• International Journal of Industrial Entomology and Biomaterials
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• v.13 no.1
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• pp.37-43
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• 2006

Enterococcus faecalis was isolated from the body fluid of dead Galleria mellonella larvae. Upon injection of E. faecalis into the hemocoel of G. mellonella, the bacteria destroyed parts of humoral defense systems in the hemolymph. In a test for the proteolytic activity of E. faecalis CS, it was confirmed that the enzyme degraded three well-known a-helical antimicrobial peptides, cecropin A, melittin and halocidin, and abolished their activities. We also determined putative cleavage sites on the primary sequences of three peptides through purification and mass analysis of peptide fragments digested by E. faecalis CS. Furthermore it was found that apolipophorin-III, recently known as a critical recognition protein for invading microbes in the hemolymph of G. mellonella, was also degraded by E. faecalis CS. Taken together, the present work shows that the protease in secretions from E. faecalis destroyed two critical humoral immune factors in the hemolymph of G. mellonella larvae. In addition, this paper demonstrates that the relationship between the host insect and the pathogenic bacteria might provide a valuable model system to study the enterococcal virulence mechanism, which may be relevant to mammalian pathogenesis.

• BMB Reports
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• v.30 no.2
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• pp.125-131
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• 1997

This work reports studies of the catalytic and structural properties of pyridoxal kinase (ATP: pyridoxal 5' -phosphotransferase, EC. 2.7.1.35), Pyridoxal kinase catalyzes the phosphorylation of vitamin $B_6$ (pyridoxal, pyridoxamine, pyridoxine) using ATP-Zn as a phosphoryl donor. The enzyme purified from brain tissues is made up of two identical subunits of 40 kDa each. Native enzyme was inhibited by a substrate analogue, pyridoxal-oxime. Limited chymotrypsin digestion of pyridoxal kinase yields two fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were isolated by DEAE ion exchange chromatography and used for binding studies with fluorescent ATP and pyridoxal analogues. The spectroscopic properties of both fluorescent pyridoxal analogue and Anthraniloyl ATP (Ant-ATP) bound to the 24 kDa fragment are indistinguishable from those of both pyridoxal analogue and Ant-ATP bound to the native pyridoxal kinase, respectively. The small 16 kDa fragment, generated by proteolytic cleavage of the kinase, does not bind any of the substrate analogues. Binding characteristics of Ant-ATP were extensively studied by measuring the changes in fluorescence spectra at various conditions. From the results presented herein, it is postulated that the structural domain associated with catalytic activity comprises approximately one-half of the molecular mass of pyridoxal kinase (24 kDa). whereas the remaining portion (16 kDa) of the enzyme contains a regulatory binding domain.

• Herbal Formula Science
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• v.25 no.3
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• pp.349-362
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• 2017

Objectives : We investigated whether the components of Ailanthus altissima induced apoptotic cell death in Jurkat acute lymphoblastic leukemia (ALL) cells. Methods : Regulation of cell proliferation is a complex process involving the regulated expression and/or modification of discrete gene products, which control transition between different stages of the cell cycle. Results : Upon treatments with Ailanthus altissima, the concentration-dependent inhibitions of cell viability were observed as compared to untreated control group. The capability of Ailanthus altissima to induce apoptosis was associated with proteolytic cleavage of specific target proteins such as poly(ADP-ribose)polymerase (PARP) and beta-catenin proteins suggesting the possible involvement of caspases. Ailanthus altissima also caused apoptosis as measured by cell morphology and DNA fragmentation. Conclusions : These results indicate that the increase of apoptotic cell death by Ailanthus altissima may be due to the inhibition of cell cycle in human Jurkat lymphocytes. Conclusively, these current and further findings will provide novel approaches to understanding and treating major diseases.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.663-669
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• 2000

