• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.143-147
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• 2008

The sodium bicarbonate cotransporter (NBC) protein is functionally expressed in salivary glands. In this experiment, we examined the role of NBC in $HCO_3^-$ formation in human parotid gland acinar cells. Intracellular pH (pHi) was measured in 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded cells. Acetazolamide (0.1 mM) and 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS, 0.5 mM) were used as specific inhibitors of carbonic anhydrase and NBC, respectively. The degree of inhibition was assessed by measuring the pHi recovery rate (${\Delta}pHi$/min) after cell acidification using an ammonium prepulse technique. In control experiments, ${\Delta}pHi$/min was $1.40{\pm}0.06$. Treatment of cells with 0.5 mM DIDS or 0.1 mM acetazolamide significantly reduced ${\Delta}pHi$/min to $1.14{\pm}0.14$ and $0.74{\pm}0.15$, respectively. Simultaneous application of DIDS and acetazolamide further reduced ${\Delta}pHi$/min to $0.47{\pm}0.10$. Therefore, DIDS and acetazolamide reduced ${\Delta}pHi$/min by 19% and 47%, respectively, while simultaneous application of both DIDS and acetazolamide caused a reduction in ${\Delta}pHi$/min of 67%. These results suggest that in addition to carbonic anhydrase, NBC also partially contributes to $HCO_3^-$ formation in human parotid gland acinar cells.

• Journal of Physiology & Pathology in Korean Medicine
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• v.20 no.5
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• pp.1211-1216
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• 2006

The effect of a chronic programme of either low- or moderate-to-high-intensity treadmill running on the activation of the Extracellular-signal regulated protein kinase (ERK1/2), Phosphorylated ERK 1/2(pERK1/2) and the Phosphorylated c-Jun N-terminal kinase(pJNK) pathways was determined in rat Back skin Hair follicle. Sprague-Dawley rats were assigned to one of three groups: (i) sedentary group(NE; n=10); (ii) low-intensity exercise group (Bm/min; LIE; n=10); and (iii) moderate-high-intensity exercise group(28m1min; HIE; n=10). The training regimens were planned so that animals covered the same distance and had similar utilization for both LIE and HIE exercise sessions. The report runs as follows; A single bout of LIE or HIE following 4 weeks of exercise led to a twofold increase in the phosphorylation of ERK2, pERK2 and a threefold increase in pJNKl, pERKl. ERKI phosphorylation in LIE Back skin sampled and pJNK2 in HIE Back skin sampled 48h after the last exercise bout was similar to sedentary values, while pJNK2 phosphorylation in LIE Back skin sampled was 70-80% lower than sedentary. 48h after the last exercise bout of LIE or HIE increased ERK2, pERKl and pJNKl expression, with the magnitude of this increase being independent of prior exercise intensity or duration. PERK1/2, pJNKl expression was increased Three- to fourfold in Back skin Hair follicle sampled 48h after the last exercise bout irrespective of the prior exercise programme, but ERKI expression in HIE Back skin sampled was approximately 90% lower than sedentary values. In conclusion, exercise-training of different jntensities/durations results in selective postexercise activation of intracellular signal pathways, which may be one mechanism regulating specific adaptations induced by diverse training programmes.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.555-561
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• 2020

