• The Plant Pathology Journal
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• v.23 no.4
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• pp.281-286
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• 2007

Interactions between viral proteins and host proteins are essential for virus replication. Especially, translation of viral genes completely depends on the host machinery. In potyviruses, interactions of genome-linked viral protein (VPg) with host translation factors including eIF4E, eIF(iso)4E, and poly(A)-binding protein (PABP) has previously been characterized. In this study, we investigated interactions between Soybean mosaic virus (SMV) viral proteins and host translation factors by yeast two-hybrid system. SMV VPg interacted with eIF4E, eIF(iso)4E, and PABP in yeast two-hybrid system, while SMV helper component proteinase (HC-pro) interacted with neither of those proteins. The interaction between SMV NIb and PABP was also detected. These results are consistent with those reported previously in other potyviruses. Interestingly, we found reproducible and specific interactions between SMV coat protein (CP) and PABP. Deletion analysis showed that the region of CP comprising amino acids 116 to 206 and the region of PABP comprising amino acids 520 to 580 are involved in CP/PABP interactions. Soybean library screening with SMV NIb by yeast two-hybrid assay also identified several soybean proteins including chlorophyll a/b binding preprotein, photo-system I-N subunit, ribulose 1,5-biphosphate carboxylase, ST-LSI protein, translation initiation factor 1, TIR-NBS type R protein, RNA binding protein, ubiquitin, and LRR protein kinase. Altogether, these results suggest that potyviral replicase may comprise a multi-protein complex with PABP, CP, and other host factors.

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.317-324
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• 2000

Candida albicans is one of the most frequently isolated fungal pathogens in human. Recently, the prevalence of candida infection has markedly increased, partially due to the increase of immunocompromised hosts. Proposed virulence factors of the pathogenic Candida are the ability to form hyphae to adhere to epithelial cell surfaces, and to secrete acid proteinases and phospholipases. We measured the relative cell surface hydrophobicity (CSH) and the ability of proteinase production (PROT), phospholipase production (PLase), adherence to host epithelium (ADH), and hyphal transition (Germ). The relative risk of virulence factors was analyzed by lethality test in murine model of hematogeneously disseminated candidal infection. According to Cox's proportional hazard analysis, the statistically significant virulence factors were PROT, ADH, and CSH. PROT was the highest risk factor of them. To evaluate the applicability for the diagnosis and treatment of Candidiasis, we examined the protective effect of the active and passive immunizations with the materials purified from virulence factors and antibodies to them in Candia-infected mice model. The mean survival times of active and passive immunized groups were slightly longer than those of non-immunized groups.

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.255-260
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• 1998

To evaluate the biological and biochemical characteristics of Trichomonas vaginalis KT9 isolate, the growth and size of trichomonads, pathogenicity in mouse, protein profiles and proteinase activity were examined after shifting the medium from TPS-1 into TYM. Generation time of trichomonads in TYM medium was 4.5 hr in comparison to TPS-1 with 7.1 hr. Size of trichomonads cultured in TPS-1 medium ($8.5{\;}{\pm}{\;}0.9{\;}{\times}{\;}6.0{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$) was significantly smaller than those in TYM medium ($10.9{\;}{\pm}{\;}1.4{\;}{\times}{\;}8.2{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$). Trichomonads cultured in TYM medium produced subcutaneous abscess in 9 out of 10 mice, whereas those in TPS-1 medium produced abscesses in 2 out of 10 mice. In SDS-PAGE, trichomonad Iysates from both media showed ten common bands. However, trichomonads in TYM medium showed additional bands of 136 kDa, 116 kDa and 40 kDa in comparison to those in TPS-1 with 100 kDa. By immunoblot with T. vaginalis-immunized rabbit sera, T. vaginalis cultivated in both TYM and TPS-1 media showed 5 common bands. and unique bands of 116 kDa. 105 kDa. and 86 kDa were observed in trichomonads in TYM while a 140 kDa band in those in TPS-1. In gelatin SDS-PAGE, trichomonads in TYM degraded gelatin stronger than those in TPS-1. Also protease activity of trichomonads in TYM was significantly higher than that of trichomonads in TPS-1 using Bz-Pro-Phe-Arg-Nan as a substrate. According to the results, it is assumed that the shift from TPS-1 into TYM medium for cultivation of T. vaginalis might modulate the biological and biochemical properties of T vaginalis in vitro.

