• Journal of Microbiology and Biotechnology
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• 제8권1호
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• pp.34-41
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• 1998

A method was developed for the controlled expression of an antimicrobial peptide magainin in Escherichia coli. A series of concatemeric magainin genes was constructed with a gene amplification vector, and fused to the 3'end of malE gene encoding the affinity ligand, E. coli maltose-binding protein (MBP). The construct directed the synthesis of the fusion protein with the magainin polypeptide fused to the C-terminus of MBP. The fusion protein was expressed in a tightly regulatable expression system which was under the control of an invertible promoter. The MBP-fused magainin monomer was expressed efficiently. However, the expression level of the MBP-fused magainin in E. coli decreased with the increasing size of multimers possibly because of the transcription and translation inhibition by the multimeric peptides. After purification using an amylose affinity column, the fusion protein was digested by factor Xa at a specific cleavage site between the monomers. The recombinant magainin had an antimicrobial activity identical to that of synthetic magainin. This experiment shows that a biologically active, antimicrobial peptide magainin can be produced by fusing to MBP, along with a promoter inversion vector system.

• 대한치과보철학회지
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• 제42권3호
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• pp.301-306
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• 2004

Statement of problem. Fibronectin mediates its biological effects by binding to integrins on cell membranes through a consensus site including the Arg-Gly-Asp (RGD) sequence within tenth type III module. Purpose. The purpose of our study was to investigate the adsorption affinity of human recombinant fibronectin peptide (hFNIII 9-10) to titanium and to investigate the effect of the surrounding ionic composition on the adsorption process. Material and methods. As for evaluating the affinity of hFNIII 9-10 to Ti, titanium disks were incubated in 40, 80 and $120{\mu}g/ml$ hFNIII 9-10 solution at $37^{\circ}C$ overnight, repectively. As for evaluating the effect of surrounding ionic concentration, hFNIII 9-10 was dissolved in distilled water, phosphate buffered saline and RPMI 1640. Optical density (O.D.) was measured in ELISA reader. Results. The results were as follows; 1. The adsorption of hFNIII 9-10 showed significantly highest mean optical density (O.D.) value in $80{\mu}g/ml$. 2. The difference of ionic composition in DW, PBS and RPMI did not influence the adsorption amount of hFNIII 9-10.

• Bulletin of the Korean Chemical Society
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• 제32권7호
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• pp.2325-2331
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• 2011

Human CDX1 and CDX2 genes play important roles in the regulation of cell proliferation and differentiation in the intestine. Hox genes clustered on four chromosomal regions (A-D) specify positional signaling along the anterior-posterior body axis, including intestinal development. Using glutathione S-transferase (GST) pulldown assays, molecular interaction measurements, and fluorescence measurements, we found that the homeodomains (HDs) of CDX1 and CDX2 directly interact with that of HOXD8 in vitro. CDX1 showed significant affinity for HOXD8, but CDX2 showed weak affinity for HOXD8. Thus far, three-dimensional structures of CDX1/2 and HOXD8 have not been determined. In this study, we developed a molecular docking model by homology modeling based on the structures of other HD members. Proteins with mutations in the HD of CDX1 (S185A, N190A, T194A, and V212A) also bound to the HD of HOXD8. Our study suggests that the HDs of CDX1/2 resemble those of HOXD8, and we provide the first insight into the interaction between the HDs of CDX1/2 proteins and those of HOXD8.

• Bulletin of the Korean Chemical Society
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• 제28권6호
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• pp.1005-1009
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• 2007

Leukotriene B4 (LTB4) is a potent chemoattractant for leukocytes and considered to be an inflammatory mediator. Human BLT2 (hBLT2) is a low-affinity G-protein coupled receptor for LTB4 and mediates pertussis toxin-sensitive chemotactic cell movement. Here, we dissected the interaction between hBLT2 and G-protein alpha subunits using GST fusion proteins containing intracellular regions of hBLT2 and various Gα protein including Gα i1, Gα i2, Gα i3, Gα s1, Gα o1, and Gα z. Among the tested Gα subunits, Gα i3 showed the highest binding to the third intracellular loop region of hBLT2 with a dissociation constant (KD) of 5.0 × 10?6 M. These results suggest that Gα i3 has the highest affinity to hBLT2, and the third intracellular loop region of hBLT2 is the major component for the interaction with Gα i3.

