• 약학회지
• /
• 제41권2호
• /
• pp.247-254
• /
• 1997

The proteinase-activated receptor (PAR-2) belongs to the family of seven transmembrane region receptors, like the thrombin receptor, it is activated by specific proteolytic clea vage of its extracellular amino terminus and a synthetic peptide (SLIGRL). The earthworm protein fraction (EPF) extracted from Lumbricus rubellus elicted dose- and endothelium-dependent relaxations in phenylephrine-contracted rat thoracic aorta, whereas heat inactivated EPF (0.5 ${\mu}g$ /ml) had no effect. In the presence of the nitric oxide synthase inhibitor NG-methyl-L-arginine (1.8 micro M), EPF (0.5 ${\mu}g$ /ml)-induced relaxations were partially inhibited. Furthermore, EPF (0.5 ${\mu}g$ /ml) dramatically caused relaxation of thrombin-desenstized rat thoracic aorta. These results indicate that EPF activates PAR-2 in vascular endothelial cell. Intravenous injection of EPF (20 mg/kg, bolus) into anesthetized rats produced a marked depressor response. EPF (0 ~ 80 ${\mu}g$ /ml, gradient) was very effective on increasing of perfusion volume in rabbit ear vessel preparations. These results imply the usefulness of EPF as a vascular smooth muscle relaxant and indicate that the activation of PAR-2 may be a mechanism of EPF on hemokinetic improvement.

• The Plant Pathology Journal
• /
• 제31권3호
• /
• pp.278-289
• /
• 2015

Indigenous strains of Trichoderma species isolated from rhizosphere soils of Tea gardens of Assam, north eastern state of India were assessed for in vitro antagonism against two important tea fungal pathogens namely Pestalotia theae and Fusarium solani. A potent antagonist against both tea pathogenic fungi, designated as SDRLIN1, was selected and identified as Trichoderma viride. The strain also showed substantial antifungal activity against five standard phytopathogenic fungi. Culture filtrate collected from stationary growth phase of the antagonist demonstrated a significantly higher degree of inhibitory activity against all the test fungi, demonstrating the presence of an optimal blend of extracellular antifungal metabolites. Moreover, quantitative enzyme assay of exponential and stationary culture filtrates revealed that the activity of cellulase, ${\beta}$-1,3-glucanase, pectinase, and amylase was highest in the exponential phase, whereas the activity of proteases and chitinase was noted highest in the stationary phase. Morphological changes such as hyphal swelling and distortion were also observed in the fungal pathogen grown on potato dextrose agar containing stationary phase culture filtrate. Moreover, the antifungal activity of the filtrate was significantly reduced but not entirely after heat or proteinase K treatment, demonstrating substantial role of certain unknown thermostable antifungal compound(s) in the inhibitory activity.

• Natural Product Sciences
• /
• 제13권2호
• /
• pp.169-173
• /
• 2007

Luteolin is a major flavonoid of Lonicera japonica and has anti-inflammatory effect. The activation of proteinase-activated receptor (PAR)-2 and -4 by trypsin appears to play a role in inflammation, In the present study, we examined the inhibitory effects of luteolin on activation of trypsin-induced human leukemic mast cells (HMC-1). HMC-1 cells were stimulated with trypsin, PAR-2 and PAR-4 agonist, in the presence or absence of luteolin. The level of TNF-${\alpha}$ secretion was measured by enzyme-linked immunosorbent assay (ELISA). The expression of tryptase and phosphorylated-extracellular signal-regulated kinase (ERK) were assessed by Westem blot analysis. Moreover, trypsin activity was measured by the substrate Bz-DL-Arg-p-nitroanilide (BAPNA). TNF-${\alpha}$ secretion and Tryptase expression in trypsin-stimulated HMC-1 cells were markedly inhibited by pretreatment of luteolin. Furthermore, the pretreatment of luteolin resulted in the reduction of ERK phosphorylation and trypsin activity. These results suggest that luteolin might has the inhibitory effects on the PAR-2 and -4-dependent inflammation.