• Development and Reproduction
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• v.5 no.1
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• pp.23-33
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• 2001

When mammalian oocytes ovulate into the oviduct, associating follicular fluid components are exposed to the oviductal environment, possibly resulting in the mutual interaction between fillicu1ar and oviductal fluids. In the Present study, we have demonstrated for the first time that components of fallicular fluid could be modified by the oviductal fluid. Gelatin zymographic analyses of human follicular fluid (hFF) obtained from IVF patients showed consistently the presence of 110 kDa gelatinase (GA110) in addition to many bands among which 62 kDa gelatinase was predominant. Addition of EDTA or phenanfhroline to the gelatinase substrate buffer during gel incubation abolished GA110 band whereas phenylmethylsulffnyl fluoride (PMSF) did not. In contrast, bovine oviductal fluid(bOF) exhibited only 62 kDa gelatinase. Surprisingly, when bOF was added to hFF in 1:1 ratio and then the mixture was incubated for 3 h at 37$^{\circ}$C, GA110 of hFF disappeared. Disappearance of GA110 by bOF was observed even within 30 min after mixing with hFF. Addition of aminophenylmercuric acetate (APMA) to hFF also abolished enzymatic activity of GA110 but increased the activityof 62 kDa gelatinase. However, APMA abolished many other gelatinases as well unlike bOF. Interestingly, treatment of hFF with EDTA for 3 h remarkably increased the enzymatic activity of GA110 but not that of other gelatinases. Addition of phenanthroline, PMSF or soybean trypsin inhibitor (SBTI) did not affect overall gelatinase activities. Again, addition of bOF to the hFF pretreated with any of the above proteinase inhibitors abolished the appearance of GA110. Human serum also showed GAI 10 of which activity was greatlyenhanced by EDTA treatment. Similar to hFF, serum GA110 also disappeared by the addition of bOF. Human granulosa cell homogenate did not reveal any appreciable gelatinase activity except 92 kDa gelatinase. Anti-human gelatinase A antibody reacted with 62 kDa gelatinase of hFF. Based upon these results, it is concluded that bOF could selectively degrade an isoform of gelatinase A present in hFF and human serum.

• Parasites, Hosts and Diseases
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• v.50 no.1
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• pp.45-51
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• 2012

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by $Fasciola$ $gigantica$ play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 $F.$ $gigantica$ metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, $IgG_1$, and $IgG_2$ ($P$<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-${\gamma}$, and TNF-${\alpha}$, revealed significant decreases ($P$<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-${\beta}$, and IL-6, showed significant increases ($P$<0.05). In conclusion, it has been found that CP released by $F.$ $gigantica$ are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

• BMB Reports
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• v.34 no.2
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• pp.109-113
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• 2001

The Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP). The three-dimensional structure of SmaPI was calculated by computer modeling using the structure complex between SMP and the Erwinia chrysanthemi inhibitor as a template. Based on this model structure, the substitution of the amino acid residues, Ala4, Leu-5, Pro-6, and Thr-7, were located at the hinge region of the N-terminal segment by site-directed mutagenesis. Although the A4R and T7A mutant SmaPIs showed a nearly full inhibitory activity, the inhibitory activity of SmaPI decreased significantly when the Leu-5 was converted to Ala, Gly, Ile, or Val. Surprisingly, the L5I and L5V mutant SmaPIs showed less inhibitory activities than the L5A mutant. From these results, we suggested that the orientations and positions of respective aliphatic groups in the side-chain of position 5 mainly affected the inhibitory activity of SmaPI. The overall side-chain hydrophobicity was only slightly affected. The side-chain of the Leu-5 residue contributed approximately 0.79 kcal/mol out of 8.44 kcal/mol to the binding of SmaPI with SMP The inhibitory activities of P6A and F6G were also severely decreased. The Pro-6 may have a critical role in maintaining the strict conformation of the N-terminal portion that may be important in the inhibitory activity of SmaPI. In conclusion, Leu-5 and Pro-6 have crucial roles in the inhibitory function of SmaPI toward SMP.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.341-349
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• 1996