TWIK-related two-pore domain K⁺ channel-2 (TREK-2) has voltage-independent activity and shows additional activation by acidic intracellular pH (pH_i) via neutralizing the E³³² in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP₂) via the alkaline K³³⁰ and triple Arg residues (R^355-357); inhibition and activation, respectively. The G³³⁴ between them appeared critical because its mutation (G³³⁴A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K³³⁰ (K³³⁰A). To further elucidate the role of putative bent conformation at G³³⁴, we compared the dual mutation forms, K³³⁰A/G³³⁴A and G³³⁴A/R^355-7A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G³³⁴A owes to a dominant influence from R^355-7. Since there are additional triple Arg residues (R^377-9) distal to R^355-7, we also examined the triple mutant (G³³⁴A/R^355-7A/R^377-9A) that showed tonic inhibition same with G³³⁴A/R^355-7A. Despite the state of tonic inhibition, the activation by acidic pH_i was preserved in both G³³⁴A/R^355-7A and G³³⁴A/R^355-7A/R^377-9A, similar to the R^355-7A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pH_i in R^355-7A, G³³⁴A/R^355-7A, and G³³⁴A/R^355-7A/R^377-9A. These results suggest that the putative bent conformation at G³³⁴ is important to set the tug-of-war between K³³⁰ and R^355-7 in the PIP₂-dependent regulation of TREK-2.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.267-276
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• 2020

T In the present study, we investigated the effect of oncogenic H-Ras on rat mdr1b expression in NIH3T3 cells. The constitutive expression of H-Ras^V12 was found to downregulate the mdr1b promoter activity and mdr1b mRNA expression. The doxorubicin-induced mdr1b promoter activity of the H-Ras^V12 expressing NIH3T3 cells was markedly lower than that of control NIH3T3 cells. Additionally, there is a positive correlation between the level of H-Ras^V12 expression and a sensitivity to doxorubicin toxicity. To examine the detailed mechanism of H-Ras^V12-mediated down-regulation of mdr1b expression, antioxidant N-acetylcysteine (NAC) and NADPH oxidase inhibitor diphenylene iodonium (DPI) were used. Pretreating cells with either NAC or DPI significantly enhanced the oncogenic H-Ras-mediated down-regulation of mdr1b expression and markedly prevented doxorubicin-induced cell death. Moreover, NAC and DPI treatment led to a decrease in ERK activity, and the ERK inhibitors PD98059 or U0126 enhanced the mdr1b-Luc activity of H-Ras^V12-NIH3T3 and reduced doxorubicin-induced apoptosis. These data suggest that Ras^V12 expression could downregulate mdr1b expression through intracellular reactive oxygen species (ROS) production, and ERK activation induced by ROS, is at least in part, contributed to the downregulation of mdr1b expression.

• Microbiology and Biotechnology Letters
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• v.26 no.3
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• pp.232-237
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• 1998

A bacterial strain producing high levels of an extracellular ${eta}$-mannanase and intracellular ${eta}$-mannosidase and ${alpha}$-galactosidase was isolated from soil. The strain isolated was identified as a strain of Sporolactobacillus sp. and designated as Sporolactobacillus sp. M20l. Synthesis of ${eta}$-mannanase by Sporolactobacillus sp. M20l was induced by sucrose, maltose, or locust bean gum. The highest induction rate was obtained with 2% locust bean gum added to the culture medium as a sole carbon source. On the other hand, induction of ${eta}$-mannosidase was observed only with locust bean gum. The optimal media for the enzyme production were established as follows: for ${eta}$-mannanase; 2% locust bean gum, 0.5% peptone, 0.2% KH$_2$PO$_4$, 80 mg/l MgSO$_4$, and 8 mg/l ZnSO$_4$ (pH 6.0), and for ${eta}$-mannosidase; 2% locust bean gum, 0.5% yeast extract, 0.2% KH$_2$PO$_4$, 80 mg/l MgSO$_4$, and 8 mg/l ZnSO$_4$ (pH 5.0). The optimal culture temperatures for production of ${eta}$-mannanase and ${eta}$-mannosidase were found to be 37$^{\circ}C$ and 3$0^{\circ}C$, respectively. Under the optimal culture conditions, the production of ${eta}$-mannanase and ${eta}$-mannosidase reached the highest levels of 10.6 units/ml and 1.35 units/ml after 30 h and 24 h cultivation, respectively.