• 미생물학회지
• /
• 제42권4호
• /
• pp.257-264
• /
• 2006

한국의 임상검체와 자연환경에서 분리된 Cryptococcus neoformans 58주에 대한 혈청형과 세포외효소 생성능에 관한 셍물학적 특성을 조사하였다. 환자로부터 분리된 임상균주 51주 중 48주는 혈청형 A (94.1%) 였으며 2주는 혈청형 B (3.92%),그리고 나머지 1주는 혈청형 D (1.96%)였다. 자연환경에서 분리된 7주는 비둘기 분변에서 분리된 것들이며 모두 혈정형 A였다. 모든 균주는 proteinase와 phnospholipase를 생성하였고, 또한 API-ZYM system을 이용한 19종류의 효소생성능 시험에서는 alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, $\alpha$-glucosidase, 그리고 $\beta$-glucosidase를 생성하였으나, N-acetyl-$\beta$-glucosarninidase는 39주 (67.2%)에서만 생성하였다. 혈청형 B로 동정된 2주와 혈청형 A로 동정된 균주중 1주는$\beta$-glucuronidase를 생성하였다. 본 연구에 사용된 총21종류의 효소 생성능 시험을 기초로 하여 생물형을 구분하였는데, 모두 4가지의 유형을 나타내었고, 또한 임상과 환경균주에서 혈정형과 생물형 특성간의 유의한 상관성를 나타내었다.

• Journal of Microbiology and Biotechnology
• /
• 제8권4호
• /
• pp.333-340
• /
• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• Asian-Australasian Journal of Animal Sciences
• /
• 제19권8호
• /
• pp.1100-1105
• /
• 2006

During involution of the mammary gland after the lactation period, the gland undergoes an extensive epithelial cell death. In our previous study, overexpression of an extracellular proteinase inhibitor (Expi) gene accelerated apoptosis of mammary epithelial cells. Here we found that expression of the cathepsin D gene was induced in the Expi-overexpressed apoptotic cells. To understand the role of cathepsin D in apoptosis, we transfected cathepsin D gene into mammary epithelial HC11 cells and established the stable cell lines overexpressing the cathepsin D gene. We found that overexpression of the cathepsin D gene partially induced apoptosis of mammary epithelial cells. Expression patterns of the cathepsin D gene were examined in mouse mammary gland at various reproductive stages. Expression of the cathepsin D gene was increased during involution stages compared to lactation stages, and highest expression levels were shown at involution on day 4. We also examined expression of the cathepsin D gene in various mouse tissues. Mammary gland at involution on day 2 showed highest levels of cathepsin D mRNA of the mouse tissues that we examined. Liver tissues showed high levels of cathepsin D expression. These results demonstrate that cathepsin D may contribute to the apoptotic process of mammary epithelial cells.

• BMB Reports
• /
• 제41권12호
• /
• pp.858-862
• /
• 2008

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a zinc-dependent proteinase found in cholesterol-rich lipid rafts on the plasma membrane. MT1-MMP hydrolyzes extracellular matrix (ECM) proteins, activates pro-matrix metalloproteinase-2 (proMMP-2) and plays an important role in ECM remodeling, cancer cell migration and metastasis. The role of caveolin-1, an integral protein of caveolae, in the activation of MT1-MMP remains largely unknown. Here, we show that the expression of caveolin-1 attenuates the activation of proMMP-2, reduces proteolytic cleavage of ECM and inhibits cell migration. We utilized the cytoplasmic tail domain deletion (${\Delta}CT$) or the E240A mutant of MT1-MMP. Co-expression of caveolin-1 with the wild-type or the ${\Delta}CT$ MT1-MMP decreased the proMMP-2 activation and inhibited collagen degradation and cell migration. Caveolin-1 had no effect on the catalytically inert E240A MT1-MMP. Our findings suggest that caveolin-1 is essential in the down-regulation of MT1-MMP activity by promoting internalization from the cell surface.

