• 제목/요약/키워드: permeabilization

검색결과 39건 처리시간 0.019초

Calcium Sensitization Induced by Sodium Fluoride in Permeabilized Rat Mesenteric Arteries

  • Yang, En-Yue;Cho, Joon-Yong;Sohn, Uy-Dong;Kim, In-Kyeom
    • The Korean Journal of Physiology and Pharmacology
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    • 제14권1호
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    • pp.51-57
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    • 2010
  • It was hypothesized that NaF induces calcium sensitization in $Ca^{2+}$-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with $\beta$-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to $Ca^{2+}$ (decreased $EC_{50}$ and increased $E_{max}$). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in $Ca^{2+}$-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and $GTP{\gamma}S$-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a $Ca^{2+}$ dependent manner in $\beta$-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.

Enzymatic Manufacture of Deoxythymidine-5'-Triphosphate with Permeable Intact Cells of E. coli Coexpressing Thymidylate Kinase and Acetate Kinase

  • Zhang, Jiao;Qian, Yahui;Ding, Qingbao;Ou, Ling
    • Journal of Microbiology and Biotechnology
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    • 제25권12호
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    • pp.2034-2042
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    • 2015
  • A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25℃ for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

Monascus sp. J101을 이용한 적색색소의 Extractive Fermentation (Extractive Fermentation of Red Pigment Using Monascus sp. JlOl)

  • 주재영;남학우;윤주천;신철수
    • 한국미생물·생명공학회지
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    • 제22권1호
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    • pp.85-91
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    • 1994
  • The characteristcs of monascus fermentation using a hyperpigment-producing mutant, Monascus sp. J101, were analyzed, and the extractive fermentations employing permeabilizing agents and resin were carried out to increase the productivity of red pigment. And the kinetic analysis was also carried out in case of the monascus fermentation using Amberlite XAD-7. The extracellular content of the red pigment produced by Monascus sp. J101 was about 17% of the total, and the production of pigment was regulated by its own product. The cell growth reached a stationary phase at 48 hours ofter inoculation, whereas the pigment production continued up to 100 hours, which showed the pattern of a mixed growth-associated type. During the fermentation, various permeabilizing agents were added to the culture medium and their effects on pigment production were examined. By adding 0.05% Triton X-100 at 48 hours of cultivation, about an 18% increase in pigment production was accomplished as compared to the control, 12% ethyle acetate and 15% for 0.05% deoxycholate, respectively. When a nonionic adsorbent, Amberlite XAD-7 was added to the culture medium at a concentration of 12.0% at 48 hours of cultivation, the pigment production was enhanced by about 48.9% as compared to the control.

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Enzyme Immobilized Reactor Design for Ammonia Removal from Waste Water

  • Song, Ju-Yeong;Chung, Soo-Bae
    • Biotechnology and Bioprocess Engineering:BBE
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    • 제2권2호
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    • pp.77-81
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    • 1997
  • Removal of nitrogen compound from waste water is essential and often accomplished by biological process. To prevent washout and to develop an efficient bioreactor, immobilization of sutibal microorganisms could be sensible approach. Strains and permeabilized cell encapsulated in cellulose nitrate microcapsules and immobilized on polystyrene films were prepared by the method described in the previous study. In the wastewater treatment system, nitrification of ammonia component is generally known as rate controlling step. To enhance the rate of nitrification, firstly nitrifying strains Nitrosomonas europaea(IFO14298), are permeabilized chemically, and immobilized on polystyrene films and secondly oxidation rates of strain system and permeabilized strain system are compared in the same condition. with 30 minute permeabilized cells, it took about 25 hours to oxidize 70% of ammonia in the solution, while it took about 40 hours to treat same amount of ammonia with untreated cells. All the immobilization procedures did not harm to the enzyme activity and no mass transfer resistance through the capsule well was shown. In the durability test of immobilized system, the system showed considerable activity for the repeated operation for 90 days. With these results, the system developed in this study showed the possibility to be used in the actual waste water treatment system.

