• Restorative Dentistry and Endodontics
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• 제25권4호
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• pp.509-526
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• 2000

It is known that injuries to the dentin have a corresponding inflammatory effect on the pulp and these inflammatory effects frequently result in pulpal pathoses due to progressive degradation of pulpal connective tissue. It was supposed that the tissue degradation in different inflammatory process was controlled by proteinase activity and antiproteinase activity. Therefore, the purpose of this study was to examine the pulp and periapical pathoses in terms of the activities of proteinase and proteinase inhibitor, 37 pulpal tissues were divided by clinical diagnostic criteria into normal pulp, acute inflamed pulp, and chronic inflamed pulp, and then those groups were subdivided by histopathological findings into 5 pulpal pathoses groups, i.e. normal pulp (P1, n=8), chronic pulpitis with fibrotic change (P2, n=2), chronic pulpitis with dystrophic calcification (P3, n=11), chronic pulpitis with pulp abscess (P4, n=7), acute pulpitis with necrotic change (P5, n=4), 26 periapical tissues were also divided by ordinary histopathological findings into 3 periapical pathoses group, i.e., granuloma (A1, n=17), cyst (A2, n=2) and abscess (A3, n=7). The activities of proteinases (cathepsin G, MMP-3) and proteinase inhibitors (${\alpha}1$-AT, TIMP-1 and, SLPI) were evaluated by RT-PCR and immunohistochemical methods. The results were as follows. 1. Generally, the intensity of immunohistochemical staining of proteinases and proteinase inhibitors increased in P2 and P5 groups compared to P1 group. 2. The immunohistochemical stain of proteinases and proteinase inhibitors was intensely detected in P2 group, showing low inflammatory reaction and low tissue degradation, but it was reduced in P3 and P4 groups, showing severe tissue degradation. 3. The distribution of proteinases and proteinase inhibitors in pulpal pathoses was consistently presented by immunohistochemical staining, while the expression of proteinase and/or proteinase inhibitors mRNAs in pulpal pathoses was occasionally detected by RT-PCR methods. 4. RT-PCR of proteinase and proteinase inhibitors was usually positive in P2, showing rare tissue degradation, but it was almost negative in P3 and P4, showing severe tissue degradation. 5. We presume that the reason why the level of proteinase and proteinase inhibitors was so sparse in RT-PCR method is due to the abrupt decrease of mRNA synthesis or degradation of synthesized mRNA of proteinase and/or proteinase inhibitors depend on the inflammatory reaction and/or on the degradation of pulp tissues(P3, P4). 6. Pulpal pathoses groups showed significant lower RT-PCR detection of proteinases and proteinase inhibitors than the periapical pathoses group(p<0.05), and there is no significant difference among the periapical pathoses groups(p>0.05).

• Parasites, Hosts and Diseases
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• 제40권2호
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• pp.93-99
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• 2002

The effect of a secretory proteinase from the pathogenic amoebae Acanthamoeba castellanii on host's defense-oriented or regulatory proteins such as immunoglobulins, interleukin-1, and protease inhibitors was investigated. The enzyme was found to degrade secretory immunoglobulin A (slgA), IgG, and IgM. It also degraded $interleukin-1{\alpha}$ ($IL-l{\alpha}$) and $IL-l{\beta}$. Its activity was not inhibited by endogenous protease inhibitors, such as ${\alpha}$2-macroglobulin, ${\alpha}l-trypsin$ inhibitor, and ${\alpha}2-antiplasmin$. Furthermore, the enzyme rapidly degraded those endogenous protease inhibitors as well. The degradation of host's defense-oriented or regulatory proteins by the Acanthanoeba proteinase suggested that the enzyme might be an important virulence factor in the pathogenesis of Acanthamoeba infection.

