• Molecules and Cells
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• v.27 no.3
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• pp.329-336
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• 2009

To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinal mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum 'Dempsey' containing an elF4E mutation ($pvr1^2$) and C. annuum 'Perennial' containing an elFiso4E mutation (pvr6). C. annuum 'Dempsey' was susceptible and C. annuum 'Perennial' was resistant to ChiVMV. All $F_1$ plants showed resistance, and $F_2$ individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five $F_2$ and 329 $F_3$ plants of 17 families were genotyped with $pvr1^2$ and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both $pvr1^2$ and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in elF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of $F_2$ plants revealed that all plants containing homozygous genotypes of both $pvr1^2$ and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of elF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.3
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• pp.335-340
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• 2012

The data used came from two trials undertaken under the same climatic conditions (spring-summer). In both trials pluriparious buffaloes were utilized similar in weight, body condition score, and milk production from the previous year. From the first trial the data used was from the sub-period 23-88 DIM provided by seven animals fed ad libitum with diet A (6.69 MJ/kg DM; 158.30 g/kg of crude protein) with a forage/concentrate ratio of 48/52. From the second trial the data used was from the sub-period 33-90 DIM provided by seven animals fed ad libitum with diet B (6.63 MJ/kg DM; 179.50 g/kg of crude protein) and by seven animals fed ad libitum with diet C (5.99 MJ/kg DM; 155.40 g/kg of crude protein), each of the diets had the same forage/concentrate ratio (53/47). A significant difference was found in milk production between group B and C (13.08 vs. 11.56 kg/d, p<0.05), an intermediate production (12.10 kg/d) was noted in group A. A significant difference was found between fat (76.58 vs. 69.24 g/kg, p<0.05), protein (46.14 vs. 43.16 g/kg, p<0.05) and casein (39.94 vs. 34.98 g/kg, p<0.05) of the milk of group B with respect to group A. The milk of group C gave fat values (71.80 g/kg), protein (45.52 g/kg) and casein (39.06 g/kg) statistically equal to those of group B. The milk of groups B and C, in respect to the milk of group A, gave values of $K_{20}$ (1.77, 1.82 vs. 3.68 min, p<0.05), statistically lower and values of $A_{30}$ (48.28, 47.27 vs. 40.64 mm, p<0.05) statistically higher. Two simple linear regressions were calculated where the independent variable (x) was the daily standardized milk production, the dependent variable (y) or the daily intake of net energy or crude protein. Equation 1) NE (MJ/d) = 74.4049+2.8308${\times}$kg of normalized milk; equation 2) CP (kg/d) = 1.4507+0.1085${\times}$kg of normalized milk, both the equations were significant (p<0.05) with determination coefficients of 0.58 and 0.50 respectively. For a production of normalized milk that varies from 9 to 13 kg, the respective energy-protein concentrations fluctuate from 6.09 to 6.78 MJ/kg DM and from 148.00 to 174.46 g/kg DM.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.56-62
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• 2000

The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Excherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderly assembled fusion protein was confirmed by modified Gm1-ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and $G_{m1}4-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process, Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.474-481
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• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.5
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• pp.616-622
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• 2011

This study evaluated the fermentation characteristics and protein degradation dynamics of wet green tea grounds (WGTG) silage. The WGTG was ensiled with distilled water (control), or lactic acid bacteria (LAB), enzyme (E), formic acid (FA) and formaldehyde (FD) prior to ensiling. Three bag silos for each treatment were randomly opened at 0, 3, 7, 14, 28 and 60 days after anaerobic storage. For all the treatments, except for FA, there was a rapid decline in pH during the first 7 days of ensiling. LAB treatment had higher lactic acid content, lower ammonia-N ($NH_3$-N) and free-amino nitrogen (FAA-N) contents than other treatments (p<0.05). E treatment had higher lactic acid, water-soluble carbohydrates (WSC) and non-protein nitrogen (NPN) content than the control (p<0.05). FA treatment had higher $NH_3$-N and FAA-N content than the control (p<0.05). FD treatment had lower NPN and FAA-N content than the control, but it did not significantly inhibit the protein degradation when compared to LAB treatment (p>0.05). Results indicate that LAB treatment had the best effect on the fermentation characteristics and protein degradation of WGTG silage.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.408-414
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• 1996

