• The KIPS Transactions:PartD
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• v.10D no.2
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• pp.261-266
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• 2003

A protein, which is a linear polymer of amino acids, is one of the most important bio-molecules composing biological structures and regulating bio-chemical reactions. Since the characteristics and functions of proteins are determined by their amino acid sequences in principle, protein sequence determination is the starting point of protein function study. This paper proposes a protein sequence prediction method based on data mining techniques, which can overcome the limitation of previous bio-chemical sequencing methods. After applying multiple proteases to acquire overlapped protein fragments, we can identify candidate fragment sequences by comparing fragment mass values with peptide databases. We propose a method to construct multi-partite graph and search maximal paths to determine the protein sequence by assembling proper candidate sequences. In addition, experimental results based on the SWISS-PROT database showing the validity of the proposed method is presented.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.10
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• pp.1454-1464
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• 2015

Four methods were adopted, including the mobile nylon bag (MNB) method, modified three-step in vitro (MTS) method, original three-step in vitro (OTS) method, and acid detergent insoluble nitrogen (ADIN) estimating method, to evaluate the intestinal digestibility of rumen undegradable protein (DRUP) of 10 types of concentrates and 7 types of roughages. After correlation analysis to determine the DRUP values using the MNB, MTS, OTS, and ADIN methods, the study aimed to find out appropriate methods to replace the MNB method due to its disadvantages such as high price, long time period, and use of a duodenal T-fistula. Three dairy cows with a permanent ruminal fistula and duodenal T-fistula were used in a single-factor experimental design. The results showed that the determined DRUP values using the MNB method for soybean meal, cottonseed meal, rapeseed meal, sunflower meal, corn germ meal, corn, rice bran, barley, wheat bran, corn fiber feed, Alfalfa (Zhao dong), Alfalfa (Long mu 801), Alfalfa (Long mu 803), grass (North), Grass (Inner Mongolia), corn silage and corn straw were 98.13%, 87.37%, 88.47%, 82.60%, 75.40%, 93.23%, 69.27%, 91.27%, 72.37%, 79.03%, 66.72%, 68.64%, 73.57%, 50.47%, 51.52%, 54.05%, and 43.84%, respectively. The coefficient of determination ($R^2=0.964$) of the results between the MTS method and the MNB method was higher than that ($R^2=0.942$) between the OTS method and the MNB method. The coefficient of determination of the DRUP values of the concentrates among the in vitro method (including the MTS and OTS methods) and the MNB method was higher than that of the roughage. There was a weak correlation between the determined DRUP values in concentrates obtained from the ADIN method and those from the MNB method, and there was a significant correlation (p<0.01) between the determined DRUP values of the roughage obtained from the MNB method and those obtained from ADIN method. The DRUP values were significantly correlated with the nutritional ingredients of the feeds. The regression equation was DRUP =100.5566+0.4169CP - 0.4344SP - 0.7102NDF - 0.7950EE ($R^2=0.8668$, p<0.01; CP, crude protein; SP, soluble protein; NDF, neutral detergent fiber; EE, ether extract). It was concluded that both the MTS method and the OTS may suitable to replace the MNB method for determining the DRUP values and the former method was more effective. Only the ADIN method could be used to predict the values of the roughages but conventional nutritional ingredients were available for all of the samples' DRUP.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1209-1223
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• 2009

