• IMMUNE NETWORK
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• 제10권5호
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• pp.164-172
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• 2010

Background: We tested the hypothesis that dengue haemorrhagic fever (DHF) is associated with a $T_H1$-skewed immune response as opposed to dengue fever (DF). Methods: We estimated intracellular (in T-cells) and serum levels of designate $T_H1/T_H2$ cytokines [interferon-${\gamma}$ (IFN-${\gamma}$), interleukin-4 (IL-4), and tumor necrosis factor-${\alpha}$] and macrophage inflammatory protein-$1{\alpha}$ (MIP-$1{\alpha}$) at admission, 48h, and day 5 in 20 adults with dengue (DF=10, DHF=10) and 10 dengue-naive healthy controls. Results: At admission, intracellular IFN-${\gamma}$/IL-4 ratio in CD4+ T-cells and proportion of MIP-$1{\alpha}$-positive CD8+ T-cells were significantly higher in patients with DHF [7.21 (5.36~10.81) vs. 3.04 (1.75~4.02); p=0.011 and 6.2% (3.2~8.2%) vs. 2.4% (2.0~3.6%); p=0.023]. The latter showed a significant positive correlation with IFN-${\gamma}$/IL-4 ratio in CD4+ T-cells (Spearman's rho=0.64; p=0.003), percentage-change in haematocrit (rho=0.47; p=0.048), and serum alanine amino-transferase level (rho=0.61; p=0.009). Conclusion: We conclude that DHF is associated with a $T_H1$-skewed immune response. Further, MIP-$1{\alpha}$ in CD8+ T-cells is an important immunologic correlate of disease severity in dengue.

• Mycobiology
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• 제37권2호
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• pp.121-127
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• 2009

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was $45^{\circ}C$ and the highest activity was exhibited in 35 to $45^{\circ}C$. The enzyme lost their activities almost completely (95${\sim}$100%) at $80^{\circ}C$ or above and as well as bellow $25^{\circ}C$.

• The Korean Journal of Physiology and Pharmacology
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• 제12권4호
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• pp.177-183
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• 2008

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and $[Ca^{2+}]_c$ of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH ($pH_e{\leq}5.5$) activated outwardly rectifying $Cl^-$ current ($I_{Cl,pH}$) with slow kinetics of voltage-dependent activation. $I_{Cl,pH}$ was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1${\mu}$M). $I_{Cl,pH}$ became more sensitive to $pH_e$ by raising temperature from $24^{circ}C$ to $37^{circ}C$. HaCaT cells also expressed $Ca^{2+}$-activated $Cl^-$ current ($I_{Cl,Ca}$), and the amplitude of $I_{Cl,Ca}$ was increased by relatively weak acidic $pH_e$ (7.0 and 6.8). Interestingly, the acidic $pH_e$ (5.0) also induced a sharp increase in the intracellular [$Ca^{2+}$] (${\triangle}[Ca^{2+}]_{acid}$) of HaCaT cells. The ${\triangle}[Ca^{2+}]_{acid}$ was independent of extracellular $Ca^{2+}$, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed ${\triangle}[Ca^{2+}]_{acid}$. In summary, we found $I_{Cl,pH}$ and ${\triangle}[Ca^{2+}]_{acid}$ in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.

• 대한소아치과학회지
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• 제25권2호
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• pp.352-367
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• 1998

