• BMB Reports
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• v.35 no.2
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• pp.236-238
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• 2002

Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDS-fibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10kDa to over 100kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

• BMB Reports
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• v.42 no.8
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• pp.523-528
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• 2009

Phosphomannose isomerase (PMI) catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate in the extracellular polysaccharide (EPS) synthesis pathway. The gene encoding PMI in Sphingomonas chungbukensis DJ77 was cloned and expressed in E. coli. The pmi gene is 1,410 nucleotides long and the deduced amino acid sequence shares high homology with other bifunctional proteins that possess both PMI and GDP-mannose pyrophosphorylase (GMP) activities. The sequence analysis of PMI revealed two domains with three conserved motifs: a GMP domain at the N-terminus and a PMI domain at the C-terminus. Enzyme assays using the PMI protein confirmed its bifunctional activity. Both activities required divalent metal ions such as $Co^{2+}$, $Ca^{2+}$, $Mg^{2+}$, $Ni^{2+}$ or $Zn^{2+}$. Of these ions, $Co^{2+}$ was found to be the most effective activator of PMI. GDP-D-mannose was found to inhibit the PMI activity, suggesting feedback regulation of this pathway.

• Membrane and Water Treatment
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• v.8 no.4
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• pp.337-353
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• 2017

Membrane fouling at different solids retention times (SRT) (7, 12 and 20 days) was studied under well-controlled conditions in a laboratory-scale aerobic submerged membrane bioreactor under constant biomass concentration using a synthetic high strength wastewater. An increase in SRT was found to improve membrane performance and this correlated to changes in the total production of bound extracellular polymeric substances (EPS), and the composition and properties of bound EPS using X-ray photoelectron spectroscopy (XPS) and Fourier transform-infrared spectroscopy (FTIR) and floc sizes. A larger amount of total bound EPS was found at the lowest SRT (7 days) tested but the ratio of proteins (PN) to carbohydrates (CH) in bound EPS increased with an increase in SRT. Similarly, the quantity of soluble microbial products (SMP) decreased with an increase in SRT and the SMP PN/CH ratio increased with an increase in SRT. SMP concentrations positively correlated to the percentage of membrane pore blocking resistance. The quantity of total bound EPS and total SMP positively corresponded to the membrane fouling rate, while the PN/CH ratio in the bound EPS and SMP negatively correlated to the membrane fouling rate. The results show that both the quantity and composition of bound EPS and SMP and floc sizes are important in controlling membrane fouling.

• Genomics & Informatics
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• v.20 no.2
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• pp.24.1-24.8
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• 2022

Hypomyelinating leukodystrophy type 2 (HLD2), is an inherited genetic disease of the central nervous system caused by recessive mutations in the gap junction protein gamma 2 (GJC2/GJA12). HLD2 is characterized by nystagmus, developmental delay, motor impairments, ataxia, severe speech problem, and hypomyelination in the brain. The GJC2 sequence encodes connexin 47 protein (Cx47). Connexins are a group of membrane proteins that oligomerize to construct gap junctions protein. In the present study, a novel missense mutation gene c.760G>A (p.Val254Met) was identified in a patient with HLD2 by performing whole exome sequencing. Following the discovery of the new mutation in the proband, we used Sanger sequencing to analyze his affected sibling and parents. Sanger sequencing verified homozygosity of the mutation in the proband and his affected sibling. The autosomal recessive inheritance pattern was confirmed since Sanger sequencing revealed both healthy parents were heterozygous for the mutation. PolyPhen2, SIFT, PROVEAN, and CADD were used to evaluate the function prediction scores of detected mutations. Cx47 is essential for oligodendrocyte function, including adequate myelination and myelin maintenance in humans. Novel mutation p.Val254Met is located in the second extracellular domain of Cx47, both extracellular loops are highly conserved and probably induce intramolecular disulfide interactions. This novel mutation in the Cx47 gene causes oligodendrocyte dysfunction and HLD2 disorder.

• Molecules and Cells
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• v.46 no.12
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• pp.757-763
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• 2023

In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.

• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.587-601
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• 2013

During the last few decades, it has become evident that the compatibility of the yeast biochemical environment with the ability to process and translate the RNA transcript, along with its capacity to modify a translated protein, are relevant requirements for selecting this host cell for protein expression in several pharmaceutical and clinical applications. In particular, Pichia pastoris is used as an industrial host for recombinant protein and metabolite production, showing a powerful capacity to meet required biomolecular target production levels in high-throughput assays for functional genomics and drug screening. In addition, there is a great advantage to using P. pastoris for protein secretion, even at high molecular weights, since the recovery and purification steps are simplified owing to relatively low levels of endogenous proteins in the extracellular medium. Clearly, no single microexpression system can provide all of the desired properties for human protein production. Moreover, chemical and physical bioprocess parameters, including culture medium formulation, temperature, pH, agitation, aeration rates, induction, and feeding strategies, can highly influence product yield and quality. In order to benefit from the currently available wide range of biosynthesis strategies using P. pastoris, this mini review focuses on the developments and technological fermentation achievements, providing both a comparative and an overall integration analysis. The main aim is to highlight the relevance and versatility of the P. pastoris biosystem to the design of more cost-effective microfactories to meet the increasing demands for recombinant membrane proteins and clinical antibodies for several therapeutic applications.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1667-1674
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• 2012