In this review, the terminology that is used to describe ileal amino acid (AA) digestibilities in piglet feed ingredients is defined. If one accepts that the determination of AA digestibilities should be based on the ileal analysis method, one should consider that ileal digesta contains variable amounts of endogenous crude protein (CP), which originates mainly from digestive secretions, sloughedoff epithelial cells and mucins. The ileal endogenous CP and AA losses are separated into basal ileal endogenous CP and AA losses ($IAAL_{B}$), which are not influenced by the feed ingredient composition, and specific ileal endogenous CP and AA losses ($IAAL_{S}$), which are induced by feed ingredient characteristics such as level and type of fiber and anti-nutritional factors (ANF). Depending how ileal endogenous CP and AA losses are considered in the measurement of CP and AA digestibilities, digestibility values are expressed as apparent (AID), standardized (SID), or true (TID) ileal digestibilities of CP and AA. The main concern associated with the use of AID values in diet formulation for pigs is that they are not additive in mixtures of feed ingredients. Consequently, the concept of standardized ileal CP and AA digestibilities was introduced by correcting AID values for basal ileal endogenous CP and AA losses ($IAAL_{B}$). The correction for both $IAAL_{B}$ and $IAAL_{S}$ yields TID values, however, routine procedures to measure $IAAL_{S}$ are not yet available. In principle, SID values should be preferred, because they represent the fundamental properties of the feed ingredient. There exist only few reports on SID of CP and AA in feedstuffs frequently used in piglet nutrition. These include soybeans (SB), soybean meal (SBM), soy proteins (SP), soy protein concentrate (SPC), soy protein isolate (SPI), corn gluten (CG), wheat gluten (WG), pea protein (PeaP), potato protein (PotP), fish meal (FM) and whey proteins (WP), but the results obtained are inconsistent. Differences in SID values within feed ingredients may, at least in part, be attributed to different processing conditions or inherent differences of the assay feed ingredients. Moreover, there is some evidence that the determination of SID values and $IAAL_{B}$ in piglets may be confounded by the dietary CP level of the assay diet, age and (or) body weight (BW), the level of feed intake or the methodological approach used to determine $IAAL_{B}$.

• Journal of Aquaculture
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• v.8 no.1
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• pp.1-19
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• 1995

In red seabream, Pagrus major the female specific protein in the vitellogenic female serum was identified by Ouchterlony's immunodiffusion test and immunoelectrophoresis. The female specific serum protein might be vitellogenin based on the results of the immunological analysis for the male and vitellogenic female sera and crude egg extracts. Also, it was identified by the immunodiffusion test that the purified yolk protein from ovarian egg extracts has antigenic identities shared with the female specific serum protein. To study the relationship between the maturational stages of gonad and plasma levels of vitellogenin, these were measured from the late resting period (January) to the vitellogenic preiod (April) by the modified enzymeimmunoassay (EIA) using antiserum against yolk protein. The level of plasma vitellogenin began to increase in February (previtellogenesis stage) and continuously increased with the ovarian growth during the vitellogenesis period (March to April). The plasma vitellogenin levels were significantly different between the females and the males in February. Validation for the modified EIA system. was tested .The absorbance curve of serial dilutions of serum from the vitellogenic female was paralleled to the standard curve of yolk protein; $109\pm5.6\%$ recovery was achieved by the modified EIA. And the intraassay coefficients of variation were less than 10% within the concentration ranging from 31.3 ng/ml to 1,000 ng/ml. These findings suggest that the sex determination in adult red seabreams could be possible by using the modified EIA as early as in February.

• Korean Journal of Food Science and Technology
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• v.50 no.1
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• pp.14-20
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• 2018

Perfluorinated compounds (PFCs) have recently been recognized as global environmental pollutants. This study was performed to develop an analytical method for determination of levels of PFCs in food by LC-MS/MS. One hundred and nine food products were divided into two groups based on their fat and protein contents (high and low), following which samples containing high fat and protein contents were pooled and subjected to pretreatment consisting of enzymatic degradation and hexane extraction. The limit of detection of 17 PFCs in the samples were in the range of 0.013-0.145 ng/g. The degrees of precision of detection for group 1 (samples with low fat and protein contents) and group 2 (samples with high fat and protein contents) were 0.8-21.1 and 1.7-28.2%, respectively, with an accuracy of 78.8-109.8% for group 1 and 80-114.5% for group 2. This study indicated that pretreatment of high fat and protein foods with enzymatic degradation and hexane extraction would improve the detection of PFCs in food.

