• International Journal of Oral Biology
• /
• 제39권4호
• /
• pp.229-236
• /
• 2014

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions ($O{_2}^{{\cdot}_-}$) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of $O{_2}^{{\cdot}_-}$ and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP ($10{\mu}M$) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-${\beta}$-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.

• 약학회지
• /
• 제41권5호
• /
• pp.615-621
• /
• 1997

Dehydropeptidase-I (DHP-I) was solubilized from porcine kidney by treatment with n-butanol and partially purified 19.25 fold by $(NH_4)_2SO_4$ precipitation, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-300 HR chromatography with an overall yield of 19.16. DHP-I showed its optimal activity at pH 7.5 and 25$^{\circ}C$. Its activity was stable under neutral and alkaline conditions, but was disappeared under acidic condition. And DHP-I was heat-labile and its activity remained at 45$^{\circ}C$ for 3hrs. The enzyme was not inhibited by dicationic ions, while its activity was increased by $Co^{2+}$(1mM) and $Zn^{2+}$ (0.1mM). The enzyme was inhibited by EDTA and N-ethylmaleimide. The relative molecular mass of DHP-I was estimated to be approximately 100kDa by gel filtration chromatography. The $K_m$ value of DHP-I for glycyldehydrophenylalanine (GDHP) was 1.98mM. DWP20418 [(1R, 5S, 6S)-6-[1-(R)-Hydroxyethyl]-1-methyl-2-[(2S, 4S)-2-(piperazinylcarbonyl)-1-(R)-hydroxyethyl)pyrrolidine-4-thio]carbapen-2-em-3-carboxylic acid], compared with meropenem (MEPM), was rather easily hydrolized by DHP-I, while it was four times more resistant than imipenem (IPM) to DHP-I.

• 미생물학회지
• /
• 제23권3호
• /
• pp.177-183
• /
• 1985

20-80%로 황산암모늄 분획한 효소액을 사용하여 Arthrobacter sp. JH-13균주가 생산하는 세포외 cytosme deaminase의 성질을 검토하였다. 검토한 기질 중, 본 효소는 cytosine과 5- f1uorocytosine을 기질로 이용하였으며, 효소의 활성에 대한 최적 pH는 8.0부근이었고, 최적 온도는 $40^{\circ}C$부근으로 나타났다. 본 효소는 0.2M의 p potassium phosphate 완충액 (pH 8.0) 보다 0.2M의 tris-HCl 완충액 (pH 8. 0) 에서 더욱 안정하였다. 온도에 대한 안정성을 검토한 결과. $50^{\circ}C$ 부근까지는 대체로 안정하였으나, $70^{\circ}C$에서는 완전히 활성을 잃었다. 또한 ImM의 $Fe^{3+},\;K^+\;Na^+$ 이온은 효소의 활성을 증가시켰으나 0.01mM의 $Co^{2+},\;Cu^{2+},\;Ni^{2+},\;Hg^{2+},\;Ag^{2+},\;Zn^{2+},\;Ba^{2+},\;Mg^{2+}$ 이온들은 효소의 활성올 강력하게 저해하였다. O.lmM의 p-mercuribenzoate, trichloroacetic acid, N-ethylmaleim mide등은 효소의 활성을 완전히 저해하였으며. O.lmM의 2-mercaptoethanol은 효소의 활성올 약간 증가시키는 것으로 나타났다.

• The Korean Journal of Physiology and Pharmacology
• /
• 제1권3호
• /
• pp.225-231
• /
• 1997

As it has been reported that the depolarization-induced norepinephrine (NE) release is modulated by activation of presynaptic $A_1-adenosine$ heteroreceptor and various lines of evidence indicate the involvement of adenylate cyclase system in $A_1-adenosine$ post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the $A_1-receptor-mediated$ control of NE release in this study. Slices from rat hippocampus were equilibrated with $[^3H]-NE$ and the release of the labelled products was evoked by electrical stimulation.(3 Hz, $5Vcm^{-1}$, 2 ms, rectangular pulses). The influence of various agents on the evoked tritium-outflow was investigated. $N^6-Cyclopentyladenosine$ (CPA), a specific $A_1-adenosine$ receptor agonist, in concentrations Tanging from 0.1 to $10{\mu}M$ decreased the $[^3H]-NE$ release in a dose-dependent mauler without any change of basal rate of release. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, $2{\mu}M$), a selective $A_1-receptor$ antagonist, inhibited the CPA effect. The responses to N-ethylmaleimide $(3&10{\mu}M)$, a SH-alkylating agent of G-protein, were characterized by increments of the evoked NE-release and the CPA effects were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.1 to $30{\mu}M$ increased the evoked and basal rate of NE release in a dose-dependent manner and the CPA effects were inhibited by forskolin pretreatment. Rolipram $(1&10{\mu}M)$, a phosphodiesterase inhibitor, did not affect the evoked NE release but reduced the CPA effect. And 8-bromo-cAMP $(100&300{\mu}M)$, a membrane permeable cAMP analogue inhibited the CPA effect significantly. These results suggest that the $A_1-adenosine$ heteroreceptor plays an important role in NE-release via nucleotide-binding protein $G_i$ in the rat hippocampus and that the adenylate cyclase system might be participated in this process.