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해양 생물 유래의 항균 펩타이드 및 작용 기작 (Antimicrobial Peptides Derived from the Marine Organism(s) and Its Mode of Action)

  • 황보미;이준영;이동건
    • 한국미생물·생명공학회지
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    • 제38권1호
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    • pp.19-23
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    • 2010
  • Recently, marine organisms are emerging as a leading group for identifying and extracting novel bioactive substances. These substances are known to possess a potential regarding not only as a source of pharmaceutical products but also their beneficial effects on humans. Among the substances, antimicrobial peptides (AMPs) specifically have attracted considerable interest for possible use in the development of new antibiotics. AMPs are characterized by relatively short cationic peptides containing the ability to adopt a structure in which cationic or hydrophobic amino acids are spatially scattered. Although a few reports address novel marine organisms-derived AMPs, their antimicrobial mechanism(s) are still remain unknown. In this review, we summarized the peptides previously investigated, such as Pleurocidin, Urechistachykinins, Piscidins and Arenicin-1. These peptides exhibited significant antimicrobial activities against human microbial pathogens without remarkable hemolytic effects against human erythrocytes, and their mode of actions are based on permeabilization of the plasma membrane of the pathogen. Therefore, the study of antimicrobial peptides derived from marine organisms may prove to be useful in the design of future therapeutic antimicrobial drugs.

Role of HIV Vpr as a Regulator of Apoptosis and an Effector on Bystander Cells

  • Moon, Ho Suck;Yang, Joo-Sung
    • Molecules and Cells
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    • 제21권1호
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    • pp.7-20
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    • 2006
  • The major event in human immunodeficiency virus type 1 (HIV-1) infection is the death of many cells related to host immune response. The demise of these cells is normally explained by cell suicide mechanism, apoptosis. Interestingly, the decrease in the number of immune cells, such as non-CD4+ cells as well as CD4+ T cells, in HIV infection usually occurs in uninfected bystander cells, not in directly infected cells. It has, therefore, been suggested that several soluble factors, including viral protein R (Vpr), are released from the infected cells and induce the death of bystander cells. Some studies show that Vpr interacts directly with adenine nucleotide translocator (ANT) to induce mitochondrial membrane permeabilization (MMP). The MMP results in release of some apoptogenic factors such as cytochrome-c (cyt-c) and apoptosis-inducing factor (AIF). Vpr also has indirect effect on mitochondria through enhancing the level of caspase-9 transcription and suppressing nuclear factor-kappa B (NF-${\kappa}B$). The involvement of p53 in Vpr-induced apoptosis remains to be studied. On the other hand, low level of Vpr expression has anti-apoptotic effect, whereas it's high level of expression induces apoptosis. Extracellular Vpr also exhibits cytotoxicity to uninfected bystander cells through apoptotic or necrotic mechanism. The facts that Vpr has cytotoxic effect on both infected cells and bystander cells, and that it exhibits both proand anti-apoptotic activity may explain its role in viral survival and disease progression.

9-Meric Peptide Analogs of Defensin-like Antimicrobial Peptide Coprisin with Potent Antibacterial Activities with Bacterial Sell Selectivites

  • Shin, Areum;Lee, Eunjung;Kim, Jin-Kyoung;Bang, Jeong-Kyu;Kim, Yangmee
    • Bulletin of the Korean Chemical Society
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    • 제35권9호
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    • pp.2809-2812
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    • 2014
  • The 43-residue defensin-like peptide coprisin, which is isolated from dung bettle, Copris tripartitus, is a potent antimicrobial peptide. In our previous work, we determined the tertiary structure of coprisin and found that alpha helical region of coprisin from residue 19 to residue 30 is important for its antimicrobial activities. Here, we designed cop12mer and cop9mer analogs of coprisin based on the tertiary structure of coprisin. To investigate the relationship between hydrophobicity and antimicrobial activities and develop the potent peptide antibiotics, we designed cop9mer-1 with substitution of $His^2$ with Trp in cop9mer. The results showed that cop9mer-1 has higher toxicities as well as improved antimicrobial activities compared to cop9mer. In order to reduce the toxicity of cop9mer-1, we designed cop9mer-2 and cop9mer-3 with substitution of $Cys^3$ with Lys or Ser. Substitution of $Cys^3$ with these hydrophilic amino acids results in lower cytotoxicities compared to cop9mer-1. Cop9mer-2 with substitution of $Cys^3$ with Lys in Cop9mer-1 showed high antibacterial activities against drug resistant bacteria without cytotoxicity. Antibiotic action of cop9mer-1 analog appears to involve permeabilization of the bacterial cell membrane while cop9mer-2 and cop9mer-3 may have different mechanism of action. These results imply that that optimum balance in hydrophobicity and hydrophilicity in these 9-meric peptides plays key roles in their antimicrobial activities as well as cytotoxicities.

