• BMB Reports
• /
• v.39 no.6
• /
• pp.709-716
• /
• 2006

Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the N-terminus region, whereas the non-catalytic region in the C-terminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domain-specific target proteins of Plk1, we constructed an N-terminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and C- termini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.

• Tissue Engineering and Regenerative Medicine
• /
• v.15 no.6
• /
• pp.761-769
• /
• 2018

BACKGROUND: Bioprinting has recently appeared as a powerful tool for building complex tissue and organ structures. However, the application of bioprinting to regenerative medicine has limitations, due to the restricted choices of bio-ink for cytocompatible cell encapsulation and the integrity of the fabricated structures. METHODS: In this study, we developed hybrid bio-inks based on acrylated hyaluronic acid (HA) for immobilizing bio-active peptides and tyramine-conjugated hyaluronic acids for fast gelation. RESULTS: Conventional acrylated HA-based hydrogels have a gelation time of more than 30 min, whereas hybrid bio-ink has been rapidly gelated within 200 s. Fibroblast cells cultured in this hybrid bio-ink up to 7 days showed >90% viability. As a guidance cue for stem cell differentiation, we immobilized four different bio-active peptides: BMP-7-derived peptides (BMP-7D) and osteopontin for osteogenesis, and substance-P (SP) and Ac-SDKP (SDKP) for angiogenesis. Mesenchymal stem cells cultured in these hybrid bio-inks showed the highest angiogenic and osteogenic activity cultured in bio-ink immobilized with a SP or BMP-7D peptide. This bio-ink was loaded in a three-dimensional (3D) bioprinting device showing reproducible printing features. CONCLUSION: We have developed bio-inks that combine biochemical and mechanical cues. Biochemical cues were able to regulate differentiation of cells, and mechanical cues enabled printing structuring. This multi-functional bio-ink can be used for complex tissue engineering and regenerative medicine.

• Journal of Microbiology and Biotechnology
• /
• v.13 no.4
• /
• pp.537-543
• /
• 2003

The mouse interferon gene (MuIFN-${\gamma}$) was cloned and then used to transform Saccharomyces cerevisiae. Expressed MuIFN-$\{gamma}$ protein (MuIFN-${\gamma}$) was successfully secreted into culture medium due to the presence oi the signal peptide of rice amylase 1A. Two different promoters fused to MuIFN-${\gamma}$ were tested: glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and a yeast hybrid ADH2-GPD (AG) promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. Using the hybrid promoter, the accumulation of MuIFN-${\gamma}$transcript was the highest after the 24 h cultivation, and then gradually decreased as the cultivation proceeded. However, both cell growth and recombinant MuIFN-${\gamma}$production reached their peaks after the 4-day cultivation. It was possible to produce 6.5 mg/l of MuIFN-${\gamma}$ without any changes in cell growth. Using GPD promoter, the MuIFN-${\gamma}$ transcript accumulation and the recombinant MuIFN-${\gamma}$ production followed the same pattern as the cell growth. However. compared to that of the hybrid promoter, the production of recombinant MuIFN-${\gamma}$ was 0.2 mg/l. The secreted MuIFN-${\gamma}$ had estimated molecular masses of 21 kDa and 23 kDa, which were larger than that of the encoded size due to glycosylation. The protection assay against the viral infection indicated that the recombinant MuIFN-${\gamma}$ was bioactive.

• Restorative Dentistry and Endodontics
• /
• v.45 no.3
• /
• pp.31.1-31.20
• /
• 2020

Matrix metalloproteinases (MMPs) are enzymes that can degrade collagen in hybrid layer and reduce the longevity of adhesive restorations. As scientific understanding of the MMPs has advanced, useful strategies focusing on preventing these enzymes' actions by MMP inhibitors have quickly developed in many medical fields. However, in restorative dentistry, it is still not well established. This paper is an overview of the strategies to inhibit MMPs that can achieve a long-lasting material-tooth adhesion. Literature search was performed comprehensively using the electronic databases: PubMed, ScienceDirect and Scopus including articles from May 2007 to December 2019 and the main search terms were "matrix metalloproteinases", "collagen", and "dentin" and "hybrid layer". MMPs typical structure consists of several distinct domains. MMP inhibitors can be divided into 2 main groups: synthetic (synthetic-peptides, non-peptide molecules and compounds, tetracyclines, metallic ions, and others) and natural bioactive inhibitors mainly flavonoids. Selective inhibitors of MMPs promise to be the future for specific targeting of preventing dentin proteolysis. The knowledge about MMPs functionality should be considered to synthesize drugs capable to efficiently and selectively block MMPs chemical routes targeting their inactivation in order to overcome the current limitations of the therapeutic use of MMPs inhibitors, i.e., easy clinical application and long-lasting effect.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.3
• /
• pp.644-647
• /
• 2017

Peptidyl prolyl cis/trans isomerases (PPIases) catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds preceding prolines. We investigated the protein-protein interaction between a 22 kDa PPIase (VaFKBP22, an FK506-binding protein) and the molecular chaperone DnaK derived from Vibrio anguillarum O1 (VaDnaK) using GST pull-down assays and a bacterial two-hybrid system for in vivo and in vitro studies, respectively. Furthermore, we analyzed the three-dimensional structure of the protein-protein interaction. Based on our results, VaFKBP22 appears to act as a cochaperone of VaDnaK, and contributes to protein folding and stabilization via its peptidyl-prolyl cis/trans isomerization activity.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.1
• /
• pp.193-196
• /
• 2004