• Animal cells and systems
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• v.3 no.3
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• pp.331-336
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• 1999

p56$^{Ick}$, a cytoplasmic protein tyrosine kinase of the src family, is non-covalently associated with the cell surface coreceptors CD4 and CD8, which are expressed on thymocytes and mature T cells. The coreceptor protein plays an important role during the differentiation of thymocytes and the activation of T cells. DNA constructs were designed to study the roles of CD4 and CD8 during the differentiation of thymocytes. One is a chimeric cDNA which consists of coding regions for the extracellular domain of CD8a and the transmembrane and cytoplasmic domain of CD4. The other is the same chimeric cDNA but with a point mutation converting Cys to Ala in the Ick-binding site to disrupt the association. We confirmed that the CD8a/CD4 chimeric molecule bound to Ick more efficiently than the wild type CD8a protein. However, the chimeric protein with the Cys$leftrightarro$Ala mutation did not associate with Ick. The results suggest a possibility that the CD8a/CD4 chimeric protein may behave like a CD4 protein in associating with Ick and that it may deliver a signal inside the cell in a similar manner, Analysing effects of the mutant CD8a/CD4 chimeric protein expression in developing thymocytes will elucidate the role of Ick during the determination of CD4/CD8 cell lineages.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.144-151
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• 2014

Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the ${\alpha}$-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.897-904
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• 2007

The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ protealytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins $(C_{LAB})$, and their half-life time $(T_{1/2})$. Moreover, it gives more realistic information on resultant activity in situ $(V_{T1/2})$ of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are general y well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1529-1535
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• 2013

Tuberculosis is a worldwide epidemic disease caused by Mycobacterium tuberculosis, with an estimated one-third of the human population currently affected. Treatment of this disease with aminoglycoside antibiotics has become less effective owing to antibiotic resistance. Recent determination of the crystal structure of the M. tuberculosis Rv3168 protein suggests a structure similar to that of Enterococcus faecalis APH(3')-IIIa, and that this protein may be an aminoglycoside phosphotransferase. To determine whether Rv3168 confers antibiotic resistance against kanamycin, we performed dose-response antibiotic resistance experiments using kanamycin. Expression of the Rv3168 protein in Escherichia coli conferred antibiotic resistance against $100{\mu}M$ kanamycin, a concentration that effected cell growth arrest in the parental E. coli strain and an E. coli strain expressing the $Rv3168^{D249A}$ mutant, in which the catalytic Asp249 residue was mutated to alanine. Furthermore, we detected phosphotransferase activity of Rv3168 against kanamycin as a substrate. Moreover, docking simulation of kanamycin into the Rv3168 structure suggests that kanamycin fits well into the substrate binding pocket of the protein, and that the phosphorylation-hydroxyl-group of kanamycin was located at a position similar to that in E. faecalis APH(3')-IIIa. On the basis of these results, we suggest that the Rv3168 mediates kanamycin resistance in M. tuberculosis, likely through phosphotransferase targeting of kanamycin.

• Macromolecular Research
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• v.14 no.2
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• pp.140-145
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• 2006

E-science refers to the large-scale science that will increasingly be carried out through distributed global collaborations enabled by the Internet. The Grid is a service-oriented architecture proposed to provide access to very large data collections, very large scale computing resources and remote facilities. Web services, which are server applications, enable online access to service providers. Web portal interfaces can further hide the complexity of accessing facility's services. The main use of synchrotron radiation (SR) facilities by protein crystallographers is to collect the best possible diffraction data for reasonably well defined problems. Significant effort is therefore being made throughout the world to automate SR protein crystallography facilities so scientists can achieve high throughput, even if they are not expert in all the techniques. By applying the above technologies, the e-HTPX project, a distributed computing infrastructure, was designed to help scientists remotely plan, initiate and monitor experiments for protein crystallographic structure determination. A description of both the hardware and control software is given together in this paper.