• The Korean Journal of Physiology and Pharmacology
• /
• 제1권3호
• /
• pp.303-313
• /
• 1997

The role of the lower esophageal sphincter(LES) is characterized by the ability to maintain tone and to relax allowing the passage of a bolus. It is known that LES relaxation during swallowing may be induced by the cessation of the tonic neural excitation and the activation of non-adrenergic, non-cholinergic(NANC) inhibitory neurons. Furthermore, it is generally accepted that the relaxation of the smooth muscle is mediated primarily by the elaboration of adenosine 3',5'-cyclic monophosphate(cyclic AMP) and guanosine 3',5'-cyclic mono-phosphate(cyclic GMP) via activation of adenylate cyclase and guanylate cyclase, respectively. It is thus possible that cyclic nucleotides might be a second messenger involved in neural stimulation-induced relaxation of LES, although a relationship between relaxation and changes in cyclic nucleotides after neural stimulation has not been established. The present study was performed to define the participation of cyclic nucleotides in the relaxation of LES of dog in response to neural stimulation. Electrical field stimulation(EFS) caused relaxation of the canine isolated LES strips in a frequency-dependent manner, which was eliminated by pretreatment with tetrodotoxin$(1{\mu}M)$, but not by atropine$(100{\mu}M)$, guanethidine$(100{\mu}M)$ and indomethacin$(10{\mu}M)$. The nitric oxide synthase inhibitors, $N^G-nitro-L-arginine$, $N^G-nitro-L-arginine$ methyl ester and $N^G-monomethyl-L-arginine$ inhibited EFS-induced relaxation. Additions of sodium nitroprusside, a nitrovasodilator and forskolin, a direct adenylate cyclase stimulant, caused a dose-dependent relaxation of LES smooth muscle. Effects of sodium nitroprusside and forskolin were selectively blocked by the corresponding inhibitors, methylene blue for guanylate cyclase and N-ethylmaleimide(NEM) for adenylate cyclase, respectively. Dibutyryl cyclic AMP and dibutyryl cyclic GMP caused a concentration-dependent relaxation of the LES smooth muscle tone, which was not blocked by NEM or methylene blue, respectively. However, both NEM and methylene blue caused significant antagonism of the relaxation in LES tone in response to EFS. EFS increased the tissue cyclic GMP content by 124%, whereas it did not affect the tissue level of cyclic AMP. Based on these results, it is suggested that one of the components of canine LES smooth muscle relaxation in response to neural stimulation is mediated by an increase of cyclic GMP via the activation of guanylate cyclase. Additionally, an activation of cyclic AMP generation system was, in part, involved in the EFS-induced relaxation.

• Korean Journal of Acupuncture
• /
• 제29권2호
• /
• pp.260-270
• /
• 2012

Objectives : Hippocampus, a region of temporal lobe, plays an important role in the pathogenic mechanisms of brain diseases such as Alzheimer's disease, depression and temporal lobe epilepsy. This research is designed to investigate hippocampal changes after acupuncture stimulation at Shinmun(HT7) using 2-dimensional gel electrophoresis(2-DE). Methods : On postnatal-day 15, rat pups were randomly devided into Normal(NOR) or HT7 group. All of Pups kept with their mothers for 7 days, but pups in HT7 group received acupuncture stimulation at HT7 daily. On postnatal-day 21, hippocampus of each rat pup was dissceted 30 minutes after last acupuncture stimulation and the protein expressions were investigated using 2-DE. Results : After acupuncture stimulation at HT7, expression of 20 proteins were significantly increased. Succinate semialdehyde dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase-like, transketolase, aconitate hydratase and phosphoglucomutase-1 were related to glucose methabolism. Eukaryotic initiation factor(eIF) 4A-II, eIF 4A-III, mitochondrial Tu translation elongation factor and chain A of crystal structure of the 70-Kda heat shock cognate protein involve in the protein synthesis in ribosome. Tubulin ${\beta}$-4 chain, tubulin T ${\beta}$-15 and tubulin ${\alpha}$-1B chain comprise cytoskeleton. Glutathione S-transferase(GST) ${\omega}$-1, GST P and GST Yb-3 can reduce oxidative stress. ${\beta}$-soluble N-ethylmaleimide-sensitive fusion protein attachment protein is required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus, glycerol-3-phosphate dehydrogenase plays a major role in lipid biosynthesis, creatine kinase U-type catalyses the conversion of creatine and consumes adenosine triphosphate to create phosphocreatine and adenosine diphosphate. Platelet-activating factor acetylhydrolase IB subunit alpha and voltage depedent anion-selective channel protein 2 were also increased. Conclusions : The results suggest that acupuncture stimulation at HT7 may enhance glucose and lipid metabolism, protein synthesis, cytoskeletal substance and anti-oxidative stress in hippocampus.