• 한국작물학회지
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• 제49권4호
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• pp.342-348
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• 2004

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the $60\%$ isopropanol extract of soybean(Glycine max [L.] Merr.) seed revealed two abundant proteins with molecular masses of 19 and 10 kDa. Amino acid analysis revealed that the isopropanol-extractable protein fraction was rich in cysteine. Two-dimensional gel electro-phoretic analysis indicated that the 19kDa and 10kDa proteins had pI of 4.2 and 4.0 respectively. Peptide mass fingerprints of trypsin digests of the two proteins obtained using matrix-assisted, laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy revealed the 19kDa protein was Kunitz trypsin inhibitor and the 10kDa protein was Bowman-Birk proteinase inhibitor. When resolved under non-denaturing conditions, the isopropanol-extracted proteins inhibited trypsin and chymotrypsin activity. Results presented in this study demonstrate that isopropanol extraction of soybean seed could be used as a simple and rapid method to obtain a protein fraction enriched in Kunitz trypsin and Bowman-Birk proteinase inhibitors. Since proteinase inhibitors are rich in sulfur amino acids and are putative anticarcinogens, this rapid and inexpensive isolation procedure could facilitate efforts in nutrition and cancer research.

• BMB Reports
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• 제29권4호
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• pp.380-385
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• 1996

Ovomucoid third domain is a serine proteinase inhibitor protein which consists of 56 amino acid residues. A fifty picosecond molecular dynamics (MD) simulation was carried out for ovomucoid third domain protein with 5 $\AA$ layer of water molecules. A comparison of main chain atoms in the MD averaged structure with the crystal structure showed that most of the backbone structures are maintained during the simulation. Investigation of the intramolecular hydrogen bondings indicated that most of the interactions between main chain atoms were conserved, whereas those between side chains were reorganized for the period of the simulation. Especially, the side chain interactions around the scissile bond of reactive site P1 (Met18) were found to be more extensive for the MD structures. During the simulation, hydrogen bonds were maintained between the side chains of Glu19 and Arg21 as well as those of Thr17 and Glu19. Extensive side chain interactions observed in the MD structures may shed light on the question of why protein proteinase inhibitors are strong inhibitors for proteinases rather than good substrates.

• 미생물학회지
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• 제29권6호
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• pp.345-352
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• 1991

A serine proteinase of molecular weight 60 kd was purified from culture supernatant of P. aeruginosa using DEAE-Trisacryl M ion-exchange and AcA 54 gel filtration column chromatography, and the properties of serine proteinase were characterized. By means of SDS-polyacrylamide gel electrophoresis, the molecular weight of the enzyme was 55 kd. The optimal pH for the activity of purified enzyme was 7.5. The activity of the purified enzyme was completely inhibited by Di-isopropylfluorophosphate(DFP) and N-.alpha.-p-tosyl-L-lysine choloromethyl detone(TLCK) but not by other proteinase inhibitors such as E-64, pepstatin A, 1, 10-phenanthroline. The purified enzyme was capable of degrading type I and type IV collagen. Antisera obtained from hymans infected with Pseudomonas aeruginosa reacted to the purified serine proteinase in immunoblots. These results indicate that the purified enzyme is trypsin-like serine proteinase and this enzyme of P. aeruginosa may play an important role in tissue damage as a spreading factor and may be useful for serodiagnosis of Pseudomonas infections.

• Parasites, Hosts and Diseases
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• 제35권1호
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• pp.39-46
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• 1997

이 연구에서는 살아있는 질편모충의 단백질녈해효소가 인체 면역글로불린을 분해하는지 알아보고 질편모충에 의한 조직세포 독성에 있어서 단백질분해효소의 역할을 시험관내에서 관찰하였다 실험에 사용한 질편모충은 질염환자로년터 분리한 KT9 분리주이었으며 세포독성을 알아보기 위한 표적세포로는 HeLa 세포를 사용하였다 질편모충 단백질분해효소가 인체 면역글로불린을 분해하는지 관찰하고자 인체의 분비 IP. 혈청 IgA 및 IgG를 살아있는 원충. 원충의 용출액 및 분비-배설 액과 DTT를 넣어 반응시켰다. 여러 계열의 단백질분해효소 저해제(aminopeptidase, serine, metallo, cystelrle계열)를 살아있는 질편모충과 미리 반응시킨 후 세척하고 면역글로불린의 분해 단백질분해효소 활성 및 조직세포독성에 미치는 영향을 관찰하였다 살아있는 질편모충은 인체의 분비 IgA. 혈청 IgA 및 IgG를 분해하였는데 질편모충 수가 증가할수록 반응시간이 길수록 분해가 더 잘 이루어졌다 질푄모충의 용출액과 분비-배설액도 분비 IgA. 혈청 IgA 및 IgG를 분해하였다. Cysteine, serine계 열의 단백질분해효소 저해제 (I-64 antipain, iodoacetic acid, iodoacetamide, TLCK)를 처리한 질편닐충은 분비 IgA의 분해를 저해하였으며. 단백질분해효소저해제로 처리한 경우 질편모충의 단백질븐해효소 활성은 감소하였고 HeLa세포에 대한 독성이 감소하였다. 이상의 성적을 종합하면 질편모충에서 분비되는 단백질분해효소는 시험관내에서 조직세포에 세포독성을 나타내며 또한 인체면역글로불린을 분해하여 숙주의 방어기전에 대한 도피물질로 작용하는 것으로 생각된다.