Intracellular pH (pHi) plays an important role in the regulation of cellular processes by influencing the acitivity of various enzymes in cells. Therefore, almost every type of mammalian cell possesses an ability to regulate its pHi. One of the most prominent mechanisms in the regulation of pHi is $Na^+/H^+$ exchanger. This exchanger has been known to be activated when cells are stimulated by the binding of agonist to the muscarinic receptors. Therefore, the aims of this study were to compare the rates of $H^+$ extrusion through $Na^+/H^+$ exchanger before and during muscarinic stimulation and to investigate the possible existence of $HCO_3^-$ transporter which is responsible for the continuous supply of $HCO_3^-$ ion to saliva. Acinar cells were isolated from the rat mandibular salivary glands and loaded with pH-sensitive fluoroprobe, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein(BCECF), for 30min at room temperature. Cells were attached onto the coverglass in the perfusion chamber and the changes in pHi were measured on the iverted microscope using spectrofluorometer. 1. By switching the perfusate from $HCO_3^-$-free to $HCO_3^-$-buffered solution, pHi decreased by $0.39{\pm}0.02$ pH units followed by a slow increase at an initial rate of $0.04{\pm}0.007$ pH units/min. The rate of pHi increase was reduced to $0.01{\pm}0.002$ pH units/min by the simultaneous addition of 1 mM amiloride and $100{\mu}M$ DIDS. 2. An addition and removal of $NH_4^+$ caused a decrease in pHi which was followed by an increase in pHi. The increase of pHi was almost completely blocked by 1mM amiloride in $HCO_3^-$-free perfusate which implied that the pHi increase was entired dependent on the activation of $Na^+/H^+$ exchanger in $HCO_3^-$-free condition. 3. An addition of $10{\mu}M$ carbachol increased the initial rate of pHi recovery from $0.16{\pm}0.01$ pH units/min to $0.28{\pm}0.03pH$ units/min. 4. The initial rate of pHi decrease induced by 1mM amiloride was also increased by the exposure of the acinar cells to $10{\mu}M$ carbachol ($0.06{\pm}0.008pH$ unit/min) compared with that obtained before carbachol stimulation ($0.03{\pm}0.004pH$ unit/min). 5. The intracellular buffering capacity ${\beta}1$ was $14.31{\pm}1.82$ at pHi 7.2-7.4 and ${\beta}1$ increased as pHi decreased. 6. The rate of $H^+$ extrusion through $Na^+/H^+$ exchanger was greatly enhanced by the stimulation of the cells with $10{\mu}M$ carbachol and there was an alkaline shift in the activity of the exchanger. 7. An intrusion mechanism of $HCO_3^-$ was identified in rat mandibular salivary acinar cells. Taken all together, I observed 3-fold increase in $Na^+/H^+$ exchanger by the stimulation of the acinar cells with $10{\mu}M$ carbachol at pH 7.25. In addition, I have found an additional mechanism for the regulation of pHi which transported $HCO_3^-$ into the cells.

• The Korean Journal of Physiology
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• 제28권2호
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• pp.169-179
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• 1994

The effect of extracellular and intracellular pH on vascular tone and $^{45}Ca$ uptake were investigated in aortic strips and dispersed single aortic smooth muscle cells of spontaneously hypertensive rats (SHR) and aged-matched Wistar-Kyoto rats (WKY). The contraction produced by a change of extracellular pH (pHo) in the range of $6.5{\sim}8.3$ was estimated by comparison with the level of vascular tone at pH 7.4. Contraction was induced below pHo 6.5 in WKY, pHo 7.1 in SHR, and over pHo 8.0 on both strains. The amplitude of contraction induced by high pHo (over pHo 7.7) was similar in SHR and WKY, but that induced by low pHo (below pHo 7.1) in SHR was greater than that in WKY. Either high pHo- or low pHo-induced contractions in WKY and SHR were not induced in the Ca-free Tyrode's solution and were induced by the addition of Ca. $^{45}Ca$ uptake increased progressively as pHo was increased from 6.8 to 8.1 in the single aortic smooth muscle cells of WKY and SHR. $NH_4Cl$ induced a gradually developing contraction in a dose-dependent manner $(5\;mM{\sim}30\;mM)$ and the removal of $NH_4Cl$ induced transient contraction was followed by profound relaxation in the aortic rings of both strains. The contractions induced by $NH_4Cl$ or by the removal of $NH_4Cl$ in SHR were significantly greater than that in WKY. These contractions were not induced in Ca-free Tyrode's solution. $^{45}Ca$ uptake was increased by $NH_4Cl$ (20 mM) and was not changed by the removal of $NH_4Cl$ (20 mM) in the aortic strips of WKY and SHR. As a summary of above results, the vascular tone of SHR was more sensitive to the change pHi and pHo than that of WKY. The contractions induced by change of extracellular or intracellular pH depended on extracellular Ca in the aorta of SHR nnd WKY. However, the Ca uptake was in accord with the changes of contraction but increase in contraction by low pH was not accompanied by an increase in Ca uptake in both strains.

• Tuberculosis and Respiratory Diseases
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• 제53권1호
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• pp.5-16
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• 2002