The SecY is a central component of the protein export machinery that mediate the translocation of secretory proteins across the plasma membrane, and has been known to be rate-limiting factor of secretion in Escherichia coli. In order to study the extracellular protein secretion in Gram-positive microorganism, we have, constructed strains harboring more than one copy of the gene for SecY. Firstly, the gene, for B. subtilis SecY and its promoter region was subcloned into pDH32 and the chimeric vector was inserted into amyE locus by homologous recombination. Secondly, low copy number vector, pCED6, was also used for subcloning the secY gene and for constructing a strain which harbors several copies of secY. The KH1 cell which harbor two copies of secY on the chromosome excreted more extracellular proteins than the wild type PB2. Moreover, the KH2 cells which harbor several copies of secY in pCED6 vector excreted more extracellular proteins than the KH1 cells. Here, we found that the capacity of protein secretion is partly controlled by the number of secY and it is suggested that SecY has also an important role in protein secretion in B. subtilis, a gram positive microorganism, as like in E. coli. This will promote the use of B. subtilis as a host for the expression of useful foreign gene and excretion of precious proteins.

• Food Science and Biotechnology
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• v.18 no.4
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• pp.889-894
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• 2009

Barley proteins are expected to have unique functional properties due to their high content of alcohol soluble protein, hordein. Since the barley proteins obtained by conventional isoelectric precipitation method cannot represent hordein fraction, barley proteins were fractionated to albumin, globulin, glutelin, and hordein with respect to extraction solvents. Functional properties and film-forming properties of solubility-fractionated barley proteins were investigated to explore their potential for human food ingredient and industrial usage. The 100 g of total barley protein comprised 5 g albumin, 23 g globulin, 45 g glutelin, and 27 g hordein. Water-binding capacities of barley protein isolates ranged from 140-183 mL water/100 g solid. Hordein showed the highest oil absorption capacity (136 mL oil/100 g), and glutelin showed the highest gelation property among the fractionated proteins. In general, the barley protein fractions formed brittle and weak films as indicated by low tensile strength (TS) and percent elongation at break (E) values. The salt-soluble globulin fraction produced film with the lowest TS value. Although films made from glutelin and hordein were dark-colored and had lower E values, they could be used as excellent barriers against water transmission.

• Korean Journal of Crystallography
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• v.5 no.1
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• pp.20-23
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• 1994

Specific binding to the oric region of E, coli chromsome by IciA protein inhibits initiation of chrorrnsomal replication in vitro by blocking the opening of this region effected by the initiator DnaA protein. The IciA protein has been suggested play a critical role in a key stage of the cell cycle. In order to study the structure-function relationship of IciA protein, we are determining the three-dimensional structure of IciA Votein by X-ray crystallography, As a first step toward its structure detumination E. coli IciA protein has been crystallized using sodium formate as a precipitant.

• BMB Reports
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• v.38 no.3
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• pp.259-265
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• 2005

Proteins must fold into their correct three-dimensional conformation in order to attain their biological function. Conversely, protein aggregation and misfolding are primary contributors to many devastating human diseases, such as prion-mediated infections, Alzheimer's disease, type II diabetes and cystic fibrosis. While the native conformation of a polypeptide is encoded within its primary amino acid sequence and is sufficient for protein folding in vitro, the situation in vivo is more complex. Inside the cell, proteins are synthesized or folded continuously; a process that is greatly assisted by molecular chaperones. Molecular chaperones re a group of structurally diverse and mechanistically distinct proteins that either promote folding or prevent the aggregation of other proteins. With our increasing understanding of the proteome, it is becoming clear that the number of proteins that can be classified as molecular chaperones is increasing steadily. Many of these proteins have novel but essential cellular functions that differ from that of more 'conventional' chaperones, such as Hsp70 and the GroE system. This review focuses on the emerging role of molecular chaperones in protein quality control, i.e. the mechanism that rids the cell of misfolded or incompletely synthesized polypeptides that otherwise would interfere with normal cellular function.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.132-137
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• 1997

A novel protein (HEAAE, High Essential Amino Acid Encoding Protein), rich in essential amino acids ($75{\%}$ of total), was designed and constructed in our laboratory. The designed peptides were analyzed by SYBLE and stable secondary and tertiary structures were predicted. The monomeric form (HEAAE-1) of the protein consists of 20 amino acid residues with four additional amino acids comprising a potential ${\beta}$-turn (HEAAE-4). Size exclusion analysis demonstrated that the monomer is self-aggregates in aqueous solution to form higher ordered multimeric structures, which are very reminiscent of natural plant storage proteins. The DNA encoding this amino acid sequence was synthesized, and from this monomeric gene fragment (heaae-1), the stable tetrameric form of the gene (heaae-4) was generated by subcloning into the E. coli expression vector pKK223-3. A clear 6 kDa polypeptide band corresponding to the molecular weight of the dimeric form (HEAAE-2) was detected. The smeared band which appeared around the molecular weight corresponding to HEAAE-4 of 11 kDa suggested that the tetramer form of this protein might be processed into smaller size products.