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Bacterial Expression of Cytochrome $b_5$ Type III Pseudogene

  • Baek, Sun-Ah;Kim, Su-Won;Kim, Jong-Won;Yoo, Min
    • Biomedical Science Letters
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    • v.18 no.3
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    • pp.310-312
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    • 2012
  • Cytochrome $b_5$ is involved in the reduction of methemoglobin back to hemoglobin, thereby maintaining normal function of the blood to carry oxygen around. Congenital abnormal condition of this enzyme causes a rare disease called methemoglobinemia. At least 4 different retropseudogenes are reported so far for cytochrome $b_5$. However, type III pseudogene has attracted most attention because it contains open reading frame in its structure. Although there is no evidence yet if this pseudogene is actually expressed in the cell or the blood the possibility of its expression needs to be elucidated. We have isolated type III pseudogene by polymerase chain reaction and cloned into pGEX-4T-1 expression vector followed by SDS-PAGE. Protein was expressed and the size of the expressed protein was 28 kDa as expected in its genetic code. This result also shows that the protein is not harmful for the viability of the microorganism. This study may contribute to the genetic diagnosis of cardiac diseases, possibly caused by cytochrome $b_5$.

Analysis of the Structure and Stability of Erythropoietin by pH and Temperature Changes using Various LC/MS

  • Chang, Seong-Hun;Kim, Hyun-Jung;Kim, Chan-Wha
    • Bulletin of the Korean Chemical Society
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    • v.34 no.9
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    • pp.2663-2670
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    • 2013
  • The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature ($25^{\circ}C$ and $40^{\circ}C$) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at $40^{\circ}C$ and pH 9, respectively.

Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal

  • Sa, Hyun Deok;Park, Ji Yeong;Jeong, Seon-Ju;Lee, Kang Wook;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • v.25 no.5
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    • pp.696-703
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    • 2015
  • A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

A Selective Compression Strategy for Performance Improvement of Database Compression (데이터베이스 압축 성능 향상을 위한 선택적 압축 전략)

  • Lee, Ki-Hoon
    • KIPS Transactions on Software and Data Engineering
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    • v.4 no.9
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    • pp.371-376
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    • 2015
  • The Internet of Things (IoT) significantly increases the amount of data. Database compression is important for big data because it can reduce costs for storage systems and save I/O bandwidth. However, it could show low performance for write-intensive workloads such as OLTP due to the updates of compressed pages. In this paper, we present practical guidelines for the performance improvement of database compression. Especially, we propose the SELECTIVE strategy, which compresses only tables whose space savings are close to the expected space savings calculated by the compressed page size. Experimental results using the TPC-C benchmark and MySQL show that the strategy can achieve 1.1 times better performance than the uncompressed counterpart with 17.3% space savings.

Molecular Characterization and Bitter Taste Formation of Tryptic Hydrolysis of 11S Glycinin

  • Kim, Mi-Ryung;Choi, Sang-Yun;Lee, Cherl-Ho
    • Journal of Microbiology and Biotechnology
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    • v.9 no.4
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    • pp.509-513
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    • 1999
  • The molecular size reduction and the formation of bitterness during a tryptic hydrolysis of soybean 11S glycinin were determined by using quantitative analysis and organoleptic evaluation. The 11S glycinin of 90% purity was prepared by cryoprecipitation and Con A Sepharose 4B affinity chromatography, and hydrolyzed with trypsin in a pH-stat reactor for 4 h. Bitterness was formed within 1 h of hydrolysis, and then slowly increased up to $3.5\times10^{-5}$ M quinine-HCl equivalent. The extent of hydrolysis (DH) was 7% at 1 h and increased up to 12% by the end of the reaction. The -amino nitrogen content increased from an initial 0.7 mM to 7 mM at the end of the period. The SDS-PAGE analysis showed that the acidic subunit of 11S glycinin was mostly hydrolyzed. The GP-HPLC analysis indicated that the bitterness was mainly contributed by the peptide fractions of molecular weights of 360-2,100 Da.

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Design of an EEPROM for a MCU with the Wide Voltage Range

  • Kim, Du-Hwi;Jang, Ji-Hye;Jin, Liyan;Ha, Pan-Bong;Kim, Young-Hee
    • JSTS:Journal of Semiconductor Technology and Science
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    • v.10 no.4
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    • pp.316-324
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    • 2010
  • In this paper, we design a 256 kbits EEPROM for a MCU (Microcontroller unit) with the wide voltage range of 1.8 V to 5.5 V. The memory space of the EEPROM is separated into a program and data region. An option memory region is added for storing user IDs, serial numbers and so forth. By making HPWs (High-voltage P-wells) of EEPROM cell arrays with the same bias voltages in accordance with the operation modes shared in a double word unit, we can reduce the HPW-to-HPW space by a half and hence the area of the EEPROM cell arrays by 9.1 percent. Also, we propose a page buffer circuit reducing a test time, and a write-verify-read mode securing a reliability of the EEPROM. Furthermore, we propose a DC-DC converter that can be applied to a MCU with the wide voltage range. Finally, we come up with a method of obtaining the oscillation period of a charge pump. The layout size of the designed 256 kbits EEPROM IP with MagnaChip's 0.18 ${\mu}m$ EEPROM process is $1581.55{\mu}m{\times}792.00{\mu}m$.

