• IMMUNE NETWORK
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• v.9 no.5
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• pp.192-202
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• 2009

Background: Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages. Methods: Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors. Results: Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-${\gamma}$ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-${\kappa}B$ and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-${\gamma}$ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3. Conclusion: These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.208-208
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• 1996

In this work we investigated coupling of the m2 and m4 subtypes of muscarinic acetylcholine receptors expressed in chinese hamster ovary (CHO) cells to activation of neuronal nitric oxide synthase (nNOS). Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an index of generation of nitric oxide (NO) in CHO cells. The agonist carbachol induced marked time and concentration-dependent enhancement of the activity of nNOS at m2 receptors. In sharp contrast, the response in CHO cells transfected with the m4 receptor gene was similar in magnitude to that observed in non-transfected cells, suggesting lack of significant coupling of m4 muscarinic receptors to NO signaling. This novel observation of functional divergence of the two muscarinic receptor subtypes at the level of activation of nNOS is quite intriguing, in light of the currently accepted dogma that they belong to the same functional class. This functional selectivity was not due to differential effects on intracellular Ca$\^$2+/ concentration, since activation of both subtypes of muscarinic receptors produced a comparable, albeit quite small, Ca$\^$2+/ signal. Taken together, our present data strongly suggest that the generally assumed functional equivalence of m2 and m4 muscarinic receptors should be carefully reexamined. These data also suggest the presence of alternate mechanisms of activation of nNOS, which might be operative in the absence of large changes in the concentration of cellular Ca$\^$2+/. The latter mechanisms are expected to be activated by m2, but not m4 muscarinic receptors. Both sets of findings are quits important in regards to refining the functional classification of muscarinic receptor subtypes and the cellular mechanisms of activation of NOS.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.206-206
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• 1996

The C-terminus ends of the second putative transmembrane domains of both m1 and m2 muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y) and threonine (T), This triplet is repeated as LYT-LYT in m2 receptors at the interface between the second transmembrane domain and the first extracellular loop. Interestingly, however, it is repeated in a transposed fashion (LYT-TYL) in the sequence of m1 receptors. In this work we employed site-directed mutagenesis to investigate the possible significance of this unique sequence diversity for determining the distinct differential drug-receptor interaction and cellular function at m1 muscarinic receptor. Mutation of the LYTTYL sequence of m1 receptors to the corresponding m2 receptor LYTLYT sequence, however, did not result in a significant change in the binding affinity of the agonist carbachol or in the affinity of the majority of a series of receptor antagonists which are able to discriminate between wild-type m1 and m2 receptors. Surprisingly, the LYTLYT ml receptor mutant demonstrated markedly enhanced coupling to activation of phospholipase C without a change in its coupling to increased cyclic AMP formation. There was also an enhanced receptor sensitivity in transducing elevation of intracellular Ca$\^$2+/. These changes were not due to alterations in the rate of receptor. desensitization or sequestration, On the other hand, the reverse LYTLYT-LYTTYL mutation in the m2 receptor did not alter its coupling to inhibition of adenylate cyclase, but slightly enhanced its coupling to stimulation of PI hydrolysis, Our data suggest that the LYTTYL/LYTLYT sequence difference between ml and n12 muscarinic receptors is not involved in determining receptor pharmacology. On the other hand, while these differences might play a role in the modulation of muscarinic receptor coupling to PI hydrolysis, they are not important for specifying coupling of various subtypes of muscarinic receptors to different cellular signaling pathways.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.928-937
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• 2016

S-Nitrosoglutathione reductase (GSNOR) metabolizes S-nitrosoglutathione (GSNO) and has been shown to play important roles in regulating cellular signaling and formulating host defense by modulating intracellular nitric oxide levels. The enzyme has been found in bacterial, yeast, mushroom, plant, and mammalian cells. However, to date, there is still no evidence of its occurrence in filamentous fungi. In this study, we cloned and investigated a GSNOR-like enzyme from the filamentous fungus Aspergillus nidulans. The enzyme occurred in native form as a homodimer and exhibited low thermal stability. GSNO was an ideal substrate for the enzyme. The apparent K_m and k_cat values were 0.55 mM and 34,100 min^-1, respectively. Substrate binding sites and catalytic center amino acid residues based on those from known GSNORs were conserved in this enzyme, and the corresponding roles were verified using site-directed mutagenesis. Therefore, we demonstrated the presence of GSNOR in a filamentous fungus for the first time.

