• Korean Journal of Microbiology
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• v.50 no.4
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• pp.275-280
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• 2014

Leucine-responsive Regulatory Protein (Lrp) from Escherichia coli is an 18.8 kDa protein composed of 164 amino acids. Wild type Lrp (Lrp Wt) does not possess any tryptophan amino acid which has strong intrinsic fluorescence, whereas the mutant Lrp R145W contains a single tryptophan at the position 145 in the leucine-responsive domain. To investigate the fluorescence character, the Lrp R145W and Lrp Wt proteins were purified. The fluorescence intensity of Lrp R145W is much higher than that of wild type protein, and the intensity of Lrp R145W was decreased by binding to its specific DNA designed from ilvIH operon and to L-leucine. In addition, the tryptophan fluorescence intensity of Lrp R145W was strongly quenched by addition of acrylamide even in the least amount of concentration as well as by urea. The data obtained from this study may give valuable information on the three dimensional structure of Lrp R145W.

• Parasites, Hosts and Diseases
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• v.38 no.2
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• pp.107-110
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• 2000

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g. HSP70-NH2-terminal region, or rT.g. HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or rT.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NHa-terminal region did not. These results suggest that T.g. HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

• Journal of the Korean Magnetic Resonance Society
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• v.16 no.2
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• pp.147-161
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• 2012

Human melanocortin-4 receptor (hMC4R) among MC-Rs, expressed in the brain, is in charge of the control on energy homeostasis and food intake. The structure and function of human MC4R have been studied to understand their essential function and roles. To investigate the structure and function, it is necessary to prepare sufficient amounts of proteins. However, their expression and purification is demanding and time-consuming due to their innate insoluble and toxic properties. The heterozygous mutations of hMC4R, exchange of Asp 90 to Asn located in second transmembrane, cause severe obesity in human. To obtain purified hMC4R wt-TM2 for structural studies, it was first over-expressed and purified by fast protein liquid chromatography (FPLC) and then solution NMR studies were performed to get high-resolution spectra. In here, we established optimized purification scheme to get more purified target peptide.

• BMB Reports
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• v.56 no.11
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• pp.618-623
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• 2023

Most cancer cells utilize glucose at a high rate to produce energy and precursors for the biosynthesis of macromolecules such as lipids, proteins, and nucleic acids. This phenomenon is called the Warburg effect or aerobic glycolysis- this distinct characteristic is an attractive target for developing anticancer drugs. Here, we found that Phosphofructokinase-1 (PFK-1) is a substrate of the Protein Phosphatase 4 catalytic subunit (PP4C)/PP4 regulatory subunit 1 (PP4R1) complex by using immunoprecipitation and in vitro assay. While manipulation of PP4C/PP4R1 does not have a critical impact on PFK-1 expression, the absence of the PP4C/PP4R1 complex increases PFK-1 activity. Although PP4C depletion or overexpression does not cause a dramatic change in the overall glycolytic rate, PP4R1 depletion induces a considerable increase in both basal and compensatory glycolytic rates, as well as the oxygen consumption rate, indicating oxidative phosphorylation. Collectively, the PP4C/PP4R1 complex regulates PFK-1 activity by reversing its phosphorylation and is a promising candidate for treating glycolytic disorders and cancers. Targeting PP4R1 could be a more efficient and safer strategy to avoid pleiotropic effects than targeting PP4C directly.

• Childhood Kidney Diseases
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• v.4 no.1
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• pp.1-5
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• 2000

Purpose: The plcr of spot urine has been uised to predict the timed urine protein excretion. Although this method reduces errors caused by variations in urine volume, it is relatively thconvenient and expensive. Recently, a more rapid and less expensive screening method with specific gravity(SG) has been reported, and we have examined whether estimated-creatinine(Cr-est) with urine 5G could be used in place of urine creatinine to predict 24-hour collected urine protein excretion in children. Methods: We had retrospectively analyzed protein, creatinine and urine SG in randomized spot urine samples of 147 patients from March 1998 till June 1998 in Korea university Guro hospital and compared the urinary protein creatinine ratio(P/Cr) with the protein estimated-creatinine ratio(P/Cr-est). We compared the correlation of urinary creatinine vs-urine 5G with the timed urine pretein excretion. Results: 1) urine SG accurately estimated urine creatinine concentration (r=0.407, P<0.001, Cr=SG x 4485.82-4482.87). 2) P/Cr correlated with urine protein excretion measured in a 24-hour urine collection (r=0.771, P<0.001, 24-hour collected urine protein : 0.338 x (P/Cr) 4+667.885). 3) P/Cr-est correlated with a 24-hour collected urine protein (r=0.723, P<0.001, 24-hour collected urine protein =0.354 x (P/Cr-est)+726.044), Conclusions: These results suggest that P/Cr-est with urine SG could be useful method for screening proteinuria in children.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.39 no.5
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• pp.489-494
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• 1994