• 한국식품저장유통학회지
• /
• 제5권3호
• /
• pp.292-298
• /
• 1998

부패하는 두부에서부터 각종 미생물 18종을 분리하여 동정한 결과, B. cereus, X. luminescens, A. calcoaceticus, Kl. pneumoniae, S. typhimurium, Pantoea sp., Bacillus sp., Cardiobacterium sp., E. coli, St. aureus, Yersinia sp. 등 11종의 각기 다른 미생물로 나타났다. 실온에서 두부의 부패 과정중 이들 미생물의 존재 함량은 B. cereus J55 23.37%, X. luminescens J48 22.37%, A. calcoaceticus J61 22.26%, X. Luminescens J48 22.73%, A. calcoaceticus J61 22.26%, Kl. pneumoniae J62 21.25%, S. typhimurium J51 2.87%, Pantoea sp. J57 2.65%, S. typhimurium J51 2.87%, Pantoea sp. J57 2.65%, Bacillus sp. J58 1.43%, Cardiobacterium sp. J54 1.26%, E. coli J53 1.20%, St. aureus J60 0.93%, Yersinia sp. J50 0.05%로 존재하였다. 이들 미생물을 각각 두부에 접종하여 두부 부패성을 조사한 결과, B. cereus J55, X. luminescens J48, Ac. calcoaceticus J61, Kl. pneumoniae J62 등 4종류의 균주는 두부부패를 진행시켰으며 심한 변패취를 내었고 액즙이 혼탁하여 미생물의 증식을 인지할 수 있었으며 또한 proteinase의 활성 및 amylase의 활성이 비교적 높게 나타났다. 그러나 나머지 7균주는 두부 부패에 큰 영향을 나타내지 않았다. 11종의 미생물에 대하여 각종 항생제 내성을 조사한 결과, amicacin, gentamicin, tobramycin 등 aminoside계 항생물질에 대해서는 높은 감수성을 나타내는 반면, penicillin G, oxacillin, cephalothin, cefazolin, cefamandole 등 $\beta$-lactamine계 항생제에 대해서 높은 내성을 가지고 있었다.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
• /
• pp.40-45
• /
• 2001

Raw starch-digesting amylase (BF-2A, M.W. 93, 000 Da) from Bacillus circulans F-2 was converted to two components during digestion with subtilisin. Two components were separated and designated as BF-2A' (63, 000 Da) and BF-2B (30, 000 Da), respectively. BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A). Moreover, the catalytic activities of original and modified enzymes were indistinguishable in $K_{m}$, Vmax for, and in their specific activity for soluble starch hydrolysis. However, its adsorbability and digestibility on raw starch was greatly decreased. Furthermore, the enzymatic action pattern on soluble starch was greatly different from that of the BF-2A. A smaller peptide (BF-2B) showed adsorb ability onto raw starch. By these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption. A similar phenomenon is observed during limited proteinase K, thermolysin, and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63- and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the protease sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive toward anti-RSDA were detected through whole bacterial cultivation. Proteins of sizes 93-, 75-, 63-, 55-, 38-, and 31-kDa were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results postulated that enzyme heterogeneity of the raw starch-hydrolysis system might arise from the endogeneous proteolytic activity of the bacterium. Truncated forms of rsda, in which the gene sequence encoding the conserved domain had been deleted, directed the synthesis of a functional amylase that did not bind to raw starch. This indicates that the conserved region of RSDA constitutes a raw starch-binding domain, which is distinct from the active centre. The possible role of this substrate-binding region is discussed.d.

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권2호
• /
• pp.153-159
• /
• 2007

In our previous study, overexpression of extracellular proteinase inhibitor (Expi) gene accelerated apoptosis of mammary epithelial cells, and induced expression of B cell activating factor (BAFF) gene. In this study, we found induction of BAFF-receptor (BAFF-R) gene expression in the Expi-transfected cells. A proliferation-inducing ligand (APRIL) gene is another TNF family member and the closest known relative of BAFF. We found induction of APRIL gene expression in the Expi-overexpressed apoptotic cells. NF-${\kappa}$B gene was also induced in the Expi-overexpressed cells. Expression patterns of BAFF and APRIL pathway-related genes were examined in in vivo mouse mammary gland at various reproductive stages. Expression levels of BAFF gene were very low at early pregnancy, increased from mid-pregnancy, and peaked at lactation, and thereafter decreased at involution stages of mammary gland. Expression of BAFF-R gene was highly induced in involution stages compared to lactation stages. Thus, expression patterns of BAFF-R gene were correlated to apoptotic status of mammary gland: active apoptosis of mammary epithelial cells occurs at involution stage of mammary gland. Expression levels of NF-${\kappa}$B gene were higher in involution stages compared to lactation stages. We analyzed mRNA levels of bcl-2 family genes from different stages of mammary development. Bcl-2 gene expression was relatively constant during lactation and involution stages. There was a slight increase in bcl-xL gene expression in involution stages compared to lactation state. Bax gene expression was highly induced in involution stage. Our results suggest that signaling pathways activated by both BAFF and ARRIL in mammary gland point towards NF-${\kappa}$B activation which causes upregulation of bax.