• Journal of Pharmacopuncture
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• v.19 no.2
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• pp.129-136
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• 2016

Objectives: The crude extracts of Scutellaria barbata D. Don (SB) have traditionally demonstrated inhibitory effects on numerous human cancers both in vitro and in vivo. Gastric cancer is one of the most common types of cancer on world. The authors investigated the effects of an ethanol extract of Scutellaria barbata D. Don (ESB) on the growth and survival of MKN-45 cells (a human gastric adenocarcinoma cell line). Methods: The MKN-45 cells were treated with different concentrations of ESB, and cell death was examined using an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Analyses of sub-G1 peaks, caspase-3 and -9 activities, and mitochondrial membrane depolarizations were conducted to determine the anti-cancer effects of SB on MKN-45 cells. Also, intracellular reactive oxygen species (ROS) generation was investigated. Results: ESB inhibited the growth of MKN-45 cells, caused cell cycle arrest, and increased the sub-G1 population. In addition, ESB markedly increased mitochondrial membrane depolarization and the activities of caspase-3 and -9. ESB exerted anti-proliferative effects on MKN-45 cells by modulating the mitogen-activated protein kinase (MAPK) signaling pathway and by increasing the generation of ROS. Furthermore, combinations of anti-cancer drugs plus ESB suppressed cell growth more than treatments with an agent or ESB, and this was especially true for cisplatin, etoposide, and doxorubicin. Conclusion: ESB has a dose-dependent cytotoxic effect on MKN-45 cells and this is closely associated with the induction of apoptosis. ESB-induced apoptosis is mediated by mitochondria-, caspase- and MAPK dependent pathways. In addition, ESB enhances ROS generation and increases the chemosensitivity of MKN-45 cells. These results suggest that treatment with ESB can inhibit the proliferation and promote the apoptosis of human gastric adenocarcinoma cells by modulating the caspase-, MAPK- and ROS-dependent pathway.

• The Journal of Korean Medicine
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• v.21 no.4
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• pp.193-203
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• 2000

Objectives : Hindered barrier function of vascular endothelium has been implicated in the initiation and progression of degenerative vascular diseases such as atherosclerosis. In this study, the effect of Sunghyangchungisan(SHCS) as a protectant against oxidant-induced destruction of endothelial barrier function was assessed. Methods : Toward this end, endothelial cells derived from the human umbilical vein were cultured as monolayers on permeable membrane filters. Endothelial permeability was monitored by measuring transendothelial electrical resistance and movement of low density lipoprotein (LDL) across the endothelial monolayer. Results : Along with increased movement of LDL, $H_2O_2$-induced increase in endothelial permeability was paralleled by a decrease in transendotheliaI electrical resistance. The effect of $H_2O_2$ was mimicked by phorbol 12-myristate 13-acetate (PMA), a potent activator of proteinkinase C. Calphostin-C, a protein kinase C inhibitor, effectively blocked the increase in endothelial permeability induced by $H_2O_2$ or PMA, indicating that activation of protein kinase C is associated with the $H_2O_2-induced$ permeability change. SHCS effectively protected the endothelial monolayer against $H_2O_2-induced$ increase in permeability, whereas, it did not affect PMA-induced change. Forskolin, a potent activator of adenylyl cyclase, antagonized $H_2O_2$ to increase endothelial permeability. In addition, in ${H_2O_2}-treated$ cens, intracenular cAMP concentration was significantly decreased, indicating that impaired cAMP production as well as activation of proteinkinase C is a mechanism underlying ${H_2O_2}>-induced$$H_2O_2$ with regard to its effect on intracellular cAMP content. However, SHCS itself did not affect resting cAMP concentration in endothelial cells. Conclusions : These results suggest that SHCS might operate as an effective protectant against oxidant-induced destruction of endothelial barrier function. The mechanism does not appear to involve direct interaction with protein kinase C- or cAMP-associated signaling mechanism.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.7
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• pp.1156-1168
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• 2007

The somatotropic (GH-IGF-I) axis consists of many hormonal and nutritional factors that control GH release from the somatotrophs in the anterior pituitary. The GH-releasing substances are GHRH and GHS (GHRP or ghrelin), while the GH release-inhibiting substances are somatostatin (SRIF), insulin-like growth factor-I (IGF-I), leptin and glucocorticoids. However, there is evidence showing that nutrition is involved in the control of the somatotropic axis. In addition, weaning is a drastic event for neonates because their alimentary and endocrine circumstances are changed due to the switch, even if gradual, from a liquid milk diet to one composed of such solids as hay and grains. The biological role of ghrelin is one of the hormonal factors that have been focused on ever since ghrelin was discovered at the end of the last century. A 27-amino acid peptide that is mainly synthesized and released from the abomasum epithelium, ghrelin has not been fully evaluated in relation to the somatotropic axis of the ruminant. It has also proven difficult even to investigate the cellular mechanisms of ghrelin action, because this hormone exerts animal-species-dependent actions via a complex set of intracellular signaling pathways. This is also the case for the action of leptin. Another substance, IGF-I, shows a partial inhibitory action on GH secretion in the ruminant. The effect of nutrition is also different among animal species. This is evident by the fact that undernutrition suppresses the circulating GH levels in rodents, but increases it in ruminants and humans. Recently, weaning has been shown to change the postprandial GH responses in ruminants; milk feeding increases, but hay and concentrate feeding suppress, the postprandial circulating GH levels. Even if the postprandial GH level is increased, the ghrelin level is decreased by milk feeding. Macronutrients also possess stimulatory and inhibitory actions on GH secretion in vivo and in vitro. These findings indicate the complexity of the control mechanisms of the somatotropic axis. In the present review, we summarize recent findings on the factors controlling the axis of the ruminant.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.31-37
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• 2020

