• Biomolecules & Therapeutics
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• v.15 no.2
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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.

• Molecules and Cells
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• v.39 no.1
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• pp.46-52
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• 2016

It is increasingly apparent that nature evolved peroxiredoxins not only as $H_2O_2$ scavengers but also as highly sensitive $H_2O_2$ sensors and signal transducers. Here we ask whether the $H_2O_2$ sensing role of Prx can be exploited to develop probes that allow to monitor intracellular $H_2O_2$ levels with unprecedented sensitivity. Indeed, simple gel shift assays visualizing the oxidation of endogenous 2-Cys peroxiredoxins have already been used to detect subtle changes in intracellular $H_2O_2$ concentration. The challenge however is to create a genetically encoded probe that offers real-time measurements of $H_2O_2$ levels in intact cells via the Prx oxidation state. We discuss potential design strategies for Prx-based probes based on either the redoxsensitive fluorophore roGFP or the conformation-sensitive fluorophore cpYFP. Furthermore, we outline the structural and chemical complexities which need to be addressed when using Prx as a sensing moiety for $H_2O_2$ probes. We suggest experimental strategies to investigate the influence of these complexities on probe behavior. In doing so, we hope to stimulate the development of Prx-based probes which may spearhead the further study of cellular $H_2O_2$ homeostasis and Prx signaling.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.1006-1012
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• 2023

In this study, we investigated the effects of sodium propionate (SP) treatment on intracellular mechanism of murine macrophages and its contribution to host immunity during Brucella abortus 544 infection. The intracellular growth assay revealed that SP inhibited Brucella replication inside the macrophages. To determine intracellular signaling involved during SP treatment after Brucella infection, we analyzed the change of five different cytokines production relevant to SP such as TNF-α, IL-10, IFN-γ, IL-1β, and IL-6, and the results indicated that the boost with IL-10 was apparent throughout the culture period for 48 h as well as IL-1β which was apparent at 24 h post-infection and IFN-γ which was apparent at 24 h and 48 h in comparison to SP untreated groups. On the other way, SP-treated cells displayed suppressed production of TNF-α and IL-6 at all time points tested and 48 h post-infection, respectively. Furthermore, we conducted western blot to establish a cellular mechanism, and the result suggested that SP treatment attenuated p50 phosphorylation, part of the NF-κB pathway. These findings indicated that the inhibitory effect of SP against Brucella infection could be attributed through induction of cytokine production and interference on intracellular pathway, suggesting SP as a potential candidate for treating brucellosis.

• Applied Biological Chemistry
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• v.36 no.6
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• pp.539-546
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• 1993

This paper describes the purification and partial characteristics of aminopeptidase from Microccus sp. LL3 to utilize the microorganism as a potential agent for industrial application for the purpose of shortening ripening period of cheddar cheese. The optimal temperature and pH for enzyme activity were $35^{\circ}C$ and 7.0, respectively for L-leucine-p-nitroanilide as substrate. The enzyme remained stable for 10 minutes up to $50^{\circ}C$. The activity of aminopeptidase was stimulated by $Mg^{++}$ ion but strongly inhibited by $Hg^{++}$, metal complexing reagents, ethylenediaminetetraacetate (EDTA) and 1,10-phenanthroline. The enzyme was thought to be metallopeptidase. This enzyme had a broad substrate specificity, but was inactive on peptide with arginine as N-terminal amino acid. An intracellular aminopeptidase from Micrococcu sp. LL3 was purified by chromatography on DEAE-Sephacel and filtration on Sepacryl S-300. The enzyme has a molecular weight of 43,500.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1452-1456
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• 2016

Overexpression of the genes encoding phosphoeneolpyruvate carboxykinase (pckA) and NAD-dependent malic enzyme (maeA) facilitates higher intracellular ATP and NAD(P)H concentrations, respectively, under aerobic conditions in Escherichia coli. To verify a hypothesis that higher intracellular energy reserves might contribute to H₂ fermentation, wild-type E. coli strains overexpressing pckA and maeA were cultured under anaerobic conditions in a glucose minimal medium. Overexpression of pckA and maeA enabled E. coli to produce 3-times and 4-times greater H₂ (193 and 284 nmol, respectively) than the wild type (66 nmol H₂). The pckA and maeA genes were further overexpressed in a hydrogenase-3-enhanced E. coli strain. The hydrogenase-3-enhanced strain (W3110+fhlA) produced 322 nmol H₂, whereas the ATP-enhanced strain (W3110+fhlA+pckA) produced 50% increased H₂ (443 nmol). Total H₂ in the NAD(P)H-enhanced strain (W3110+fhlA+maeA) was similar to that in the control strain at 319 nmol H₂. Possible explanations for the contribution of the increased cellular energy reserves to the enhanced hydrogen fermentation observed are discussed based on the viewpoint of metabolic engineering strategy.