Development of Candida albicans Biofilms Is Diminished by Paeonia lactiflora via Obstruction of Cell Adhesion and Cell Lysis

  • Lee, Heung-Shick;Kim, Younhee
    • Journal of Microbiology and Biotechnology
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    • 제28권3호
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    • pp.482-490
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    • 2018
  • Candida albicans infections are often problematic to treat owing to antifungal resistance, as such infections are mostly associated with biofilms. The ability of C. albicans to switch from a budding yeast to filamentous hyphae and to adhere to host cells or various surfaces supports biofilm formation. Previously, the ethanol extract from Paeonia lactiflora was reported to inhibit cell wall synthesis and cause depolarization and permeabilization of the cell membrane in C. albicans. In this study, the P. lactiflora extract was found to significantly reduce the initial stage of C. albicans biofilms from 12 clinical isolates by 38.4%. Thus, to assess the action mechanism, the effect of the P. lactiflora extract on the adhesion of C. albicans cells to polystyrene and germ tube formation was investigated using a microscopic analysis. The density of the adherent cells was diminished following incubation with the P. lactiflora extract in an acidic medium. Additionally, the P. lactiflora-treated C. albicans cells were mostly composed of less virulent pseudohyphae, and ruptured debris was found in the serum-containing medium. A quantitative real-time PCR analysis indicated that P. lactiflora downregulated the expression of C. albicans hypha-specific genes: ALS3 by 65% (p = 0.004), ECE1 by 34.9% (p = 0.001), HWP1 by 29.2% (p = 0.002), and SAP1 by 37.5% (p = 0.001), matching the microscopic analysis of the P. lactiflora action on biofilm formation. Therefore, the current findings demonstrate that the P. lactiflora ethanol extract is effective in inhibiting C. albicans biofilms in vitro, suggesting its therapeutic potential for the treatment of biofilm-associated infections.

Lactobacillus plantarum 299v Surface-Bound GAPDH: A New Insight Into Enzyme Cell Walls Location

  • Saad, N.;Urdaci, M.;Vignoles, C.;Chaignepain, S.;Tallon, R.;Schmitter, J.M.;Bressollier, P.
    • Journal of Microbiology and Biotechnology
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    • 제19권12호
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    • pp.1635-1643
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    • 2009
  • The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

대두(Glycine max L.) 현탁배양 세포와 원형질체 내로의 외부 Calmodulin의 도입 (Introduction of Calmodulin into Suspension-Cultured Cells and Protoplasts of Soybean (Glycine max L.))

  • Hyun Sook CHAE;Kyu Chung HUR;In Sun YOON;Bin G. KANG
    • 식물조직배양학회지
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    • 제21권6호
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    • pp.363-367
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    • 1994
  • 식물 세포의 신호전달 기작에 있어 calmodulin (CaM)의 역할을 조사하기 위하여 외부기원의 CaM을 식물 세포 내로 주입하였다. 이를 위하여 bovine testis로부터 CaM을 분리, 정제하여 SDS-PAGE상에서 순수함을 확인하였고, 이를 세포 내로 주입한 후 실제 주입 여부를 확인하기 위해 정제된 CaM을 미리 fluorescein isothiocyanate (FITC)로 표식하여 사용하였다. 또한, FITC-CaM complex의 농도에 따른 fluorescent intensity를 측정하여 주입된 CaM의 농도를 결정하였다. 대두 현탁 배양 세포를 saponin (0.1 mg/mL)이 포함된 배지에서 15분간 배양하여 permeabilization시키고,FITC-CaM (1mg/mL)을 30분간 처리한 후 형광현미경으로 조사했을 때 세포질에서 강한 형광이 나타났으며, 이 결과로 외부의 calmodulin이 세포 내로 주입되었음을 확인할 수 있었다. 대두 현탁 배양 세포에 3가지의 세포벽 분해 효소를 처리하여 원형질체를 분리하였고, 이 원형질체를 FITC-CaM이 들어있는 완충용액에 넣고 electroporation 하였을때 capacitance와 field strength가 증가할수록 원형질체의 생존률은 감소하였으나, 세포 내의 fluorescent intensity는 증가하였다. 이것은 외부의 calmodulin이 electroporation에 의해 대두의 원형질체 내로 도입되었음을 의미하는 것이다.

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