Near Infrared Reflectance Spectroscopy(NIRS) has been used as a tool for the rapid, accurate, protein assay of malting barley. NIRS used in this study was filter type instruments, Neotec 102. The objective of this study was to obtain the best calibration equation, for the rapid, ease and accurate protein content analysis of malting barley using NIRS system. The optimum wavelength for protein content analysis used NIRS were 2095nm, 2095/1941nm, 2095/1941/2282nm, 2905/1941/2282/2086nm, respectively. Mean protein content with this calibration equation in NIRS analysis was 10.59%, while 10.60% in Micro-Kjeldahl one. The range of protein content in Micro-Kjeldahl was 8.66~12.66% and that in NIRS was 8.80~12.35%. When 18 other varieties produced in 1992 were analysed with 2095nm, 2095/1941nm, 2095/1941/2282nm, 2095/1941/2282/2086nm equation, standard deviation of difference (SDD)and standard error of performence(SEP) and $R^2$ values were 0.47, 0.43, 0.95, respectively. Both the mean protein content by Micro-Kjeldahl and by NIRS was 10.25%. With this equation, analysied 31 varities produced in 1993, SDD and SEP and r values were 0.69, 0.67, 0.91, respectively, and that bias value was 0.65. In this analysis, mean protein content by Micro-Kjeldahl was 10.17% and by NIRS was 10.81%. The range of protein content in Micro-Kjeldahl was 7.58~14.29%, What that in NIRS was 8.63~13.93%. After adjusted bias in the best calibration equation, mean protein content of Micro-Kjeldahl was 10.17% and that of NIRS was 10.09%, without variance of SDD, SEP and r values.

• Journal of Microbiology and Biotechnology
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• v.26 no.1
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• pp.190-196
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• 2016

In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

• Korean Journal of Veterinary Research
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• v.40 no.1
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• pp.86-93
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• 2000

Paratuberculosis (Johne's disease), a chronic enteritis produced by Mycobacterium paratuberculosis, affects a large proportion of ruminants in all continents and causes important economic losses. The identification of well-characterized and species-specific components of M paratuberculosis would provide the means to improve the specificity and sensitivity of immunodiagnostic assays for Johne's disease. The aims of this study were to express the recombinant C-terminal of 34kDa protein (rC34P) of M paratuberculosis in E coli and to investigate the effectiveness of this protein in detecting antibodies to the native protein in sera from paratuberculosis infected cattle. The C-terminal of the gene encoding the 34kDa protein was amplified by polymerase chain reaction from the chromosomal DNA of M paratuberculosis (ATCC 19698) and cloned into vector pGEX-4T-2. Then, cloned plasmid was transformed into E coli DH5${\alpha}$ and the rC34P was overexpressed. The rC34P was purified by affinity chromatography and gel filtration. The rC34P was examined antigenicity by Western blot. The rC34P was reactive with culture positive bovine serum and hyperimmune rabbit anti-M paratuberculosis serum but was not reactive with culture negative bovine serum and tuberculin positive bovine serum in Western blot. In conclusion, the rC34P produced in this study is expected as a useful candidate for antigen in serological diagnosis of Johne's disease.

• Journal of Photoscience
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• v.9 no.2
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• pp.106-109
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• 2002

Archaeal rhodopsins possess retinal molecule as their chromophores, and their light-energy and light-signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of protein then leads to functional processes, proton pump in bacteriorhodopsin (bR) and transducer activation in phoborhodopsin (pR). It is known that sensory rhodopsins can pump protons in the absence of their transducers. Thus, there should be common and specific features in their protein structural changes for function. In this paper, our r ecent studies on pR from Natronobacterium pharaonis (ppR) by means of low-temperature Fourier-transform infrared (FTIR) spectroscopy are compared with those of bR. In particular, protein structural changes upon retinal photoisomerization are studied. Comparative investigation of ppR and bR revealed the similar structures of the polyene chain of the chromophore and water-containing hydrogen-bonding network, whereas the structural changes upon photoisomerization were more extended in ppR than in bR. Extended protein structural changes were clearly shown by the assignment of the C=O stretch of Asnl05. FTIR studies of a ppR mutant with the same retinal binding site as in bR revealed that the Schiff base region is important to determine their colors.

• Journal of Microbiology
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• v.45 no.5
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• pp.418-421
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• 2007

The nucleotide at position 791(G791) of E. coli 16S rRNA was previously identified as an invariant residue for ribosomal function. In order to characterize the functional role of G791, base substitutions were introduced at this position, and mutant ribosomes were analyzed with regard to their protein synthesis ability, via the use of a specialized ribosome system. These ribosomal RNA mutations attenuated the ability of ribosomes to conduct protein synthesis by more than 65%. A transition mutation (G to A) exerted a moderate effect on ribosomal function, whereas a transversion mutation (G to C or U) resulted in a loss of protein synthesis ability of more than 90%. The sucrose gradient profiles of ribosomes and primer extension analysis showed that the loss of protein-synthesis ability of mutant ribosomes harboring a base substitution from G to U at position 791 stems partially from its inability to form 70S ribosomes. These findings show the involvement of the nucleotide at position 791 in the association of ribosomal subunits and protein synthesis steps after 70S formation, as well as the possibility of using 16S rRNA mutated at position 791 for the selection of second-site revertants in order to identify ligands that interact with G791 in protein synthesis.