We previously identified a new heparinase inhibitor fungal metabolite, named CRM646-A, which showed inhibition of heparinase and telomerase activities in an in vitro enzyme assay and antimetastatic activity in a cell-based assay. In this study, we elucidated the mechanism by which CRM646-A rapidly induced nucleus condensation, plasma membrane disruption and morphological changes by increasing intracellular Ca²⁺ levels. Furthermore, PD98059, a mitogen-activated protein kinase (MEK) inhibitor, inhibited CRM646-A-induced nucleus condensation through ERK1/2 activation in rat 3Y1 fibroblast cells. We identified CRM646-A as a Ca²⁺ ionophore-like agent with a distinctly different chemical structure from that of previously reported Ca²⁺ ionophores. These results indicate that CRM646-A has the potential to be used as a new and effective antimetastatic drug.

• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1184-1194
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• 2020

Melanin is a major factor that darkens skin color as one of the defense systems to prevent the harmful effects of UV light. However, darkened skin from the localized or systemic accumulation of melanin is viewed in many cultures as an esthetic problem. Consequentially, searching for anti-melanogenic agents from natural sources is very popular worldwide. Previous screening of fermented rice products, obtained from various rice cultivars fermented with different sources of loog-pang (Thai traditional fermentation starter), revealed that the highest ability to reduce the melanin content in B16F10 melanoma cells was from unpolished black rice fermented with a defined starter mixture of microbes isolated from loog-pang E11. The aim of this study was to investigate the mechanism of the fermented unpolished black rice (FUBR) on the inhibition of melanogenesis in B16F10 melanoma cells. The strongest reduction of cellular melanin content was found in the FUBR sap (FUBRS). The melanin reduction activity was consistent with the significant decrease in the intracellular tyrosinase activity. The FUBRS showed no cytotoxic effect to B16F10 melanoma or Hs68 human fibroblast cell lines. It also significantly reduced the transcript and protein expression levels of tyrosinase, tyrosinase-related protein 1 (TYRP-1), TYRP-2, and microphthalmia-associated transcription factor. Furthermore, it induced a significantly increased level of phosphorylated ERK, p38 and Akt signaling pathways, which likely contributed to the negative regulation of melanogenesis. From these results, a model for the mechanism of FUBRS on melanogenesis inhibition was proposed. Moreover, these results strongly suggested that FUBRS possesses anti-melanogenesis activity with high potential for cosmeceutical application as a skin depigmenting agent.

• Journal of Korean Neurosurgical Society
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• v.42 no.5
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• pp.392-399
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• 2007

Objective : Arsenic trioxide ($As_2O_3$) has been used as an anticancer agent in traditional Chinese medicine for thousand years and berberine is an isoquinoline alkaloid present that has indicated significant antimicrobial activity. We have examined the combined anticancer effects of $As_2O_3$ and berberine against the human neuroblastoma (HNB) SH-SY5Y cells in vitro, and to elucidate underlying molecular mechanism. Methods : HNB SH-SY5Y cells were treated with $2\;{\mu}M\;As_2O_3$ and $75\;{\mu}g/ml$ berberine, and their survival, cell death mechanism as well as synergistic cytotoxic effects were estimated by using MTT assay, DAPI staining, agarose gel electrophoresis, flow cytometric analysis, and western blot analysis. Results : The combined treatment of two drugs also markedly decreased cell viability. The cytotoxic effects of two drugs were revealed as apoptosis characterized by chromatin condensation, DNA fragmentation, and the loss of mitochondrial membrane potential. The apoptotic cytotoxicity was accompanied by activation of caspase-3 protease as well as decreased the expression of Bcl-2, Bid, and Bcl-x/L. In addition, the cells treated with combination of two drugs also showed significantly increased intracellular reactive oxygen species levels and lipid peroxidation compared to cells $As_2O_3$or berberine only. Conclusion : Combined treatment of $As_2O_3$ with berberine induced activation of apoptotic signaling pathways in HNB SH-SY5Y cells. These results suggest that the possibility of the combined treatment of two chemotherapeutic agents with low concentration improving cytotoxic effect for cancer cells with minimal side effects.