연구배경 : 말초혈액 단핵구와 폐포 대식세포가 림프구에 의하여 활성화된 후 탐식된 세포내 결핵균을 제거하는 최종 효과기로 작용하지만 결핵균은 이러한 세포 내에서조차 살아남아 증식한다. 본 연구에서는 인체 감염의 경우와 유사하도록 낮은 감염률(결핵균:포식세포 1:10)을 이용하여 MAC(Mycobacterium avium)과 Mycobaterium tuberculosis $H_{37}Ra$에 대한 단핵구와 폐포 대식세포의 림프구 의존적 결핵균 살해능의 정도를 측정하고자 하였다. 방 법 : 정상인과 폐결핵 환자의 말초혈액 단핵구를 Ficoll gradient 방법을 이용하여 분리하고, 정상인의 폐포 대식세포를 기판지폐포 세척액에서 분리 하여 96well plate에 $1{\times}10^5$ 씩 분주하고 결핵균주 MAC과 $H_{37}Ra$에 낮은 감염률(결핵균:단핵구 1:10)로 감염시켰다. 일부는 비부착세포(림프구)를 림프구:포식세포 10:1 비율로 첨가하여 $37^{\circ}C$, 5% $CO_2$ 배양기에 배양하였다. 1일, 4일, 및 7일째 각각 수확하여 middlebrook 7H10/OADC agar plate에 접종 후 집락이 형성될 때까지 $37^{\circ}C$, 5% $CO_2$ 배양기에 배양하였다. 결핵균의 수는 lysate의 $m{\ell}$(세포수 $10^6$)당 CFU로 표시하였고, 결핵균의 살해능은 log killing ratio[logKR=$log_{10}$(Final CFU/Initial CFU)]로 표시하였다. 배양 1일째 상층액에서 TNF-${\alpha}$, 배양 4일째 상층액에서 ${\gamma}$-IFN을 Elisa kit를 사용하여 측정하였다. 결 과 : 단핵구의 세포내 결핵균 살해능은 정상대조군에 비하여 결핵환자군에서 ${\Delta}logKR$가 MAC -0.4, $H_{37}Ra$ -0.2 정도로 유의하게 증가되어 있었다(p<0.05). 정상대조군에서 폐포 대식세포의 결핵균 살해능은 단핵구에 비하여 ${\Delta}$logKR가 MAC과 $H_{37}Ra$ 모두 -0.2정도로 증가된 경향을 보였다. 단핵구와 폐포 대식세포내 결핵균 살해능은 림프구 의존적으로 림프구 첨가시의 ${\Delta}logKR$는 MAC -0.4, $H_{37}Ra$ -0.5 정도로 포식세포 단독에 비하여 유의하게 증가되었다 (p<0.05). ${\gamma}$-IFN은 말초 단핵구와 폐포 대식세포 단독배양시 분비량이 경미하였으나 림프구 첨가시에는 유의한 분비의 증가를 보였다. 결 론 : 본 연구는 낮은 결핵균 감염률을 이용한 인체 포식세포의 세포내 결핵균 감염 모델이다. 결핵균 살해능은 림프구 의존적이며, 포식세포의 결핵균 탐식 후 7일까지의 배양에서 결핵환자의 단핵구와 PPD 양성 정상인의 폐포 대식세포에 림프구를 첨가한 경우에만 실질적인 균 증식의 제한을 볼 수 있었다.

• Clinical and Experimental Pediatrics
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• 제46권1호
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• pp.56-66
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• 2003

목 적 : 제대정맥은 모체의 혈액을 태아로 운반하여 산소와 영양물질을 공급하는 유일한 통로이다. 이러한 제대 혈류의 장애가 있을 시 자궁내 성장제한, 임신성 고혈압 등을 초래할 수 있다. 제대-태반 혈관의 내피세포 손상을 유발하는 원인 중 amiloride 유도체들을 중심으로 내피세포에 미치는 amiloride 유도체들의 작용을 밝히고, 세포 내 이온농도 변화와 apoptosis 간의 관계를 규명하고자 하였다. 방 법 : 인간 제대정맥 내피세포는 Clonetics로부터 구입하였으며, 내피세포 성장에 필요한 여러 성장인자가 포함된 배지에서 배양하였다. MTT 방법과 flow cytometry 방법을 이용하여 세포독성 효과 및 apoptosis를 확인하였다. 세포 내 이온농도 변화를 측정하기 위해서는 각각의 목적에 적합한 형광염료들을 세포 내에 부하시켜 놓은 뒤, 형광 현미경과 연결된 영상분석장치를 이용하여 관찰하였다. 결 과 : 1) Amiloride 유도체들은 농도 의존적으로 HUVEC의 사멸을 나타내었으며, 각각의 정도는 HMA($IC_{50}$; $11.2{\mu}M$), MIA($13.6{\mu}M$)>EIPA($30.8{\mu}M$)>>amiloride($106{\mu}M$) 순이었다. 2) 세포주기 분석결과 apoptosis의 특징적인 sub $G_0/G_1$ ploidy peak를 나타냈으며, 이러한 작용은 caspase 억제제에 의해 감소되었다. 3) Annexin-V와 propidium iodide 이중 염색 결과 apoptosis로 진행된 세포의 비율(73.0%)을 대조군(11.3%)에 비해 현저히 증가시켰다. 4) HMA에 의한 apoptosis 효과는 세포외액의 pH를 높이거나, $NH_4Cl$을 투여하여 세포내액의 pH를 높일 경우 크게 증가하였다. 5) HMA는 농도 의존적으로 세포내 주요 이온인 $K^+$ 및 $Cl^-$의 세포내 농도를 감소시켰으며, 이러한 효과 역시 세포외액의 pH를 높일수록 현저하게 증가하였다. 결 론 : 이상의 결과들로 미루어 볼 때, HUVEC에서 amiloride 유도체들에 의한 apoptosis 과정에 세포내 주요 이온 농도 감소가 일부 관여하고 있을 것이라 생각된다.