• Journal of Microbiology and Biotechnology
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• v.19 no.4
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• pp.351-357
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• 2009

Natural wild-type strains of Bacillus subtilis are extensively used in agriculture as biocontrol agents for plants. This study examined two antagonist B. subtilis strains, KB-1111 and KB-1122, and the results illustrated that KB-1122 was a more potent inhibitor of the indicator pathogen than KB-1111. Thus, to investigate the intrinsic differences between the two antagonist strains under normal culture conditions, samples of KB-1111 and KB-1122 were analyzed using MALDI-TOF-MS. The main differences were related to 20 abundant intracellular and 17 extracellular proteins. When searching the NCBI database, a number of the differentially expressed proteins were identified, including 11 cellular proteins and 10 secretory proteins. Among these proteins, class III stress-response-related ATPase, aconitate hydratase, alpha-amylase precursor, and a secretory protein, endo-l, 4-beta-glucanase, were differentially expressed by the two strains. These results are useful to comprehend the intrinsic differences between the antagonism of KB-1111 and KB-1122.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.524-528
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• 1989

A $\beta$-Galactosidase producing strain, Alkalophilic Bacillus sp, YS-309, has been isolated from soil sample. The strain was capable of producing large amount of intracellular $\beta$-galactosidase in the alkaline media rather than in the neutral media. The preferable medium composition has been determined to be as follows: 0.5% lactose, 0.5% yeast extract, 0.5% soybean meal, 0.1% KH$_2$PO$_4$, 0.02% MgSO$_4$7$H_2O$ 0,0.6% Na$_2$CO$_3$ (pH 9.9). The enzyme was produced by lactose or IPTG as in-ducer. But both Enzyme synthesis and cellular growth were decreased when lactose was added at the higher concentrations than 1.5% (v/v).

• BMB Reports
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• v.40 no.6
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• pp.1009-1015
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• 2007

In this communication, we report the efficacy of $\beta$-carotene towards differentiation and apoptosis of leukemia cells. Dose ($20{\mu}M$) and time dependence (12 h) tests of $\beta$-carotene showed a higher magnitude of decrease (significance p < 0.05) in cell numbers and cell viability in HL-60 cells than U937 cells but not normal cell like Peripheral blood mononuclear cell (PBMC). Microscopical observation of $\beta$-carotene treated cells showed a distinct pattern of morphological abnormalities with inclusion of apoptotic bodies in both leukemia cell lines. When cells were treated with $20{\mu}M$ of $\beta$-carotene, total genomic DNA showed a fragmentation pattern and this pattern was clear in HL-60 than U937 cells. Both the cell lines, on treatment with $\beta$-carotene, showed a clear shift in $G_1$ phase of the cell cycle. In addition the study also revealed anti-oxidant properties of $\beta$-carotene since there was reduction in relative fluorescent when treated than the control at lower concentration. Collectively this study shows the dual phenomenon of apoptosis and differentiation of leukemia cells on treatment with $\beta$-carotene.

• Bulletin of the Korean Chemical Society
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• v.31 no.11
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• pp.3241-3246
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• 2010

NHERF ($Na^+/H^+$ exchanger regulatory factor) and E3KARP (NHE3 kinase A regulatory protein) play important roles in membrane targeting, trafficking and sorting of ion channels, transmembrane receptors and signaling proteins in many tissues. Each of these proteins contains two PDZ (PSD-95/Dlg-1/ZO-1) domains, which mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The interaction between NHERF and E3KARP was investigated by surface plasmon resonance spectroscopy (BIAcore), fluorescence measurement, His-tagged pull-down experiment, and size-exclusion column (SEC) chromatography. BIAcore experiments revealed that NHERF bound to E3KARP with an apparent $K_D$ of 7 nM. Fluorescence emission spectra of the NHERF-E3KARP complex suggested that the tight interaction between these proteins was accompanied by significant conformational changes in one or both. The CD spectra of NHERF and E3KARP show that the conformational changes of these proteins were dependent on pH and temperature. These results implicate that the NHERF-E3KARP complex allows intracellular signaling complexes to form through PDZ-PDZ interactions.