• Parasites, Hosts and Diseases
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• 제36권4호
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• pp.261-268
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• 1998

질편모충의 시스테인 단백분해효소가 숙주-기생충 관계에서 어떤 역할을 하는지 알아보기 위해 질편모충을 대량배양하여 초음파분쇄한 후 초원심분리하여 조추출물을 얻었고, activated Thiol-Sepharose 4B, Bacitracin Sepharose affinity chromatography, Sephacryl S-200 HR gel filtration 등을 이용하여 단백분해효소를 정제하였다. SDS-PAGE로 정제도를 확인하여 이들 효소의 생화학적 특성, 그리고 인체 면역글로불린 및 헤모글로빈 분해능을 관찰하였다. 정제된 단백분해효소는 0.1 M sodium phosphate (pH 6.0)에서 최적 활성을 나타내었으며, SDS-PAGE에서 분자량은 60 kDa이었고 gel filtration에서 native 분자량은 62 kDa이었다. 정제된 단백분해효소는 시스테인 계열 억제제인 E-64, IAA, NEM에 의해서 활성이 억제되었으며, 메탈로, 세린, 아스파틱 계열 억제제에 의해서는 활성이 억제되지 않았다. $Hg^{2+}$ 이온에 의해 활성이 억제되었고 시스테인 계열 활성제인 DTT에 의해서는 2배 이상의 활성을 보여 정제된 단백분해효소가 시스테인 계열임을 알 수 있었다. 정제된 단백분해효소와 serum IgG, serum IgA, secretory IgA를 반응시켰을 때 면역글로불린이 분해되었고 헤모글로빈과 반응시켰을 때도 헤모글로빈을 분해하였다. 이상의 결과로 보아 질편모충에서 정제한 60 kDa acidic 시스테인 단백분해효소는 인체의 IgG, IgA 및 헤모글로빈 등을 분해하여 숙주의 면역기작을 회피하며 영양대사에 이용할 것으로 생각된다.

• 한국어업기술학회:학술대회논문집
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• 한국어업기술학회 2001년도 춘계 수산관련학회 공동학술대회발표요지집
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• pp.148-149
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• 2001

Protein inhibitors are proteins or peptides capable of inhibiting catalytic activities of proteolytic enzymes. They are grouped primarily as either serine, cysteine, aspartic or metallto-proteinase inhibitors. Pretense inhibitors have been hewn since the end of the last century in nematodes and human blood serum, and their ubiquitous distribution in microorganisms, animals and plants has been widely documented. (omitted)

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권5호
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• pp.402-407
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• 2006

Vascular endothelial cells release proteinases that degrade the extracellular matrix (ECM), thus enabling cell migration during angiogenesis and vasculogenesis. Sildenafil citrate stimulates the nitric oxide-cyclic guanosine monophosphate pathway through inhibition of phosphodiesterase type V (PDE5). In this report, we examined the mechanisms underlying sildenafil citrate-induced cell migration using cultured mouse aortic endothelial cells (MAECs). Sildenafil citrate induced migration and proteinase secretion by murine endothelial cells. Sildenafil citrate induced the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9, which is inhibited by $NF-{\kappa}B$ inhibitors. Sildenafil citrate also induced the secretion of plasmin, which is inhibited by PI 3'-kinase inhibitors. It is suggested that sildenafil citrate-induced migrating activity in endothelial cells may be accomplished by increased secretion of proteinases.

• BMB Reports
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• 제29권5호
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• pp.455-461
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• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.