• 대한약리학회지
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• 제31권1호
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• pp.39-51
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• 1995

갑상선 기능 항진증 심장에서의 ${\beta}-adrenoceptor$의 역할은 잘 알려져 있으나 ${\alpha}-adrenoceptor$에 대해서는 알려져 있지 않은 바, 저자 등은 갑상선 기능 항진증 기니픽 심장의 유두근에서 일반 미세 전극과 이온-선택적 미세 전극을 이용하여 세포내 $Na^+$과 $H^+$ 활성도에 대한 phenylephrine의 영향을 연구하였다. Phenylephrine ($10^{-5}$ 또는 $3{\times}10^{-5}M$)에 의한 ${\alpha}_1-adrenoceptor$ 자극은 다양한 활동전위의 변동, 수축기 막전위의 과분극 ($1.5{\pm}0.1mM$), 세포내 활성도 증가 ($0.4{\pm}0.15mM$), 현저한 수축력 증가 ($220{\pm}15%$) 그리고 세포내 pH의 증가 ($0.06{\pm}0.002\;unit$)를 일으켰고, 이와 같은 변동이 prazosin과 atenolol에 의해 차단되었다. 그래서 이들 효과가 ${\alpha}_1-adrenoceptor$를 경유함을 알 수 있었고, 역시, 세포내 $Na^+$ 활성도와 수축력 증가 효과가 $Na^{+}-H^{+}$ 교환기 억제제인 ethylisopropylamiloride로 차단됨으로 보아 ${\alpha}_1-adrenoceptor$ 자극은 $Na^{+}-H^{+}$ 교환기를 자극하여 세포내 $a^{i}_{Na}$와 pH를 증가시킴을 시사한다. 이는 ${\alpha}_1-adrenoceptor$ 자극에 의한 세포내 $Na^+$감소와 초기 수축력 감소 효과를 나타내는 정상 기니픽 심장과는 매우 다른 결과로 갑상선 기능 항진증 심장에서 ${\alpha}_1-adrenoceptor$는 매우 중요한 기능을 갖고 있음을 의미한다.

• 한국미생물·생명공학회지
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• 제34권4호
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• pp.318-322
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• 2006

알칼리와 고온에 대한 내성을 가진 invertase를 대량생산하기 위하여 호알칼리성이며 고온성 세균인 Bacillus sp. TA-11의 세포내 invertase유전자를 pUC 19벡터를 이용하여 E. coli HB101에 클로닝 시키고 발현시켜 invertase를 강력하게 생산하는 재조합 E. coli (pYC17)를 얻었다. 재조합 E. coli(pYC17)를 이용한 invertase 생산 최적조건을 검토한 결과 E. coil(pYC17)를 0.25% sucrose, 0.5% yeast extract, 0.1% $K_2HPO_4$와 0.1% $KH_2PO_4$을 함유한 SY배지(초기 pH 9.0)에 접종하여 37$^{\circ}C$에서 9시간 배양하였을 때 친주보다도 많은 47.7 U/ml-cell free extract의 invertase가 생산되었다.

• Biomolecules & Therapeutics
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• 제15권2호
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• pp.73-77
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• 2007

N,N-Dimethyl-D-ribo-phytosphingosine (DMPH) is an N-methyl derivative of sphingosine. In the present paper, we studied effects of DMPH on intracellular Ca$^{2+}$ concentration, pH, glutamate uptake, and cell viability in human 1321N1 astrocytes. DMPH increased intracellular Ca$^{2+}$ concentration and cytosolic pH significantly in a dose-dependent manner. DMPH also inhibited glutamate uptake by 1321N1 astrocytes. Finally, treatment of cells with DMPH for 24 h reduced viability of cells largely and concentration-dependently. In summary, DMPH increased intracellular Ca$^{2+}$ concentration and pH, inhibited glutamate uptake and evoked cytotoxicity in 1321N1 astrocytes. Our observations with DMPH in the 1321N1 astrocytes would enhance understanding of DMPH actions in the brain.