• BMB Reports
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• 제33권5호
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• pp.370-378
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• 2000

Incubation of purified laminin1-nidogen1 complexes with $[{\gamma}-^{32}P]-ATP$ in the presence of the catalytic subunit of the protein kinase A (cAMP-dependent protein kinase) resulted in the phosphorylation of the alpha chain of laminin-1 and of the nidogen-1 molecule. Aminoacid electrophoresis indicated that phosphate was incorporated on serine residues. The phosphorylation effect of laminin-1 on the process of self assembly was studied by turbidometry. In these experiments, the phosphorylated laminin-1 showed a reduced maximal aggregation capacity in comparison to the non-phosphorylated molecule. Examination of the laminin-1 network under the electron microscope showed that the phosphorylated sample formed mainly linear extended oligomers, in contrast to controls that formed large and dense multimeric aggregates. Heparin binding on phosphorylated laminin-1 in comparison to controls was also tested using solid-phase binding assays. The results indicated an enhanced heparin binding to the phosphorylated protein. The results of this study indicate that laminin1-nidogen1 is a substrate for protein kinase A in vitro. This phosphorylation had an obvious influence on the lamininl-nidogen1 network formation and the heparin binding capacity of this molecule. However, further studies are needed to investigate whether or not this phenomenon could play a role in the formation of the structure of basement membranes in vivo.

• 한국식품영양과학회지
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• 제29권3호
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• pp.493-499
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• 2000

골감소증 환자의 혈청중에 존재하는 IGFBP-5의 존재와 변화를 검토한 결과, 골감소즈 환자에서는 정상 대조군에 비해 IGFBP-5가 감소하였는데 이 변화는 IGFBP-5의 분해효소가 작용하기 때문인 것으로 나타났다. IGFBP-5에 작용하는 단백질분해효소 저해제중 metallo계인 EDTA 및 1,10-phenanthroline과 seriner계인 aprotinin, heparin, heparin cofactor 2(HC2), heparine+HC2가 IGFBP-5에 대해 저해효과가 크므로 metalloprotease이면서 serine protease의 성질을 가지는 효소들이 IGFBP-5에 작용하였다. IGF-I과 IGF-II 그리고 insulin은 효소 활성에 아무런 영향이 없었다. IGFBP-5의 zymography에서 정상인과 골감소증 환제어서 180 kDa 크기의 band가 나타났고, gelatin zymography에서 정상 대조군의 경우 66 kDa과 97 kDa 정도의 band가 확인되었고 골감소증 환자의 경우는 69 kDa의 band가 확인되었다.

• Reproductive and Developmental Biology
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• 제31권3호
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• pp.133-137
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• 2007

본 연구는 정자 내의 phsopholipid hydroxide glutathion peroxidase (PHGPx) mRNA 발현 수준, heparin-binding protein (HBP) mRNA 발현 수준, 수태율 그리고 산자수 사이의 관계를 조사하고자 실시하였다. 수태율과 산자수 사이에 있어서 상관관계는 나타나지 않았다. PHGPx mRNA 발현 수준에 있어 산자수가 10두 이상 군에서 $(2,414.7{\pm}400.7)$ 8두미만 군보다 $(1,875.8{\pm}311.2)$ 높게 나타났으나 유의적인 차이는 없었다. HBP mRNA 발현 수준에 있어서도 산자수 10두 이상 군에서 $(2,255.9{\pm}360.8)$ 8두 미만 군보다 $(2,155.4{\pm}378.0)$ 약간 높은 결과를 보였으나, 유의적인 차이는 인정되지 않았다. PHGPx mRNA 발현 수준과 산자수 사이의 관계는 (r=0.206) 정의 상관관계를 보였으나, 유의한 상관관계는 나타나지 않았다. 본 실험의 결과, PHGPx와 HBP의 발현수준이 수태율, 산자수와 유의한 상관관계를 보이지 않았기 때문에 PHGPx와 HBP가 정자의 수정능력을 예측하기에는 미흡한 면이 있다.

• Preventive Nutrition and Food Science
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• 제4권2호
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• pp.139-144
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• 1999

Lipoprotein lipase (LPL) I san enzyme that catalyzed the hydrolysis of triacylglycerols of chylomicrons and VLDL to produce 20acylglycerols and fatty acids. The enzyme, LPL, is localized on the surface of the capillary endothelium and is widely distributed in extrahepatic tissues including heart, skeletal muscle and adipose tissue. LPL has been isolated from boving milk by affinity chromatography on heparin-separose in 2 M NaCL, 5mM barbital buffer, pH 7.4. To elucidate the lipid-binding regin, LPL was digested with trypsin and then separated by gel filtration. Lipid binding region of LPL has been investigated by recombining LPL peptides with DMPC vesicles. Proteolytic LPL fragments with DMPC were reassembled and stabilized by cholate. Lipid-binding region of LPL was identified by a PTH-automated protein sequencer, as AQQHYPVSAGYTK. The analysis of the secondary structure of the lipid-binding peptides revealed a higher probability of $\alpha$-helix structure compared to the whole LPL protein. The prediction of hydrophobicity of lipid -binding region was highly hydrophobic (-1.1) compared to LPL polypetide(-0.4).