The fur (ferric uptake regulation) expression vector pMON2064 was modified to produce a Fur-fusion expression vector. A kinker site, factor Xa cleavage site, and several restriction endonuclease sites were introduced to facilitate easy cloning and isolating of the fusion protein. The resulting fusion expression vector, pMONSTER, was then used to make fusion expression vector, pMONSTER, was then used to make fusion proteins with $\beta$-galactosidase and the protease of the human immunodeficiency virus type 1 (HIV-1 PR). Strain SW4020 harboring the Fur $\beta$-galactosidase fusion vector produced blue colonies on a 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactoside plate and the resulting 133 kDa fusion protein reacted with an anti-Fur antibody. The strain harboring the Fur-HIV-1 PR fusion vector produced a 29 kDa fusion protein, which also reacted with an anti-Fur antibody. The Fur-HIV-1 PR fusion protein was purified by a single column application that was designed to isolate the Fur protein. The purified Fur-HIV-1 PR fusion protein digested with factor Xa cleaved a recombinant Gag protein to release smaller fragments, including a p24 capsid protein. The Fur-HIV-1 PR fusion protein itself did not exhibit any proteolytic activity.

• International Journal of Oral Biology
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• v.41 no.4
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• pp.175-181
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• 2016

Tyrosol, a phenylethanoid and a derivative of phenethyl alcohol, possesses various biological properties, such as anti-oxidative and cardioprotective activity. Olive oil is the principal source of tyrosol in the human diet. However, so far the anti-cancer activity of tyrosol has not yet been well defined. This study therefore undertakes to examine the cytotoxic activity and the mechanism of cell death exhibited by tyrosol in KB human oral cancer cells. Treatment of KB cells with tyrosol induced the cell growth inhibition in a concentration- and a time-dependent manner. Furthermore, the treatment of tyrosol induced nuclear condensation and fragmentation of KB cells. Tyrosol also promoted proteolytic cleavage of procaspase-3, -7, -8 and -9, increasing the amounts of cleaved caspase-3, -7, -8 and -9. In addition, tyrosol increased the levels of cleaved PARP in KB cells. These results suggest that tyrosol induces the suppression of cell growth and cell apoptosis in KB human oral cancer cells, and is therefore a potential candidate for anti-cancer drug discovery.

• Archives of Pharmacal Research
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• v.24 no.5
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• pp.446-454
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• 2001

Peptide fragments, isolated from proteolytic cleavage of thyroglobulin at specific sites, were examined for the iodination of tyrosine residues. The 50 kDa polypeptide, which was prepared from digestion of bovine thyroglobulin and continuous preparative SDS-PAGE, was subjected to reduction with DTT and alkylation with iodoacetic acid to generate S-car-boxymethylated peptide derivative, which was further hydrohysed by endoproteinase-Asp-N. Peptide products were separated by RP-HPLC, and each fraction was analyzed by LC/ESI-MS and MALDI-MS analyses. Based on the specificity of endoproteinase-Asp-N andthe mass spectra data, a peptide fragment turned out to correspond to a peptide, DALCCVKCPEGSYFQ (1438-1452), characterized by the presence of a thioredoxin box (CVKC) and a tyrosine residue. In addition, another peptide fragment (1453-1465) containing a thioredoxin box (CIPC) and a tyrosine residue was also observed. However, any evidence of iodination of the tyrosine residue present in these peptides was not provided. Meanwhile, tyrosine residues in the peptides, DVEEALAGKYLAGRFA (1366-1381) and DYSGLLLAFQVFLL (1290-1303) were found to be iodinated; mono- or diiodinated tyrosine residues, characteristic of a hormogenic site, existed in both peptides. In addition, the tyrosine residue in the peptide (1218-1252), corresponding to a hormonogenic site was also iodinated. Thus, there was a sharp difference of the susceptibility to oxidative iodination between the tyrosine residue in a hormonogenic site and that in a thioredoxin region. From these results, it is suggested that polypeptide region adjacent to tyrosine residues may govern the susceptibility of tyrosine to oxidative iodination.