• Natural Product Sciences
• /
• 제26권3호
• /
• pp.207-213
• /
• 2020

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn²⁺, Na²⁺, Ba²⁺ and Ni ²⁺ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca²⁺, Mg²⁺ and Zn²⁺ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H₂O₂. The activity was increased by dithiothreitol up to a concentration of 80 μM and inactivated at 140 μM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 μL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

• The Korean Journal of Physiology and Pharmacology
• /
• 제8권3호
• /
• pp.141-146
• /
• 2004

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins, composed of two presynaptic membrane proteins [synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin] and a presynaptic vesicular protein [vesicle-associated membrane protein (VAMP)], serve as a core of exocytotic fusion machinery, which can be affected by ischemia. Synaptic protein in core region, striatum and cortex has been shown to alter after focal ischemia, however, little is known in hippocampus. Hippocampus is remote from ischemic core, but it is one of the most vulnerable regions. Using immunohistochemistry, the present study was undertaken to investigate the alteration of expression of SNAP-25, syntaxin, and VAMP in the hippocampus of rats which were subjected to middle cerebral artery occlusion (MCAO) for 2h and allowed to reperfuse. At 2 weeks of reperfusion, the SNAP-25 and syntaxin immunoreactivity was increased in the stratum oriens of the CA1 and the stratum lucidum of the CA3 in the ipsilateral hippocampus. However, VAMP immunoreactivity didn't show significant change. These results demonstrate that the level of the presynatpic plasma membrane proteins (SNAP-25 and syntaxin) in the rat hippocampus is more sensitively affected by focal ischemia than that of the synaptic vesicle protein (VAMP).

• Journal of Nutrition and Health
• /
• 제39권4호
• /
• pp.323-330
• /
• 2006

Peripheral insulin resistance in obese/type II diabetes animals results from an impairment of insulin-stimulated glucose uptake into skeletal muscle. Insulin stimulate the translocation of GLUT4 from intracellular location to the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) is implicated in mediation of fusion of GLUT4-containing vesicle with the plasma membrane. Present study investigated regulatory effects of Rhodiola sachalinensis administration and exercise training on the expression of GLUT4 protein and SNAREs protein in skeletal muscles of obese Zucker rats. Experimental animals were randomly assigned into one of five groups ; lean control(LN), obese control(OB), exercise-treated(EXE), Rhodiola sachalinensis-treated(Rho), combine of Rho & EXE (Rho-EXE). All animals of exercise training (EXE, Rho-EXE) performed treadmill running for 8 weeks, and animals of Rho groups (Rho, Rho-EXE) were dosed daily by gastric gavage during the same period. After experiment, blood were taken for analyses of glucose, insulin, and lipids levels. Mitochondrial oxidative enzyme (citrate synthase, CS ; $\beta$-hydroxyacyl-CoA dehydrogenase, $\beta$-HAD) activity were analysed. Skeletal muscles were dissected out for analyses of proteins (GLUT4, VAMP2, syntaxin4, SNAP23). Results are as follows. Exercise and/or Rhodiola sachalinensis administration significantly reduced body weight and improved blood lipids (TG, FFA), and increased insulin sensitivity. Endurance exercise significantly increased the activity of mitochondrial enzymes and the expression of GLUT4 protein, however, administration of Rhodiola sachalinensis did not affect them. The effect of exercise and/or Rhodiola sachalinensis administration on the expression of SNARE proteins was unclear. Our study suggested that improvement insulin sensitivity by exercise and/or Rhodiola sachalinensis administration in obese Zucker rats is independent of expression of SNARE proteins.

• 생약학회지
• /
• 제35권3호통권138호
• /
• pp.250-254
• /
• 2004

Helicobacter pylori (H. pyroli) infection it associated with type B gastritis, peptic uler disease, and gastric cancer. The vacuolation of cells induced by H. pylori is thought to be essential for the initiation and maintenance of gastric infection. The roles of H. pylori cytotoxin, urease, and ammonia in the vacuolation of HeLa cells were determined. H. pylori toxin induced vacuolation of HeLa cells. Korean and Japanese radishes significantly prevented the vacuolation of HeLa cells induced by H. pylori toxin. The urease activity in vacuolated cells was also decreased with Korean and Japanese radishes. H. Pylori toxin-induced vacuolation was inhibited by vacuolar type ATPase inhibitors (bafilomycin and N-ethylmaleimide). However, further investigation is required to determine the mechanisms of radish for the inhibition of vacuole formation of eukaryotic cells in response to the H. pylori toxin.