• BMB Reports
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• v.40 no.5
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• pp.723-730
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• 2007

Kinesin is an ATP-driven microtubule motor protein that plays important roles in control of microtubule dynamics, intracellular transport, cell division and signal transduction. The kinesin superfamily is composed of numerous members that are classified into 14 subfamilies. Animal kinesins have been well characterized. In contrast, plant kinesins have not yet to be characterized adequately. Here, a novel plant-specific kinesin gene, GhKCH2, has been cloned from cotton (Gossypium hirsutum) fibers and biochemically identified by prokaryotic expression, affinity purification, ATPase activity assay and microtubule-binding analysis. The putative motor domain of GhKCH2, $M_{396-734}$ corresponding to amino acids Q396-N734 was fused with 6$\times$His-tag, soluble-expressed in E. coli and affinity-purified in a large amount. The biochemical analysis demonstrated that the basal ATPase activity of $M_{396-734}$ is not activated by $Ca^{2+}$, but stimulated 30-fold max by microtubules. The enzymatic activation is microtubule-concentration-dependent, and the concentration of microtubules that corresponds to half-maximum activation was about 11 ${\mu}M$, much higher than that of other kinesins reported. The cosedimentation assay indicated that $M_{396-734}$ could bind to microtubules in vitro whenever the nucleotide AMP-PNP is present or absent. As a plant-specific microtubule-dependent kinesin with a lower microtubule-affinity and a nucleotide-independent microtubule-binding ability, cotton GhKCH2 might be involved in the function of microtubules during the deposition of cellulose microfibrils in fibers or the formation of cell wall.

• BMB Reports
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• v.31 no.3
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• pp.274-280
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• 1998

An inositol monophosphatase (IMPase) was purified to homogeneity from rat duodenal mucosa for the first time and its enzymatic properties were investigated. Rat duodenal mucosa peculiarly exhibited the highest IMPase activity among various rat tissues examined. By means of ammonium sulfate precipitation, followed by Q-Sepharose, polylysine agarose, reactive-red agarose column chromatography, Uno-Q FPLC, and Bio-Silect FPLC, duodenal IMPase was purified 223-fold to a specific activity of 13.6 U/mg protein. The molecular mass of the native enzyme was estimated to be 48,000 Da on gel filtration. The subunit molecular mass was determined by SDS-PAGE to be 24,000 Da. These results indicate that duodenal IMPase is a dime ric protein made up of identical subunits. Rat duodenal IMPase has distinct properties from brain IMPase. It has a broad spectrum of substrate specificity and is insensitive to $Li^+$. Duodenal IMPase does not absolutely require $Mg^{2+}$ for its catalytic activity. Furthermore, duodenal IMPase is less stable to heat than brain enzyme. It is suggested that the rat duodenal mucosa needs a large amount of IMPase whose properties are quite different from that of the brain enzyme.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.3
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• pp.331-343
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• 1998

Sublethal dose of bacterial lipopolysaccharide (LPS) would induce protection against cardiac ischemic/reperfusion (I/R) injury. This study examines the following areas: 1) the temporal induction of the cardio-protection produced by LPS; and 2) the relations between a degree of protection and the myocardial prostacyclin ($PGI_2$) production. Rats were administered LPS (2 mg/kg, i.v.), and hearts were removed 1, 4, 8, 14, 24, 48, 72,and 96 h later. Using Langendorff apparatus, haemodynamic differences during 25 min of global ischemia/30 min reperfusion were investigated. The concentration of $PGI_2$ in aliquots of the coronary effluent was determined by radioimmunoassay as its stable hydrolysis product $6-keto-PGF1_{\alpha}$ and lactate dehydrogenase release were measured as an indicative of cellular injury. LPS-induced cardiac protection against I/R injury appeared 4 h after LPS treatment and remained until 96 h after treatment. $PGI_2$ release increased 2-3 fold at the beginning of reperfusion compared to basal level except in hearts treated with LPS for 48 and 72 h. In hearts removed 48 and 72 h after LPS treatment, basal $PGI_2$ was increased. To determine the enzymatic step in relation to LPS-induced basal $PGI_2$ production, we examined prostaglandin H synthase (PGHS) protein expression, a rate limiting enzyme of prostaglandin production, by using Western blot analysis. LPS increased PGHS protein expression in hearts at 24, 48, 72, 96 h after LPS treatment. Induction of PGHS expression appeared in both isotypes of PGHS, a constitutive PGHS-1 and an inducible PGHS-2. To identify the correlationship between $PGI_2$ production and the cardioprotective effect against I/R injury, indomethacin was administered in vivo or in vitro. Indomethacin did not inhibit LPS-induced cardioprotection, which was not affected by the duration of LPS treatment. Taken together, our results suggest that $PGI_2$ might not be the major endogenous mediator of LPS-induced cardioprotection.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.1
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• pp.1-14
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• 2014