초기배의 성판정은 대상가축의 성을 선발하는 수단으로써 뿐만아니라 인간의 유전적 질병의 조기진단법으로서 매우 가치가 크다. 체외수정 소 초기배의 성을 결정하기 위해 PCR과 PRINS를 이용하였으며 성판정에 이용된 8 세포${\sim)$배반포기 초기배는 체외수정후 난관상피세포와의 공배양에 의해 생산되었다. 초기배 의 DNA는 $200{\mu}g/ml$ proteinase K가 함유된 PCR lysis buffer에 하나의 초기배를 부유한 후 $50^{\circ}C$에서 1시간동안 처리하여 준비하였다. 중기 염색체 spreads는 초기배를 nocodazole로 처리한 후 air-drying 방법을 이용하여 준비하였다. 가능한 false positive signals을 배제하기 위해 소특이 및 Y 염색체 특이 primers를 이용하여 PCR을 수행한 결과, 웅성 초기배에서는 두 개의 증폭산물 (소특이 및 Y 염색체 특이)이 합성된 반면 자성 초기배에서는 하나의 증폭산물만 합성되었다. 한편 중기염색체상의 Y 염색체를 동정하기 위해 FISH와 PRINS를 수행한 결과, FISH에서보다 PRINS에서 더 강한 Y 염색체 특이 형광 signals이 탐지되었다. 이러한 결과는 PCR에 의한 체외생산 소 초기배의 신속정확하고 효율적인 성판정이 가능함을 보여주었다. 또한 PRINS를 통해 PCR 에 이용된 Y 특이 probe의 신뢰성이 염색체 수준에서 확인되었다.

• Tuberculosis and Respiratory Diseases
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• v.72 no.1
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• pp.93-97
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• 2012

Propylthiouracil (PTU) is one of the most common drugs used in the treatment of Graves' disease. There are a number of side effects found with PTU use including fever, rash, arthralgia, and flu-like symptoms. Recently antineutrophil cytoplasmic antibodies (ANCA) positive vasculitis after PTU treatment was reported as a rare side effect, which can cause diffuse alveolar hemorrhage and glomerulonephritis. A 45-year-old woman with Graves' disease had been treated with PTU for five months, complained of hemoptysis due to pulmonary alveolar hemorrhage causing anemia, and also had hematuria. Simple chest X-ray and HRCT showed bilateral consolidation and bronchoalveolar lavage fluid revealed alveolar hemorrhage. A serologic test was positive for ANCA against myeloperoxidase and proteinase-3. Such findings suggested that the presence of PTU induced ANCA positive vasculitis. Cessation of PTU and the administration of high dose steroids improved the clinical manifestation, radiologic and serologic findings. We observed ANCA titer serially for 6 years. During the follow up period, ANCA titer decreased slowly and stayed within the acceptable upper normal limit.

• Archives of Pharmacal Research
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• v.28 no.7
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• pp.816-822
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• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.559-568
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• 2007

Recently, copy number variations (CNV) of genes or genomic segments have been intensively studied and various analysis methods have been developed. In this study, quantitative oligonucleotide ligation assay (qOLA) was applied to investigate CNV of KIT gene in the Landrace breed. A combined assay using qOLA and pyrosequencing, 6 genotype classes, I1/I1 or I3/i (IBe), I1/I2 or I3/IP, I1/I3, I1/IP or I2/i (IBe), I2/I2and I2/IP, were identified from 44 Landrace pigs. Genotype assignment using grouping features of measurements on a scatter plot showed 100% agreement with those using a statistical assignment by PROC FASTCLUS procedure implemented in the SAS package. Two versions (3100 and 3130) of ABI sequencers gave the same genotyping results, indicating there was no influence on qOLA by different versions of instrument, however, the means of standard deviation and coefficient of variation from the qOLA on a ABI 3130 (2.33 and 4.10) was lower than those from the qOLA on a ABI 3100 (2.67 and 4.81). Effect of proteinase K treatment on the PCR product followed by qOLA was very clear because noise peaks were disappeared and the observed ration fit better to the reference ratio corresponding to each genotype.