Objective: To explore the differential protein expression profile in EC304 gastric cancer cells induced by alphastatin. Methods: Cultured EC304 cells in the exponential phase of growth were randomly divided into alphastatin and control groups. Total proteins were extracted and the two dimensional electrophoresis (2-DE) technique was applied to analyze differences in expression with ImageMaster 2D Platinum 5.0 software. Proteins were identified using the MASCOT database and selected differently expressed proteins were characterised by western blotting and immunofluorescence. Results: $1350{\pm}90$ protein spots were detected by the ImageMaster software in the 2-DE gel images from the control and alphastatin groups. The match rate was about 72-80% for the spectrum profiles, with 29 significantly different protein spots being identified, 10 upregulated, 16 downregulated, two new and one lost. The MASCOT search scores were 64-666 and the peptide matching numbers were 3-27 with sequence coverage of 8-62%. Twenty-three proteins were checked by mass spectrometry, including decrease in Nm23 and profilin-2 isoform b associated with the regulation of actin multimerisation induced by extracellular signals. Conclusion: The proteome in EC304 cells is dramatically altered by alphastatin, which appears to play an important role in modulating cellular activity and anti-angiogenesis by regulating protein expression and signal transduction pathways through Nm23 and profilin-2 isoform b, providing new research directions for anti-angiogenic therapy of gastric cancer.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.1
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• pp.19-25
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• 2001

We have studied the effects of excitatory amino acids on the expression of the c-fos and c-jun mRNA in rat C6 glioma cells. The glutamate, $N-methyl-_D-aspartate$ (NMDA), and kainic acid (KA) increased c-fos mRNA level in a concentration-dependent manner. However, they did not affect c-jun mRNA level. In addition, forskolin and phorbol 12-myristate 13-acetate (PMA) increased c-fos mRNA level. Furthermore, PMA increased c-jun mRNA level whereas forskolin downregulated c-jun mRNA level. The glutamate, NMDA and KA, at a concentration of 0.25 mM, did not affect the basal c-fos and c-jun mRNA levels, and also did not affect forskolin- and PMA-induced responses. Furthermore, both forskolin and PMA itself increased the phosphorylation of ERK (extracellular signal regulated kinase) and CREB (cyclicAMP responsible element binding protein) proteins. The KA, NMDA, and glutamate did not affect forskolin- induced increase of ERK and CREB phosphorylation. The KA decreased PMA-induced increase of phosphorylation of ERK and CREB proteins, whereas glutamate and NMDA did not affect the phosphorylation of ERK and CREB proteins induced by PMA. These findings suggest that, in C6 glioma cells, c-fos mRNA induction induced by EAAs is not mediated by phosphorylation of ERK and CREB proteins.

• Clinical and Experimental Pediatrics
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• v.62 no.3
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• pp.95-101
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• 2019

Purpose: Increased apoptosis was recently found in the hypertrophied left ventricle of spontaneously hypertensive rats (SHRs). Although the available evidence suggests that apoptosis can be induced in cardiac cells by various insults including pressure overload, cardiac apoptosis appears to result from an exaggerated local production of angiotensin in adult SHRs. Altered expressions of Bcl associated X (Bax), Bcl-2, chemokine receptor (CCR)-2, monocyte chemoattractant protein (MCP)-1, transforming growth factor $(TGF)-{\beta}1$, phosphorylated extracellular signal-regulated kinases (PERK), and connexin 43 proteins, and kallikrein mRNA were investigated to explore the effects of losartan on the SHR model. Methods: Twelve-week-old male rats were grouped as follows: control (C), SHR (hypertension: H), and losartan (L; SHRs were treated with losartan [10 mg/kg/day] for 5 weeks). Western blot and reverse transcription polymerase chain reaction assays were performed. Results: Expression of Bax, CCR-2, MCP-1, $TGF-{\beta}1$, PERK, and connexin 43 proteins, and kallikrein mRNA was significantly increased in the H group compared to that in the C group at weeks 3 and 5. Expression of Bax, CCR-2, MCP-1, $TGF-{\beta}1$, and connexin 43 proteins and kallikrein mRNA was significantly decreased after losartan treatment at week 5. PERK protein expression was significantly decreased after losartan treatment at weeks 3 and 5. Bcl-2 protein expression was significantly decreased in the H group compared to that in the C group at weeks 3 and 5. Conclusion: Losartan treatment reduced expression of Bax, CCR-2, MCP-1, $TGF-{\beta}1$, PERK, and connexin 43 proteins, and kallikrein mRNA in SHRs, along with decreased inflammation and apoptosis.

• International Journal of Oral Biology
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• v.33 no.3
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• pp.105-111
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• 2008

Thrombospondins (TSP-1, TSP-2) are secretory extracellular glycoproteins that are involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. The present study was undertaken to elucidate the involvement of thrombospondins in the adhesion of osteoblast-like cells using the TSP-1 or TSP-2 antisense MG63 and MC3T3-E1 cell lines. For downregulation of TSPs expression, we prepared antisense constructs for TSP-1 and TSP-2 using the pREP4 an episomal mammalian expression vector, which be able to produce the specific antisense oligonucleotides around chromosome. MG63 and MC3T3-E1 osteoblast-like cells were transfected with the antisense constructs and nonliposomal Fugene 6, and then selected under hygromycin B (50 ${\mu}g/ml$) treatment for 2 weeks. Western blot analysis revealed that expression of the TSP proteins was downregulated in the antisense cell lines. The cell adhesion assay showed that adhesive properties of TSP-1 and TSP-2 antisense MG63 cells on the polystyrene culture plate were reduced to 17% and 21% of the control cells, respectively, and those of the TSP-1 and TSP-2 antisense MC3T3-E1 cells also decreased to 19% and 27% of control, respectively. Adhesion of TSP-1 and TSP-2 antisense MC3T3-E1 cells on Type I collagen-coated culture plate decreased to 27% and 76%, respectively. These results indicate that TSP-1 and TSP-2 proteins may have an important role in adhesion of osteoblast-like cells to extracellular matrix.