During long standing hyperglycaemic state in diabetes mellitus, glucose forms covalent adducts with the plasma proteins through a non-enzymatic process known as glycation. Protein glycation and formation of advanced glycation end products (AGEs) play an important role in the pathogenesis of diabetic complications like retinopathy, nephropathy, neuropathy, cardiomyopathy along with some other diseases such as rheumatoid arthritis, osteoporosis and aging. Glycation of proteins interferes with their normal functions by disrupting molecular conformation, altering enzymatic activity, and interfering with receptor functioning. AGEs form intra- and extracellular cross linking not only with proteins, but with some other endogenous key molecules including lipids and nucleic acids to contribute in the development of diabetic complications. Recent studies suggest that AGEs interact with plasma membrane localized receptors for AGEs (RAGE) to alter intracellular signaling, gene expression, release of pro-inflammatory molecules and free radicals. The present review discusses the glycation of plasma proteins such as albumin, fibrinogen, globulins and collagen to form different types of AGEs. Furthermore, the role of AGEs in the pathogenesis of diabetic complications including retinopathy, cataract, neuropathy, nephropathy and cardiomyopathy is also discussed.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.3
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• pp.271-278
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• 2001

Oxidative stress and methylglyoxal (MG), a reactive dicarbonyl metabolites produced by enzymatic and non-enzymatic reaction of normal metabolism, induced aldose reductase (AR) expression in rat aortic smooth muscle cells (SMC). AR expression was induced in a time-dependent manner and reached at a maximum of 4.5-fold in 12 h of MG treatment. This effect of MG was completely abolished by cyclohemide and actinomycin D treatment suggesting AR was synthesized by de novo pathway. Pretreatment of the SMC with N-acetyl-L-cysteine significantly down-regulated the MG-induced AR mRNA. Furthermore, DL-Buthionine-(S,R)-sulfoximine, a reagent which depletes intracellular glutathione levels, increased the levels of MG-induced AR mRNA. These results indicated that MG induces AR mRNA by increasing the intracellular peroxide levels. Aminoguanidine, a scanvenger of dicarbonyl, significantly down-regulated the MG-induced AR mRNA. In addition, the inhibition of AR activities with statil, an AR inhibitor, enhanced the cytotoxic effect of MG on SMC under normal glucose, suggesting a protective role of AR against MG-induced cell damages. These results imply that the induction of AR by MG may contribute to an important cellular detoxification of reactive aldehyde compounds generated under oxidative stress in extrahepatic tissues.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.2
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• pp.165-175
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• 2001

Background: Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve $K_{ATP}$ channel activation. However, it was demonstrated that isoflurane inhibited $K_{ATP}$ channel activities in the inside-out patch mode. To explain this discrepancy, the present investigation tested the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurnae-induced cardioprotection via $K_{ATP}$ channel activation during myocardial ischemia and reperfusion. Methods: Single ventricular myocytes were isolated from rabbit hearts by an enzymatic dissociation procedure. Patch-clamp techniques were used to record single-channel currents. $K_{ATP}$ channel activities were assessed before and after the application of TFA with the inside-out patch mode. Results: TFA enhanced channel activity in a concentration-dependent fashion. The concentration of TFA for half-maximal activation and the Hill coefficient were 0.03 mM and 1.2, respectively. TFA did not affect the single channel conductance of $K_{ATP}$ channels. Analysis of open and closed time distributions showed that TFA increased burst duration and decreased the interburst interval without changes in open and closed time distributions shorter than 5 ms. TFA diminished ATP sensitivity of $K_{ATP}$ channels in a concentration-response relationship for ATP. Conclusions: TFA, a metabolite of isoflurane, enhanced $K_{ATP}$ channel activity in a concentration-dependent fashion. These results imply that TFA could mediate isoflurane-induced cardioprotection via $K_{ATP}$ channel activation during myocardial ischemia and reperfusion.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.4
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• pp.355-366
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• 1997