• 대한약리학회지
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• 제18권2호
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• pp.33-44
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• 1982

전해질농도(電解質濃度)를 달리하고 morphine, naloxone 또는 morphine+naloxone을 가(加)하거나 가(加)하지 않은 modified Krebs-Henseleit bicarbonate buffer용액(溶液)에 흰쥐의 뇌절편(腦切片)을 incubate하여 저온처리(低溫處理)와 media내(內) 전해질조성(電解質組成)이 $(^3H)-morphine$의 saturable binding에 미치는 영향(影響)을 관찰(觀察)하여 다음과 같은 성적(成績)을 얻었다. 1) 저온처리(低溫處理)는 $(^3H)-morphine$의 saturable binding을 현저(顯著)히 증가(增加)시켰고, maximal saturable binding은 증가(增加)시키나 $K_D$치(値)에는 영향(影響)을 미치지 못하였다. 2) 저온처리시(低溫處理時) media내 morphine과 naloxone은 $(^3H)-morphine$ 의 maximal saturable binding 과 $K_D$치(値)를 증가(增加) 시켰다. 3) 저온처리시(低溫處理時) media내(內) $K^+$감소(減少), $Mg^{++}$제거 또는$Mn^{++}$첨가(添加)는 $(^3H)-morphine$의 saturable binding을 현저(顯著)히 증가(增加)시켰고, $Na^+$감소(減少), $Ca^{++}$증가(增加)는 saturable binding에 영향(影響)을 미치지 못하였다. 4) 저온처리시(低溫處理時) media내(內) morphine에 의한 $(^3H)-morphine$의 saturable binding증가(增加)는 media내(內) $Na^+$감소(減少), $K^+$감소(減少), $Mg^{++}$제거 또는 $Mn^{++}$첨가(添加)로는 영향(影響) 받지 않았으나 $Ca^{++}$증가(增加)로 억제(抑制)되었고, media내(內) naloxone에 의한 saturable binding증가(增加)는 $Mn^{++}$첨가(添加)는 영향(影響)받지 않았으나 media내(內) $Na^+$감소(減少), $K^+$감소(減少), $Ca^{++}$증가(增加) 또는 $Mg^{++}$제거로 억제(抑制)되었다. 이상(以上) 실험성적(實驗成績)은 저온처리(低溫處理) 및 저온처리시(低溫處理時) media내(內) morphine 또는 naloxone이 opiate수용체(受容體)의 양적(量的)인 변동(變動)과 또는 친화력(親和力)의 변동(變動)을 유발(誘發)하며, media내(內) 전해질조성(電解質組成)이 이들 변동(變動)에 영향(影響)을 미침을 시사(示唆)한다.

• 대한약리학회지
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• 제30권1호
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• pp.125-136
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• 1994

갑각류 골격근의 SR에서 칼슘유리 channel protein complex의 성격을 규명하기 위해 민물가재 및/또는 바다가재의 SR vesicles을 분리하여 $^{45}Ca$ 유리, $[^3H]ryanodine$결합, 및 immunoblot 실험을 실시하여 다음과 같은 결과를 얻었다. 1.민물가재 SR의 $[^3H]ryanodine$결합 실험에서 민물가재 SR의 maximal binding site및 affinity모두 바다가재에서 보다 낮았으나, high affinity binding site이었다. Extravesicles 칼슘농도를 증가시켰을 때 $[^3H]ryanodine$결합은 약간 증가되었으나, AMP나 AMP와 caffeine을 동시에 첨가하였을 때는 현저히 증가되었다(p<0.05). 이런 증가 현상은 $MgCl_2$나 tetracaine으로 유의성 있게 억제되었으나(p<0.001), ruthenium red에 의해서는 약간 억제되었다. 2.민물가재 SR을 전기영동하였을 때 바다가재의 ryanodine receptor band (HMWBr)와 비슷하나 포유류의 것(HMWBS) 보다는 약간 빠른 mobility를 나타낸다. 3.바다가재 HMWBr에 대한 polyclonal Ab를 이용한 민물가재, 바다가재 및 토끼 골격근의 칼슘유리 channel간의 면역학적 교차반응에서 민물가재와 바다가재의 칼슘유리 channel 간에는 교차반응이 있었으나, 포유류의 것과는 아무런 반응이 없었다. 4.민물가재 SR에서 $^{45}Ca$유리는 extravesicles의 칼슘농도 증가에 따라서 증가되었고, 낮은 외부 칼슘 농도에서 바다가재 보다 빠르게 일어났으나, AMP와 caffeine에 의해 영향을 받지 않았고, $MgCl_2$와 tetracaine으로 약간($3{\sim}8%$) 그리고 고농도의 ruthenium red로 중등도(23%) 억제되었다. 이상의 실험성적으로 갑각류 칼슘유리 channel protein은 포유류의 것과는 기능적으로나 면역학적으로 매우 다른 특징을 가지고 있고, 민물가재와 바다가재 칼슘유리 channel은 서로 유사한 특징을 갖지만, 민물가재의 칼슘유리 channel이 바다가재의 것보다 외부칼슘에 예민한 기능을 갖는 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제24권9호
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• pp.1199-1203
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• 2011

The objective of this study was to examine the binding potential of sperm to the epithelium of the sperm storage tubules (SST) in vitro and in vivo to assess the functional maturation of fowl sperm. Sperm from the testis, epididymis, as well as the proximal, middle and distal vas deferens were incubated in vitro with either the uterovaginal junction (UVJ)- or infundibular tissue containing SST at $39^{\circ}C$ for 30 min. Aliquots of sperm were also artificially inseminated into the uteri of hens, and the UVJ and infundibulum were collected 24 h post artificial insemination (AI). After incubation and AI, tissues were washed to remove loosely adhered sperm and subjected to fluorescence staining with 4', 6-diamidino-2-phenylindole, dihydrochloride (DAPI) for counting the number of bound sperm per 0.25 mm2 of surface area. Sperm from the testis, epididymis, and the three segments of the vas deferens exhibited their differential (p<0.05) binding capacity, which increased gradually from the testicular to distal vas deferens sperm under both in vitro and in vivo conditions. Existing similar trend, sperm, regardless of their source had a lesser affinity to bind to the epithelium of the infundibular SST than to the UVJ-SST. These experimental results suggested that fowl sperm may undergo gradual changes in the process of functional maturation, whereby they gain the ability to bind to the epithelium of the SST during their passage through the male reproductive tract (MRT).

