• Biotechnology and Bioprocess Engineering:BBE
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• 제10권3호
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• pp.180-185
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• 2005

Cytosine deaminase (cytosine aminohydrolase, EC 3.5.4.1) stoichiometrically catalyzes the hydrolytic deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively. Amino acid residues located in or near the active sites of the intracellular cytosine deaminase from chromobacterium violaceum YK 391 were identified by chemical modification studies. The enzymic activity was completely inhibited by chemical modifiers, such as 1mM NBS, chloramine-T, $\rho-CMB,\;\rho-HMB$ and iodine, and was strongly inhibited by 1mM PMSF and pyridoxal 5'-phosphate. This chemical deactivation of the enzymic activity was reversed by a high concentration of cytosine. Furthermore, the deactivation of the enzymic activity by $\rho-CMB$ was also reversed by 1mM cysteine-HCI, DTT and 2-mercaptoethanol. These results suggested that cysteine, tryptophan and methionine residues might be located in or near the active sites of the enzyme, while serine and lysine were indirectly involved in the enzymic activity. The intracellular cytosine deaminase from C violaceum YK 391 was assumed to be a thiol enzyme.

• 미생물학회지
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• 제38권4호
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• pp.218-218
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• 2002

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• Journal of Microbiology
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• 제38권4호
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• pp.218-223
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• 2000

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.251-257
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• 2003

Metabolic engineering has become a new paradigm for the more efficient production of desired bioproducts. Metabolic engineering can be defined as directed modification of cellular metabolism and properties through the introduction, deletion, and modification of metabolic pathways by using recombinant DNA and other molecular biological tools. During the last decade, metabolic flux analysis(MFA) has become an essential tool fur metabolic engineering. By MFA, the intracellular metabolic fluxes can be quantified by the measurement of extracellular metabolite concentrations in combination with the stoichiometry of intracellular reactions and mass balances. The usefulness and functionality of MFA are demonstrated by applying to metabolic pathways in E. coli. First, a large-scale in silico E. coli model is constructed, and then the effects of carbon sources on intracellular flux distributions and succinic acid production were investigated on the basis of the uptake and secretion rates of the relevant metabolites. The results indicated that succinic acid yields increased in order of gluconate, glucose and sorbitol. Acetic acid and lactic acid were produced as major products rather than when gluconate and glucose were used carbon sources. The results indicated that among three carbon sources available, the most reduced substrate is sorbitol which yields efficient succinic acid production.

• BMB Reports
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• 제43권10호
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• pp.649-655
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• 2010

Alzheimer's disease (AD) is the most common age-dependent neurodegenerative disorder and shows progressive memory loss and cognitive decline. Intraneuronal filaments composed of aggregated hyperphosphorylated tau protein, called neurofibrillary tangles, along with extracellular accumulations of amyloid $\beta$ protein (A$\beta$), called senile plaques, are known to be the neuropathological hallmarks of AD. In light of recent studies, epigenetic modification has emerged as one of the pathogenic mechanisms of AD. Epigenetic changes encompass an array of molecular modifications to both DNA and chromatin, including transcription factors and cofactors. In this review, we summarize how DNA methylation and changes to DNA chromatin packaging by post-translational histone modification are involved in AD. In addition, we describe the role of SIRTs, histone deacetylases, and the effect of SIRT-modulating drugs on AD. Lastly, we discuss how amyloid precursor protein (APP) intracellular domain (AICD) regulates neuronal transcription. Our understanding of the epigenomes and transcriptomes of AD may warrant future identification of novel biological markers and beneficial therapeutic targets for AD.

• 한국자기공명학회논문지
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• 제23권3호
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• pp.67-72
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• 2019

Caveolae are small plasma membrane invaginations that play many roles in signal transduction, endocytosis, mechanoprotection, lipid metabolism. The most important protein in caveolae is the integral membrane protein, caveolin, which is divided into three families such as caveolin 1, caveolin 2, and caveolin 3. Caveolin 1 and 3 are known to incorporate palmitate through linkage to three cysteine residues. Regulation of the protein palmitoylation cycle is important for the cellular processes such as intracellular localization of the target protein, membrane association, conformation, protein-protein interaction, and activity. However, the detailed aspect of individual palmitoylation has not been studied. In the present work, the role of each lipid modification at three cysteines was studied by NMR. Our results suggest that each lipid modification at the natively palmitoylation site has its own roles. For example, lipidations to C106 and C129 are play a role in structural stabilization, however, interestingly, lipid modification to C116 interrupts the structural stabilization.