• Fisheries and Aquatic Sciences
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• v.10 no.2
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• pp.60-67
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• 2007

Myostatin (MSTN; also known as GDF8) is a member of the transforming growth factor ${\beta}-superfamily$ of proteins. MSTN negatively regulates mammalian skeletal muscle growth and development by inhibiting myoblast proliferation. Mice and cattle possessing mutant MSTN alleles display a 'double muscling' phenotype characterized by extreme skeletal muscle hypertrophy and/or hyperplasia. We isolated the full-length cDNA of a novel MSTN gene from S. schlegeli muscle tissue and examined its expression pattern in various tissues. The full-length gene (GenBank DQ423474) consists of 1941bp with an open reading frame of 1134 bp, encoding 377 amino acids that show 62-92% amino acid similarity to other vertebrate MSTNs. The predicted protein contains a conserved proteolytic cleavage site (RXRR) and nine conserved cysteine residues at the C terminus. RT-PCR revealed that the unprocessed and prodomain myostatin mRNAs were predominantly present in muscle, with limited expression in other tissues. However, the mature myostatin mRNA was highly expressed in brain and muscle, intermediately expressed in the gills, intestine, heart, and kidney, and weakly expressed in the liver and spleen.

• Preventive Nutrition and Food Science
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• v.7 no.2
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• pp.123-127
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• 2002

Ninety nine extracts from Korean plants were screened for their inhibitory activities on human immunodeficiency virus (HIV) type 1 pretense by an HPLC method. The pretense inhibitory activities were determined by incubating the extracts in reaction mixtures containing pretense and substrate (His-Lys-Ala-Arg-Val-Leu-(p-NO$_2$- Phe)-Glu-Ala-Nle-Ser-NH$_2$) to perform proteolytic cleavage reactions. Of the extracts tested, the water extracts of Viburnum awabuki (stem and leaves) and Distylium racemosum (leaves) had the highest pretense inhibitory activities at a concentration of 100ug/mL. Activity-guided fractionation, revealed that the n-butanol fraction of the V. awabuki extract and the ethyl acetate fraction from the D. racemosum extract had the greatest inhibitory activity on HIV-1 pretense.

• Korean Journal of Pharmacognosy
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• v.33 no.1 s.128
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• pp.29-34
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• 2002

Albizzia julibrissin belonging to the family Leguminosae has been used for the treatment of contusion, sore throat, amnesia, and insomnia in Oriental traditional medicine. The water extract of A. julibrissin induced apoptosis in Jurkat T-acute lymphoblastic leukemia (ALL) cells as measured by cell morphology. The capability of this herb medicine to induce apoptosis was associated with proteolytic cleavage of specific target protein such as beta-catenin protein suggesting the possible involvement of caspases. The purpose of the present study is also to investigate the effect of A. julibrissin on cell cycle progression. Our results showed that GI checkpoint related gene products (cyclin D1, cyclin dependent kinase 4, retinoblastoma, E2F1) were decreased in their protein levels in a dose-dependent manners after treatment of the extract. These results indicate that the increase of apoptotic cell death by A. julibrissin may be due to the inhibition of cell cycle progression in wild type p53-lacking Jurkat cells.

• YAKHAK HOEJI
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• v.45 no.6
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• pp.730-738
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• 2001

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs in many different cell types in a wide variety of organisms. Ajbizzia julibrissin belonging the family Leguminosae has been used for the treatment of contusion, sore throat, amnesia, and insomnia in oriental traditional medicine. This study investigates whether the water extract off julibrissin induce apoptotic cell death in Jurkat T-acute lymphoblastic leukemia (ALL) cells. Jurkat cells were increased inhibitions of cell viability in a concentration-dependent manner by A julibrissin. This herbal medicine also caused apoptosis as measured by cell morphology and DNA fragmentation. The capability oft julibrissin to induce apoptosis was associated with proteolytic cleavage of specific target protein such as poly (ADP-ribose) polymerase (PARP) protein suggesting the possible involvement of caspases. Our result skewed that Bcl-2 and Bax protein levels were not changed in all A julibrissin-treated groups compared to control group. These results suggest that A julibrissin-mediated apoptosis is independent with Bcl-2 related signaling pathway in this cells.