Although cerebellar Purkinje cells display spontaneous electrical activity in vivo and in slice experiments, the mechanism of the spontaneous activity generation has not been clearly understood. The aim of this study was to investigate whether cerebellar Purkinje cells of postnatal rats generate spontaneous electrical activity without synaptic inputs. Dissociated cerebellar Purkinje cells were used for reducing synaptic inputs in the present study. Cerebellar Purkinje cells with dendrites were dissociated from postnatal rats using enzymatic treatment followed by mechanical trituration. Spontaneous electrical activities were recorded from dissociated cells without any stimulus using whole-cell patch clamp configuration. Two types, spontaneously firing or quiescent, of dissociated Purkinje cells were observed in postnatal rats. Both types of cells were identified as Purkinje cells using immunocytochemical staining technique with anti-calbindin after recording. Spontaneously active cells displayed two patterns of firing, repetitive and burst firings. Two thirds of dissociated Purkinje cells displayed repetitive firing and the rest of them did burst firing under same recording condition. Repetitive firing activities were maintained even after further isolation using either physical or pharmacological techniques. Neither high magnessium solution nor excitatory synaptic blockers, AP-5 and DNQX, block the spontaneous activity. These results demonstrate that spontaneous electrical activity of isolated cerebellar Purkinje cells in postnatal rats is generated by intrinsic membrane properties rather than synaptic inputs.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.2
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• pp.149-156
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• 2013

Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with $Ca^{2+}$-free solution or thapsigargin (a $Ca^{2+}$-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external $Ca^{2+}$ influx and $Ca^{2+}$ release from internal stores in a PLC and PLD dependent manner.

• Biomolecules & Therapeutics
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• v.21 no.3
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• pp.196-203
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• 2013

Recent accumulating studies have reported that hypoxic preconditioning during ex vivo expansion enhanced the self-renewal or differentiation of various stem cells and provide an important strategy for the adequate modulation of oxygen in culture conditions, which might increase the functional bioactivity of these cells for cardiac regeneration. In this study, we proposed a novel priming protocol to increase the functional bioactivity of cardiac progenitor cells (CPCs) for the treatment of cardiac regeneration. Firstly, patient-derived c-$kit^+$ CPCs isolated from the atrium of human hearts by enzymatic digestion and secondly, pivotal target molecules identified their differentiation into specific cell lineages. We observed that hCPCs, in response to hypoxia, strongly activated ERK phosphorylation in ex vivo culture conditioning. Interestingly, pre-treatment with an ERK inhibitor, U0126, significantly enhanced cellular proliferation and tubular formation capacities of CPCs. Furthermore, we observed that hCPCs efficiently maintained the expression of the c-kit, a typical stem cell marker of CPCs, under both hypoxic conditioning and ERK inhibition. We also show that hCPCs, after preconditioning of both hypoxic and ERK inhibition, are capable of differentiating into smooth muscle cells (SMCs) and cardiomyocytes (CMs), but not endothelial cells (ECs), as demonstrated by the strong expression of ${\alpha}$-SMA, Nkx2.5, and cTnT, respectively. From our results, we conclude that the functional bioactivity of patient-derived hCPCs and their ability to differentiate into SMCs and CMs can be efficiently increased under specifically defined culture conditions such as short-term hypoxic preconditioning and ERK inhibition.

• Journal of Ginseng Research
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• v.48 no.1
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• pp.1-11
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• 2024

Fresh ginseng is prone to spoilage due to its high moisture content. For long-term storage, most fresh ginsengs are dried to white ginseng (WG) or steamed for hours at high temperature/pressure and dried to form Korean Red ginseng (KRG). They are further processed for ginseng products when subjected to hot water extraction/concentration under pressure. These WG or KRG preparation processes affect ginsenoside compositions and also other ginseng components, probably during treatments like steaming and drying, to form diverse bioactive phospholipids. It is known that ginseng contains high amounts of gintonin lysophosphatidic acids (LPAs). LPAs are simple lipid-derived growth factors in animals and humans and act as exogenous ligands of six GTP-binding-protein coupled LPA receptor subtypes. LPAs play diverse roles ranging from brain development to hair growth in animals and humans. LPA-mediated signaling pathways involve various GTP-binding proteins to regulate downstream pathways like [Ca²⁺]_i transient induction. Recent studies have shown that gintonin exhibits anti-Alzheimer's disease and antiarthritis effects in vitro and in vivo mediated by gintonin LPAs, the active ingredients of gintonin, a ginseng-derived neurotrophin. However, little is known about how gintonin LPAs are formed in high amounts in ginseng compared to other herbs. This review introduces atypical or non-enzymatic pathways under the conversion of ginseng phospholipids into gintonin LPAs during steaming and extraction/concentration processes, which exert beneficial effects against degenerative diseases, including Alzheimer's disease and arthritis in animals and humans via LPA receptors.