• Journal of Chest Surgery
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• v.43 no.4
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• pp.345-355
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• 2010

Background: Extracellular and intracellular pH ($pH_o$ and $pH_i$), which can be changed in various pathological conditions such as hypoxia, affects vascular contractility. To elucidate the mechanism to alter vascular contractility by pH, the effects of pH on reactivity to vasocontracting agents, intracellular $Ca^{2+}$ influx, and $Ca^{2+}$ sensitivity in vascular smooth muscle were examined. Material and Method: Isometric contractions in rat superior mesenteric arteries (SMA) were observed. Intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) was recorded by microfluorometer using Fura-2/acetoxylmethyl ester in muscle cells. $pH_o$ was increased from 7.4 to 7.8 or decreased to 6.9 or 6.4. $pH_i$ was decreased by applying $NH_4^+$ or propionic acid or modulated by changing $pH_o$ after increasing membrane permeability using $\beta$-escin. Result: Decreases in $pH_o$ from 7.4 to 6.9 or 6.4 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the right and significantly increased half maximal effective concentration (EC50) to NE or SE. Increase in $pH_o$ from 7.4 to 7.8 shifted concentration-response curve by norepinephrine (NE) or serotonin (SE) to the left and significantly reduced EC50 to NE or SE. NE increased $[Ca^{2+}]_i$ in cultured smooth muscle cells from SMA and the increased $[Ca^{2+}]_i$ was reduced by decreases in $pH_o$. NE-induced contraction was inhibited by $NH_4^+$, whereas the resting tension was increased by $NH_4^+$ or propionic acid. When the cell membrane of SMA was permeabilized using ${\beta}$-escin, SMA was contracted by increasing extracellular $Ca^{2+}$ concentration from 0 to $10{\mu}M$ and the magnitude of contraction was decreased by a decrease in $pH_o$ and vice versa. Conclusion: From these results, it can be concluded that a decrease in $pH_o$ might inhibit vascular contraction by reducing the reactivity of vascular smooth muscle to vasoactive agents, $Ca^{2+}$ influx and the sensitivity of vascular smooth muscle to $Ca^{2+}$.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.3
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• pp.139-142
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• 2002

To examine intracellular signaling of human $S1P_3\;(hS1P_3),$ a sphingosine 1-phosphate (S1P) receptor in plasma membrane, $hS1P_3$ DNA was transfected into RH7777 rat hepatoma cell line, and the inhibition of forskolin-induced cAMP accumulation and activation of MAP kinases by S1P were tested. In $hS1P_3$ transformants, S1P inhibited forskolin-induced activation of adenylyl cyclase activity by about 80% and activated MAP kinases in dose-dependent and pertussis-toxin (PTX) sensitive manners. In oocytes expressing $hS1P_3$ receptor, S1P evoked $Cl^-$ conductance. These data suggested that PTX-sensitive G proteins are involved in $hS1P_3-mediated$ signaling, especially the positive action of S1P in cell proliferation. The potential advantages of rat hepatoma cells for the research of sphingosine 1-phosphate receptor are discussed.

• Reproductive and Developmental Biology
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• v.41 no.4
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• pp.65-70
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• 2017

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that intracellular reactive oxygen species (ROS) in mammalian oocytes were reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing $200{\mu}m$ $H_2O_2$ or $H_2O_2$ with $50{\mu}m$ ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress state, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II (MII) stage development was significantly reduced in $H_2O_2$ group compared to non-treated control group $61.84{\pm}1.42%$ and 80.00%, respectively; p<0.05) and it was slightly recovered by treatment of ALA ($69.76{\pm}1.67%$; p<0.05). The intracellular GSH levels was decreased in $H_2O_2$ groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, $H_2O_2$ treatment increased intracellular ROS level in oocytes and $H_2O_2$-induced ROS was decreased by treatment of ALA (p<0.05). Our findings suggested that ALA treatment under oxidative stress condition improve oocyte maturation via elevated GSH and reduced ROS levels in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

• Journal of Ginseng Research
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• v.25 no.1
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• pp.53-58
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• 2001

To clarify a significant difference between red-colored phenomena (RCP) and microbes isolated from rhizosphere soil of healthy ginseng (HES) and red-colored ginseng (RCS), we have examined growth and cellulase activities of the microbes according to pH variation and iron status. The soil microbes could not grow at pH 3.0 on the YEB medium. The growth of bacterium isolated from RCG at pH from 5.0 to 9.0 showed small differences and the growth of bacterium HES was lower than that of others. The growth of bacteria from RCS and surface soil (SUS) at pH 5.0 were also lower than that of pH 7.0 and pH 9.0. However, the bacteria isolated from red-colored ginseng (RCG) and RCS are able to grow on the medium contained 2 mM Fe$\^$3+/ at pH 3.0. Furthermore, the growth of bacterium from RCG increased about two times in the medium contained iron at pH 7.0 compared with minus iron. The cellulase activity of isolated bacteria increased two times in the medium contained 2 mM Fe$\^$3+/ compared with minus iron. The activity of extra-cellular cellulase was higher by one hundred times than that of intracellular level. The cellulase activity of the bacterium from RCS at pH 5.0 was higher by two times than that of pH 7.0. Especially, intracellular activity of the bacterium from RCS on the medium contained 2mM Fe$\^$3+/ increased about six to seven times compared with control (minus iron). Also, extra-cellular activity increased about eleven to twelve times compared with control. These results indicate that the soil microbes seem to be related iron redoxidation by proton extrusion and with cell wall digestion by secreted cellulase.