenol compounds by the polyphenol oxidase (PPO) enzyme family. These oxidized pigment compounds were measured quantitatively using a fluorospectrophotometer and, through chelation deactivation and heat inactivation, were confirmed to be enzymatic browning products of reactions by a metalloprotein tyrosinase in the PPO family. Furthermore, a transcript analysis of the identified putative PPO-coding genes in the different growth phases showed that tyrosinase and laccase isoenzymes were highly expressed in the mushroom fruiting body, and these could be potential PPOs involved in the enzymatic browning reaction. A metabolite profiling analysis of two different growth phases also revealed a number of potential enzymatic browning substances that were grouped into amino acids and their derivatives, phenolic compounds, and purine and pyrimidine nucleobases. In addition, these analyses also demonstrated that the mushroom contained a relatively high amount of natural antioxidant compounds that can effectively decrease the browning reaction via PPO-inhibitory mechanisms that inhibit tyrosinase and scavenge free radicals in the fruiting body. Altogether, these results contribute to an understanding of the metabolites and PPO enzymes responsible for the enzymatic browning reaction of H. erinaceus.
Induced extracellular production of stilbenes in grapevine cell culture medium by elicitation with methyl jasmonate and stevioside
No full textWe report the high production of stilbenes, including resveratrol and viniferin, in grapevine (Vitis labruscana L.) cell cultures through elicitation with methyl jasmonate (MeJA) and stevioside (STE). Methyl-β-cyclodextrin (MeβCD) is widely used as a solubilizer for resveratrol production. For the first time, we used STE as a solubilizer for stilbene production in plant cell cultures. MeJA was most effective elicitor in activating VvSTS expression and stimulating stilbene biosynthesis in grapevine cell cultures. The maximum concentration of δ-viniferin (892.2 mg/L) production with a small amount of trans-resveratrol (12.2 mg/L) was observed in the culture medium by co-treatment of cells with MeJA and STE, whereas the highest level of trans-resveratrol (371.9 mg/L) with a slight amount of δ-viniferin (11.5 mg/L) was accumulated in the culture medium of cells treated with MeJA and MeβCD. However, neither trans-resveratrol nor δ-viniferin were significantly elevated within the cells by the applications. Notably, predominant production of δ-viniferin and trans-resveratrol was observed in shake and static flask culture medium, respectively, by co-treatment of MeJA and STE. Furthermore, stilbene compounds of resveratrol, ε-viniferin, and δ-viniferin were mainly produced in a 3-L bioreactor culture following elicitation of cells with MeJA and STE. These results provide new strategies for conditional, high-level production of resveratrol and viniferin in cell cultures by utilizing the solubilizing properties of STE or MeβCD.
Which pathologic staining method can visualize the hyperacute infarction lesion identified by diffusion MRI?: a comparative experimental study
No full textBackground: The study aimed to establish a staining method that could delineate the macroscopic lesion boundary of a hyperacute infarction depicted by diffusion-weighted MRI (DWI) and to validate the infarction boundary by comparing different staining methods.
New Method: Thirteen rats with 1？-h middle cerebral artery (MCA) infarction were included. Five different staining methods (Hematoxylin and eosin (H&E), Nissl, 2,3,5-triphenyltetrazolium hydrochloride (TTC), microtubule associated protein 2 (MAP2), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stains) were used to identify whether the hyperacute infarction could be histopathologically identified. Dice indices were compared to evaluate similarities in the lesion area ascertained by DWI and the staining methods. Through macroscopic lesion delineation, each region was subdivided into abnormal regions in all three stains (ROIA), abnormal in two stains (ROIB), and abnormal in only one (ROIC). Microscopic cellular changes were evaluated and graded according to each region.
Results: Mean Dice indices of the H&E stain were significantly higher than those of the Nissl- and MAP2-stained specimens (0.83？±？0.052, 0.58？±？0.107, and 0.56？±？0.059, respectively; p？=？0.000). Grading scores for ROIs in the DWI abnormal lesions varied by region: ROIA exhibited the most severe damage [median (IQR), 3 (1)], followed respectively by ROIB [median (IQR), 2 (0)] and ROIC [median (IQR), 1 (0)]
Comparison with existing methods: H&E stain best reflects 1？h hyperacute DWI abnormal lesions.
Conclusions: H&E stain allowed for the macroscopic delineation of the 1？h DWI-abnormal lesions, while MAP2 and Nissl stains could only partially depict lesions.
The establishment of new protein expression system using N starvation inducible promoters in Chlorella
No full textChlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-CSF) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-CSF, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codon-optimized hG-CSF sequence, hG-CSF polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella.