• Journal of Life Science
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• v.19 no.9
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• pp.1226-1231
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• 2009

Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

• Food Science and Preservation
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• v.16 no.6
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• pp.1008-1012
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• 2009

We investigated the biological activities of ethanol extracts obtained from five endive cultivars. Free radical-scavenging activities of extracts from Redoria, Halifax, Vintor, Kibora, and Metafora cultivars were 94.2%, 60.8%, 66.6%, 70.0% and 67.4%, respectively. The extract from Redoria showed the highest activity among all the tested samples including BHT (Butylated hydroxyanisole) and vitamine C, known as antioxidant. Endive extracts slightly (17.5 - 38.6%) inhibited $\alpha$-glucosidase activity. However, there is no inhibitory activity against $\alpha$-amylase. In terms of proteolytic activity, all endive extracts showed strong activity than pancreatin (used as a positive control), and the extract of Redoria also exhibited stronger activity than other extracts as well as free radical scavenging activity. These results indicate that endive, especially the Redoria cultivar, can be used as a natural resource for development of antioxidants or materials aiding digestion.

• Culinary science and hospitality research
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• v.19 no.5
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• pp.136-145
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• 2013

Currently many studies aimed at enhancing efficacy of medicinal food on biological activity using bioconversion technology including fermentation process. In this study, the quality characteristics and antioxidative activity of fermented Crataegi fructus was investigated. The antioxidant activity of fermented Crataegi Fructus was assessed by various radical scavenging assays using DPPH (2,2-Diphenyl-1-picrylhydrazyl), FRAP (Ferric ion reducing antioxidant power), Reducing power and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)). Moisture content of fermented Crataegi Fructus was $39.3{\pm}0.06%$. Contents of crude ash, crude protein, and crude fat were $0.20{\pm}0.01$, $1.77{\pm}0.04$, and $1.40{\pm}0.59%$, respectively. Moreover, the hunter's color values of fermented Crataegi Fructus were 79.24 (lightnees), 1.58 (redness), and 31.25 (yellowness), respectively. Total phenolic contents of fermented Crataegi Fructus were $3,015{\pm}250$ GAE ${\mu}g/g$. The antioxidative activities of fermented Crataegi Fructus significantly increased in a dose dependent manner. In addition, fermented Crataegi Fructus slightly (10.4%) inhibited ${\alpha}$-glucosidase activity; however, there was no inhibitory activity against ${\alpha}$-amylase. In terms of proteolytic activity, fermented Crataegi Fructus showed a strong activity than pancreatin (used as a positive control). These results indicate that fermented Crataegi Fructus can be used as a natural resource for material aiding digestion.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.4
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• pp.351-362
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• 1998

Microbial growth efficiency in the rumen was studied in sheep given hourly, 31.25 g oaten chaff with either 0.31 and 0.88 g urea or 1.88 and 5.63 g casein (exp. 1) and 33.33 g oaten chaff with 1.04 casein or 0.3, 0.6 and 0.9 g urea or the mixture of the casein and urea (exp. 2). Concentrations of ruminal fluid ammonia increased with increasing nitrogenous supplements. Organic matter digestibility in sacco in the rumen was not different irrespective of N sources. Isoacids and valeric acid increased with increasing ingested casein but decreased with increasing urea intake. Peptide and amino acid pools in ruminal fluid increased with increasing ammonia concentrations (exp. 2) suggesting that proteolytic activity and transportation of peptides and amino acids across microbial membrane of rumen microbes may be regulated by the metabolite mechanism (intracellular amino acids and $NH_4{^+}$, respectively). Densities of total viable and cellulolytic bacteria in ruminal fluid increased with increasing ammonia levels but that of small Entodinia decreased. The density of fungal sporangia growth on oat leaf blades decreased with increasing ammonia concentrations but appeared to remain constant in the presence of casein. Efficiency of net microbial cell synthesis was 15-28% higher when ammonia concentrations increased from 100 to above 200 mg N/l regardless of N sources. In conclusion, supplementation of preformed protein had no effect on rumen digestion and microbial growth efficiency. This could not be accounted for its effect on ruminal fluid ammonia. Increased microbial growth efficiency with increasing ammonia levels may be due to a reduction in the turnover of microbial cells within the rumen.

• Bulletin of the Korean Chemical Society
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• v.27 no.7
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• pp.1001-1004
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• 2006

The m-calpain activity hydrolyzing a fluorogenic substrate of N-Succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcourmarin (LLVY-AMC) was significantly stimulated by more than two-fold in the presence of 5$\mu$M $\alpha$synuclein at $15{^{\circ}C}$. The stimulation was also confirmed with azocasein. The stimulation of the peptide hydrolyzing activity required structural intactness of $\alpha$-synuclein since the C-terminally or N-terminally modified proteins such as $\beta$-synuclein, $\alpha$-syn1-97, and $\alpha$-syn61-140 did not increase the proteolytic activity. Instead, however, the N-terminally truncated $\alpha$-syn61-140 was shown to drastically suppress the calpain activity. Since the N-terminal truncation was known to be the primary cleaving event of calpain-mediated proteolysis of $\alpha$-synuclein and the $\alpha$-syn61-140 has been demonstrated to be resistant against the calpain digestion, it has been proposed that the intracellular calpain activity could be regulated in a reciprocal manner by $\alpha$-synuclein and its proteolyzed C-terminal fragment. Based on the results, a possible physiological function of $\alpha$-synuclein has been suggested as a calpain regulator which contains both stimulatory and inhibitory activities.

• Bulletin of the Korean Chemical Society
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• v.33 no.9
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• pp.2883-2889
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• 2012

LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric antimicrobial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and ${\alpha}$-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.1
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• pp.49-54
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• 2011

A case of Behcet's syndrome found in a 22 years old male was is reported along with the histological and immunohistochemical studiesfindings. He The patient had had received prolonged medical therapy of anti-inflammatory agents for the conjunctival ulcer, genital ulcer, oral ulcer, and intestinal ulcers since the first onset of the Behcet's syndrome about approximately 7 years agoearlier. Recently, he felt the athickening of both corners of his lip causing that caused mouth opening difficultydifficulties. A plastic rReconstructive surgery was performed to enlarge the size of the oral orifice by multiple Z-plasty incisions, and finallywhich resulted in proper enlargement of the circumferential length of the lip. During the operation, a scar-like thick fibrous tissue was obtained and examined pathologically. In theThe microscopic observation the revealed the submucosal lesion was to be diffusely fibrosed with the a distribution of sclerotic collagen bundles. Particularly, sSeveral foci of collagen degeneration were found observed in the deep connective tissue, and the degenerating collagen bundles were gradually lost their fibrillar appearance. In the immunohistochemical observations, the foci of collagen degeneration was were strongly positive for IgK, but almost negative for TNF${\alpha}$ lysozyme, and MMP-3. Taken together, it was presumed that tThe submucosal fibrosis was presumed to have firstly presented in this study was probablybeeninduced by the prolonged anti-inflammatory therapy, which may inhibit the removal of sclerosed collagen bundles by the cell-mediated immunity and proteolytic digestion of macrophages, and that it was secondarily aggravated by the deposition of immunoglobulins derived from an autoimmune origin. Therefore, even after the successful plastic surgery of the lip to ameliorate the mouth opening difficulty, the recurrent submucosal fibrosis of lip should be carefully managed in the follow-up treatment.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.7
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• pp.951-957
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• 2015

Four rumen fistulated swamp buffaloes were randomly assigned according to a $4{\times}4$ Latin square design to investigate the effects of Eucalyptus (E. Camaldulensis) leaf meal (ELM) supplementation as a rumen enhancer on feed intake and rumen fermentation characteristics. The dietary treatments were as follows: T1 = 0 g ELM/hd/d; T2 = 40 g ELM/hd/d; T3 = 80 g ELM/hd/d; T4 = 120 g ELM/hd/d, respectively. Experimental animals were kept in individual pens and concentrate was offered at 0.3% BW while rice straw was fed ad libitum. The results revealed that voluntary feed intake and digestion coefficients of nutrients were similar among treatments. Ruminal pH, temperature and blood urea nitrogen concentrations were not affected by ELM supplementation; however, ELM supplementation resulted in lower concentration of ruminal ammonia nitrogen. Total volatile fatty acids, propionate concentration increased with the increasing level of EML (p<0.05) while the proportion of acetate was decreased (p<0.05). Methane production was linearly decreased (p<0.05) with the increasing level of ELM supplementation. Protozoa count and proteolytic bacteria population were reduced (p<0.05) while fungal zoospores and total viable bacteria, amylolytic, cellulolytic bacteria were unchanged. In addition, nitrogen utilization and microbial protein synthesis tended to increase by the dietary treatments. Based on the present findings, it is suggested that ELM could modify the rumen fermentation and is potentially used as a rumen enhancer in methane mitigation and rumen fermentation efficiency.

• Animal cells and systems
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• v.4 no.3
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• pp.273-278
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• 2000

Transforming growth factor-$\beta$ (IGF-$\beta$)s are multifunctional small polypeptides synthesized in most cell types. TGF-$\beta$ exerts pivotal effects on both bone formation and resorption. In addition, increasing lines of evidence implicate TGF-$\beta$ as a potential coupling factor between these two processes during bone remodeling. In the present study, the expression form and the activation mechanism of latent-TGF-$\beta$ were investigated using specific antibodies for each isoform. TGF-$\beta$s were observed to be synthesized and accumulated in a large amount in cultured osteoblastic cells. The estimated molecular weights of intracellular TGF-$\beta$2 and -$\beta$3 were 49 and 55 kDa, respectively. Based on proteolytic digestion study and immunofluorescence observation, these precursor forms seemed to be accumulated in distinct intracellular compartments. To examine whether the internal pool of TGF-$\beta$ was possiblely regulated by external signals, their biological activites were examined in a conditioned media of this cell. Although the intact conditioned media did not contain detectable TGF-$\beta$ activity, heat-treatment or acid-activation of the conditioned media revealed significant TGF-$\beta$ activity. Furthermore, in the presence of estrogen, this activity was dramatically diminished. It is known that activation of latent TGF-$\beta$ can be achieved by different chemical and enzymatic treatments, or by incubation with certain cell types. This extracellular activation was suggested as a key step in the regulation of TGF-$\beta$ activity. In addition to these extracellular activation, this study suggests that the synthesis and intracellular processing are important regulation steps for TGF-$\beta$ action. In addition, this regulation Is specific for TGF-$\beta$ type 2, because the change was not observed in TGF-$\beta$3 in osteoblastic cell line.

• Food Science of Animal Resources
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• v.34 no.3
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• pp.362-371
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• 2014

This study utilized commercially available proteolytic enzymes to prepare egg-white protein hydrolysates (EPHs) with different degrees of hydrolysis. The antioxidant effect and functionalities of the resultant products were then investigated. Treatment with Neutrase yielded the most ${\alpha}$-amino groups (6.52 mg/mL). Alcalase, Flavourzyme, Protamex, and Ficin showed similar degrees of ${\alpha}$-amino group liberation (3.19-3.62 mg/mL). Neutrase treatment also resulted in the highest degree of hydrolysis (23.4%). Alcalase and Ficin treatment resulted in similar degrees of hydrolysis. All hydrolysates, except for the Flavourzyme hydrolysate, had greater radical scavenging activity than the control. The Neutrase hydrolysate showed the highest 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity ($IC_{50}=3.6mg/mL$). Therefore, Neutrase was identified as the optimal enzyme for hydrolyzing egg-white protein to yield antioxidant peptides. During Neutrase hydrolysis, the reaction rate was rapid over the first 4 h, and then subsequently declined. The $IC_{50}$ value was lowest after the first hour (2.99 mg/mL). The emulsifying activity index (EAI) of EPH treated with Neutrase decreased, as the pH decreased. The EPH foaming capacity was maximal at pH 3.6, and decreased at an alkaline pH. Digestion resulted in significantly higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTS radical scavenging activity. The active peptides released from egg-white protein showed antioxidative activities on ABTS and DHHP radical. Thus, this approach may be useful for the preparation of potent antioxidant products.