• Journal of Microbiology
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• 제39권4호
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• pp.314-320
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• 2001

Macrophages stimulated by lipopolysaccharide(LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS rsponsive element(LPE) We detected 3 specific bands termed B1, B2 and 3 formed by the interaction of the LPE and proteins found in LPS-stimulated RAW 367.7 cells. An additional band B4 was determined to be an Ap-1 binding protein. The B1 band appears within 1 hour of LPS nuclear extracts from LPS-stimulation, and this protein kinase enhances B1 and formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction purified Ser/Thr specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the trgulation of the LRE-dependent Rants experssion: two binding factors that bind directly to the target sequences. and two factors that control their binding. The future purification and characterization of these binding pro-teins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

• IMMUNE NETWORK
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• 제11권2호
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• pp.123-133
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• 2011

Background: Mycobacterium tuberculosis (Mtb) heparin binding hemagglutinin (HBHA) is an Ag known to evoke effective host immune responses during tuberculosis infection. However, the molecular basis of the host immune response to HBHA has not been fully characterized. In this study, we examined the molecular mechanisms by which HBHA can induce the expression of proinflammatory cytokines in macrophages. Methods: HBHA-induced mRNA and protein levels of proinflammatory cytokines were determined in bone marrow-derived macrophages (BMDMs) using RT-PCR and ELISA analysis. The roles of intracellular signaling pathways for NF-${\kappa}B$, PI3-K/Akt, and MAPKs were investigated in macrophage proinflammatory responses after stimulation with HBHA. Results: HBHA robustly activated the expression of mRNA and protein of both TNF-${\alpha}$ and IL-6, and induced phosphorylation of NF-${\kappa}B$, Akt, and MAPKs in BMDMs. Both TNF-${\alpha}$ and IL-6 production by HBHA was regulated by the NF-${\kappa}B$, PI3-K, and MAPK pathways. Furthermore, PI3-K activity was required for the HBHA-induced activation of ERK1/2 and p38 MAPK, but not JNK, pathways. Conclusion: These data suggest that mycobacterial HBHA significantly induces proinflammatory responses through crosstalk between the PI3-K and MAPK pathways in macrophages.

• Asian-Australasian Journal of Animal Sciences
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• 제29권9호
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• pp.1247-1255
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• 2016

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, $40{\mu}g/mLs$ semen were standardized to find out the optimum dose and $20{\mu}g/mLs$ was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with $20{\mu}g/mL$ SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/$32.7{\mu}M$/$10^9$ spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species.

• 한국수정란이식학회지
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• 제10권1호
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• pp.15-22
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• 1995

Since the turn of the century, scientists have earnestly sought to develop a single laboratory assay or combination of laboratory assays which accurately predict the fertility of a semen sample. Most of these assays have focused on evaluating physical characteristics of sperm such as motility, viability, acrosomal integrity and morphology. In recent years new approches have been used to assess the functional aspects of a sperm that are needed to reach the oocyte, fertilize it and contribute to successful embryo development. Among these techniques are the ability of sperm to undergo a heparin induced acrosome reaction and in vitro fertilization, and the affinity of sperm to bind heparin binding protein. Intensification of research efforts in the area of control of sperm fertilizing ability should be a high priority, in view of undoubted benifits both to our basic understanding of sperm fertilizing ability and to our ability to modify it for Al industry.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.211.2-211.2
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• 2003

Glycosaminolycans (GAGs). such as heparin and heparan sulfate, are highly charged molecules and are of great biological importance. Protein-GAGs interactions play prominent roles in cell-cell recognition and cell growth. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, is a novel member of glycosaminoglycan families. It showed antitumor activity by the inhibition of angiogenesis. (omitted)

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.57-62
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• 1999

Light-regulated translation of chloroplast mRNAs requires nuclear-encoded trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. A set of four proteins (60, 55, 47, and 38 kDa) that bind to the 5'-UTR of the psbA mRNA had been identified in C. reinhardtii. 47 kDa protein (RB47) was found to encode a chloroplast poly (A)-binding protein (cPABP) that specifically binds to the 5'-UTR of the psbA mRNA, and essential for translation of this mRNA, cDNA encoding 60 kDa protein (RB60) was isolated, and the amino acid sequence of the encoded protein was highly homologous to plants and mammalian protein disulfide isomerases (PDI), normally found in the endoplasmic reticulum (ER). Immunoblot analysis of C. reinhardtii proteins showed that anti-PDI recognized a distinct protein of 56 kDa in whole cell extract, whereas anti-rRB60 detected a 60 kDa protein. The ER-PDI was not retained on heparin-agarose resin whereas RB60 was retained. In vitro translation products of the RB60 cDNA can be transported into C. reinhardtii chloroplast in vitro. Immunoblot analysis of isolated pea chloroplasts indicated that higher plant also possess a RB60 homolog. In vitro RNA-binding studies showed that RB60 modulates the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP using redox potential or ADP-dependent phosphorylation. Site-directed mutagenesis of -CGHC- catalytic site in thioredoxin-like domain of RB60 is an unique PDI located in the chloroplast of C. reinhardtii, and suggest that the chloroplast PDI may have evolved to utilize the redox-regulated thioredoxin like domain as a mechanism for regulating the light-activated translation of the psbA mRNA.