• Journal of Pharmaceutical Investigation
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• 제32권4호
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• pp.285-290
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• 2002

In order to assay the efficacy of newly synthesized antiviral compounds, in vitro studies of their active intracellular phosphorylated metabolites were established as compared with Zidovudine (ZDV). Antiviral base analogs require intracellular phosphorylation prior to the inhibition of HIV replication. Therefore, antiviral drugs concentrations in plasma have not reflected any direct relationship with activity or toxicity. A method has been developed to measure the concentration of total phosphorylated metabolites inside peripheral blood mononuclear cells using modified commercial radioimmunoassay (RIA). ZDV 5'-monophosphate was synthesized and used as a procedural control for RIA modification. PBMCs were isolated from whole blood and incubated with ZDV for 20 h to allow metabolic phosphorylation. Viable cells were extracted overnight with 60% methanol. After evaporation, the extract was reconstituted in Tris buffer. Samples were split into two fractions, one of which was treated with alkaline phosphatase (AP) to liberate phosphate groups. Concentrations of phosphorylated metabolites were determined by subtracting thε concentration of non-AP-treated fraction from that of the treated fraction. Recovery of phosphorylated ZDV from cell extracts was approximately 90%, and reproducibility was acceptable (coefficients of variation <15% for concentrations${\geq}$0.25 ng/mL). Intracellular concentrations $(0.135{\sim}5.019\;nmole/10^6\;cells)$ followed a nonlinear dose-response relationship over the range $0.015{\sim}2.996mM$ extracellular ZDV, with concentration-dependant saturation.

• KSBB Journal
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• 제29권5호
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• pp.328-335
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• 2014

Vehicle toxicity is one of the main obstacles for intracellular delivery of bioactive compounds. Silk fibroin is a natural polymer proven to have high biocompatibility since being used as suture material. In this report, fibroin microspheres were prepared without any chemical modification or cross-linking not to affect its biocompatibility. The microspheres were taken up by more than 90% of 3T3 cells. Cellular uptake continued after medium replenishment with a different-colored fluorescent microsphere, suggesting that simultaneous ingestion and exocytosis occurred. Cellular uptake of fibroin microspheres did not affect cell viability. Intracellular trafficking of the microspheres using lysosome-specific fluorescent dye revealed that fibroin microspheres were localized both in the cytoplasm and in the lysosome. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of large cargos, such as mixtures of proteins, nutrients or artificial organelles.

• Journal of Yeungnam Medical Science
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• 제38권2호
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• pp.83-94
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• 2021

The global obesity epidemic and the growing elderly population largely contribute to the increasing incidence of type 2 diabetes. Insulin resistance acts as a critical link between the present obesity pandemic and type 2 diabetes. Naturally occurring reactive oxygen species (ROS) regulate intracellular signaling and are kept in balance by the antioxidant system. However, the imbalance between ROS production and antioxidant capacity causes ROS accumulation and induces oxidative stress. Oxidative stress interrupts insulin-mediated intracellular signaling pathways, as supported by studies involving genetic modification of antioxidant enzymes in experimental rodents. In addition, a close association between oxidative stress and insulin resistance has been reported in numerous human studies. However, the controversial results with the use of antioxidants in type 2 diabetes raise the question of whether oxidative stress plays a critical role in insulin resistance. In this review article, we discuss the relevance of oxidative stress to insulin resistance based on genetically modified animal models and human trials.

• Applied Science and Convergence Technology
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• 제24권6호
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• pp.284-288
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• 2015

Anionic small molecules are hard to penetrate the cell membranes because of their negative charges. Encapsulation of small molecules into liposome particles can provide target specific delivery of them. In our previous study, siRNA could be efficiently encapsulated into liposome particles using an asymmetric preparation method of liposomes. In this study, the same method was applied for encapsulation of small anionic fluorescent chemicals such as calcein and indocyanine green (ICG). More than 90% fluorescent chemicals were encapsulated in the asymmetric liposome particles (ALPs). No intracellular fluorescent signal was observed in the tumor cells treated with the unmodified calcein/ALPs and ICG/ALPs, whereas the surface modification with a cell-penetrating polyarginine peptide (R8 or R12) allows cellular uptake of the ALPs. The results demonstrate that the ALPs encapsulating small anionic drugs will be useful for target-specific delivery after modification of target-specific ligands.