• Journal of Microbiology and Biotechnology
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• 제20권9호
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• pp.1283-1287
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• 2010

In eukaryotes, eRF3 participates in translation termination and belongs to the superfamily of GTPases. In this work, the dissociation constants for nucleosides bound to Euplotes octocarinatus eRF3 in the presence and absence of eRF1a were determined using fluorescence spectra methods. Furthermore, a GTP hydrolyzing assay of eRF3 was carried out using an HPLC method, and the kinetic parameters for GTP hydrolysis by eRF3 were determined. Consistent with data from humans, the results showed that eRF1a promoted the binding of GTP to eRF3 and the GTP hydrolyzing activity of eRF3. However, in contrast to the lack of GTP binding in the absence of eRF1 in human eRF3, the E. octocarinatus eRF3 was able to bind GTP by itself. The nucleotide binding affinity of the E. octocarinatus eRF3 also differed from the human data. A structure model and amino acid sequence alignment of potential G domains indicated that these differences may be due to valine 317 and glutamate 452 displacing the conserved glycine and lysine involved in GTP binding.

• Archives of Pharmacal Research
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• 제14권2호
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• pp.181-187
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• 1991

The muscarinic antagonist 1-[benzilic 4, 4'-$[^3H]$ QUINUCLIDINYL BENZILATE $([^3H]$ QNB) bound to a single class of muscarinic receptors with high affinity in rabbit ileal membranes. The $K_D\;and\;B_{ max}$ values for $([^3H]$ QNB calculated from analysis of saturation isotherms were 52.5 pM AND 154 fmol/mg, respectively. Chlopheniramine (CHP), histamine $H_1$ blocker, increased $K_D$ vlue for $([^3H]$QNB without affecting the binding site concentrations and Hill coefficient. The $K_i$ value of CHP for inhibition of $([^3H]$QNB binding in ileal membranes was 1.44\mu{M}$ and the pseudo-Hill coefficient for CHP was close to unit. In the functional assay carbachol, muscarinic agonist, increased the contractile force of ileum with $ED_{50}$ value of $0.11\mu{M}$. CHP caused the rightward shift of the dose-response curve to carbachol. The $pA_2$ value of CHP determined from Schild analysis of carbacholinduced contraction was 5.77 and the slope was unity indicating competitive antagonism with carbachol. The dissociation constant $(K_i)$ of CHP obtained in competitive experiments with $([^3H]$ QNB was similar to the $K_A$ value (1.69 \mu{M)}$ of CHP as inhibitor of carbachol induced contraction in rabbit ileum. This result suggest that the binding of $H_i$ blocker. CHP, vs $([^3H]$QNB to muscarinic receptors in ileal membranes represents an interaction with a receptor of physiological relevance.

• Archives of Pharmacal Research
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• 제9권1호
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• pp.29-38
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• 1986

The effects of ginseng saponins on the sarcolemmal $Na^{+}$, $K^{+}$-ATPase were compared to gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 to elucidate whether the effects are due to the membrane distruption, using a highly enriched preparation of cardiac sarcolemma prepared from dog ventricular myocardium. About 26% and 29% of vesicles in the preparation, enriched in ouabain-sensitive $Na^{+}$, $K^{+}$-ATP ase, $\beta$-adrenergic and muscarinic receptors are rightside-out and inside-out orientation, respectively. Ginseng saponins (triol>total> diol) inhibited $Na^{+}$, $K^{+}$-ATP ase activity, $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H]ouabain binding of sarcolemmal vesicles. However, gypsophila saponin, SDS (0.4$\mu$g/$\mu$g protein) and Triton X-100 (0.6 $\mu$g/$\mu$g protein) caused about 1.35 and 1.40-fold increase in $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H] oubain binding, respectively. Especially, the activating effect of gypsophila saponin on membrane Na+, K+ ATPase was detected at gypsophila saponin to sarcolemmal protein ratios as high as 100. Low dose of ginseng saponin (3$\mu$g/$\mu$g protein) decreased the phosphorylation sites and the concentration of ouabain binding sites (Bmax) without affecting the turnover number and affinity for ouabain binding, while gypsophila saponin, SDS(0.4 ug/ug protein), ahd Triton X-100 (0.6$\mu$g/$\mu$g protein) increased the Bmax. The results suggest that ginseng saponins cause a decrease in the number of active sites by interacting directly with $Na^{+}$, $K^{+}$-ATPase before disruption of membrane barriers of sarcolemmal vesicles.