The peptide Arg-Gly-Asp (RGD) was immobilized through their amino terminus to ends of a sugar bearing copolymer, producing a functional hybrid copolymer. Poly(N-p-vinylbenzyl-D-maltonamide-co-6-(p-vinylbenzamido)-hexanoic acid-g-GRGDS) [p(VMA-co-VBGRGDS)] promoted the attachment and growth of NIH fibroblast cells. The interaction between fibroblast cells and p(VMA-co- VBGRGDS) copolymer resulted in effective cell attachment, proliferation, and morphological changes by introduction of a GRGDS sequence. Moreover, when pretreated with soluble RGD monomer, attachment of fibroblast cells was suppressed approximately 50% from that of the p(VMA-co-VBGRGDS) surface.

• Korean Journal of Pharmacognosy
• /
• v.54 no.1
• /
• pp.16-20
• /
• 2023

KSM-2690 B (1), a peptide-polyketide hybrid compound, was discovered from an actinomycete strain (CJD 1) isolated from Dong-Baek hill on Jeju Island, Republic of Korea. The chemical structure of 1 was identified by using NMR, MS, and UV spectroscopic analyses. Careful analysis of 1D and 2D NMR data revealed that KSM-2690 (1) has an oxazole ring, a β-lactone-γ-lactam spirocycle ring, and both triene and diene structures. KSM-2690 B (1) showed inhibitory activities against E. coli at 200 ㎍/mL.

• Journal of Applied Biological Chemistry
• /
• v.61 no.1
• /
• pp.83-91
• /
• 2018

Advances in bacterial and fungal genome mining uncover a plethora of cryptic secondary metabolite biosynthetic gene clusters. Guided by the genome information, targeted transcriptional derepression could be employed to determine the product of a cryptic gene cluster and to explore its biological role. Monascus spp. are food grade filamentous fungi popular in eastern Asia and several genome data belong to them are now available. We achieved transcription activation of a cryptic fungal polyketide synthase-nonribosomal peptide synthase gene Mpfus1 in Monascus purpureus ${\Delta}MpPKS5$ by inserting Aspergillus gpdA promoter at the upstream of Mpfus1 through double crossover gene replacement. The gene cluster with Mpfus1 show a high similarity to those for the biosynthesis of conjugated polyene derivatives with 2-pyrrolidone ring and the mycotoxin fusarin is the representative member of this group. The ${\Delta}MpPKS5$ is incapable of producing azaphilone pigment, providing an excellent background to identify chromogenic and UV-absorbing compounds. Activation of Mpfus1 resulted in a yellow hue on mycelia and its methanol extract exhibit a maximum absorption at 365 nm. HPLC analysis of the organic extracts indicated the presence of a variety of yellow compounds in the extract. This implies that the product of MpFus1 is metabolically or chemically unstable. LC-MS analysis guided us to predict the MpFus1 product and to propose that the Mpfus1-containing gene cluster encode the biosynthesis of a desmethyl analogue of fusarin. This study showcases the genome mining in Monascus and the possibility to unveil new biological activities embedded in it.

• Bulletin of the Korean Chemical Society
• /
• v.33 no.10
• /
• pp.3233-3240
• /
• 2012

We demonstrated the combined applications of online protein digestion using trypsin immobilized enzyme reactor (IMER) and dual tandem mass spectrometry with collisionally activated dissociation (CAD) and electron transfer dissociation (ETD) for tryptic peptides eluted through the trypsin-IMER. For the trypsin-IMER, the organic and inorganic hybrid monolithic material was used. By employing the trypsin-IMER, the long digestion time could be saved with little or no sacrifice of the digestion efficiency, which was demonstrated for standard protein samples. For three model proteins (cytochrome c, carbonic anhydrase, and bovine serum albumin), the tryptic peptides digested by the IMER were analyzed using LC-MS/MS with the dual application of CAD and ETD. As previously shown by others, the dual application of CAD and ETD increased the sequence coverage in comparison with CAD application only. In particular, ETD was very useful for the analysis of highly-protontated peptide cations, e.g., ${\geq}3+$. The combination approach provided the advantages of both trypsin-IMER and CAD/ETD dual tandem mass spectrometry applications, which are rapid digestion (i.e., 10 min), good digestion efficiency, online coupling of trypsin-IMER and liquid chromatography, and high sequence coverage.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.3
• /
• pp.229-237
• /
• 2009

Among 12 marine bacterial strains from the China coast that exhibited interesting bioactivity (positive for both antimicrobial and cytotoxic activities), only four strains, namely, NJ6-3-1, NJ6-3-2, NB-6, and YTHM-17, had a KS domain or A domain when screened for PKS and NRPS genes using a PCR. Interestingly, two of these strains belonging to Pseudoalteromonas and associated with the marine sponge Hymeniacidon perleve were positive for both PKS and NRPS, whereas the other two strains of Pseudoalteromonas did not have a PKS or NRPS gene. A molecular phylogeny analysis and DGGE analysis of the Pseudoalteromonas sp. indicated that they had a specific affinity with the host marine sponge Hymeniacidon perleve. Furthermore, an analysis of a partial sequence of Pseudoalteromonas sp. NJ6-3-2 isolated from the marine sponge Hymeniacidon perleve obtained from genomic walking using a computational approach indicated a relatively complete PKS module including auxiliary domains (DH, KR, and Cy).