Spatio-Temporal Index Structure for Trajectory Queries of Moving Objects in Video (비디오에서 이동 객체의 궤적 검색을 위한 시공간 색인구조)

  • Lee, Nak-Gyu;Bok, Kyoung-Soo;Yoo, Jae-Soo;Cho, Ki-Hyung
    • The KIPS Transactions:PartD
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    • v.11D no.1
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    • pp.69-82
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    • 2004
  • A moving object has a special feature that it's spatial location, shape and size are changed as time goes. These changes of the object accompany the continuous movement that is called the trajectory. In this paper, we propose an index structure that users can retrieve the trajectory of a moving object with the access of a page. We also propose the multi-complex query that is a new query type for trajectory retrieval. In order to prove the excellence of our method, we compare and analyze the performance for query time and storage space through experiments in various environments. It is shown that our method outperforms the existing index structures when processing spatio-temporal trajectory queries on moving objects.

Characterization of a Bacteriocin Produced by Enterococcus sp. T7 Isolated from Humans

  • Moon, Hi-Seong;Jeong, Jong-Jin;Ji, Geun-Eog;Kim, Jong-Sang;Kim, Jeong-Hwan
    • Journal of Microbiology and Biotechnology
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    • v.10 no.4
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    • pp.507-513
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    • 2000
  • A bacteriocin-producing organism, Enterococcus sp. T7, was isolated from human fecal samples. Bacteriocin T7, named tentatively as the bacteriocin, was produced by Enterococcus sp. T7 and it inhibited some strains of Lactobacillus. Staphylococcus, Enterococcus, and Streptococcus, but not all the lactococci and gram-negative bacteria tested. Bacteriocin T7 inhibited the growth of Listeria monocytogenes Scott A, but the degree of inhibition was less than those for other sensitive gram-positive vacteria. Bacteriocin T7 in MRS broth started to produce at the middle of the exponential growth phase and the inhibitory activity reached its maximum level during the stationary growth phase. Bacteriocin T7 was stable against heat treatments, pH variations (pH 2-10), and exposure to organic solvents. The molecular weight of bacteriocin T7 was estimated to be 6.500 Da by SDS-PAGe. All these facts, including physico-chemical stabilities, small molecular size, and inhibition of Kisteria monocytogenes, indicate that bacteriocin T7 is likely to be a member of the class IIa bacteriocins.

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Isolation and Analysis of the argG Gene Encoding Argininosuccinate Synthetase from Corynebacterium glutamicum

  • Ko, Soon-Young;Kim, Sei-Hyun;Lee, Heung-Shick;Lee, Myeong-Sok
    • Journal of Microbiology and Biotechnology
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    • v.13 no.6
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    • pp.949-954
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    • 2003
  • The argG gene of Corynebacterium glutamicum encoding argininosuccinate synthetase (EC6345) was cloned and sequenced. The gene was cloned by heterologous complementation of an Escherichia coli arginine auxotrophic mutant (argG/sup -/). The cloned DNA fragment also complements E. coli argD, argF, and argH mutants, suggesting a clustered organization of the genes in the chromosome. The coding region of the argG gene is 1,206 nucleotides long with a deduced molecular weight of about 44 kDa, comparable with the predicted size of the expressed protein on the SDS-PAGE. Computer analysis revealed that the amino acid sequence of the argG gene product had a high similarity to that of Mycobacterium tuberculosis and Streptomyces clavuligerus. Two conserved sequence motifs within the ArgG appear to be ATP-binding sites which correspond to 2 of the 3 conserved regions found in sequences of all known argininosuccinate synthetases.

Properties of Bac W42, a Bacteriocin Produced by Bacillus subtilis W42 Isolated from Cheonggukjang

  • Kindoli, Salum;Lee, Hwang A;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • v.22 no.8
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    • pp.1092-1100
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    • 2012
  • Ten Bacillus strains with antimicrobial activities were isolated from Cheonggukjang produced at different parts in Korea. They all inhibited Listeria monocytogenes ATCC 19111 and nine inhibited Bacillus cereus ATCC 14579. Four isolates (W42, H27, SKE 12, and K21) showing strong inhibiting activities were identified as B. subtilis. B. subtilis W42 was the most inhibiting strain. The antimicrobial activity of culture supernatant from B. subtilis W42 was destroyed completely by proteinase K treatment, indicating that a bacteriocin was the responsible agent. The bacteriocin, Bac W42, was most stable at pH 7 and stable between pH 3-6 and 8-9. Bac W42 was stable up to $80^{\circ}C$. BHI (brain heart infusion) and TSB (tryptic soy broth) were the best media for the activity (320 AU/ml) followed by LB (160 AU/ml). Bac W42 was partially purified by column chromatographies. The specific activity was increased from 1,151.2 AU/ml to 9,043.5 AU/ml and the final yield was 26.3%. Bac W42 was 5.4 kDa in size as determined by SDS-PAGE. Bac W42 showed bactericidal activity against L. monocytogenes ATCC 19111.