Seoul Lee

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.

Reabsorption of neutral amino acids mediated by amino acid transporter LAT2 and TAT1 in the basolateral membrane of proximal tubule.

In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.

Regulation of contractile activity by magnolol in the rat isolated gastrointestinal tracts.

This study examined the pharmacological property of magnolol, a phenolic compound purified from Magnolia officinalis, on the GI motility using the rat isolated gastrointestinal (GI) strips. Magnolol (0.3-30 microM) dose-dependently stimulated the tone and amplitude of spontaneous contractions in ileum longitudinal muscles. Magnolol at 3 microM significantly increased the contractions of jejunum longitudinal and colon circular muscles, but not the longitudinal muscle contractions in fundus, antrum and colon. Pretreatment of ileum strips with either atropine (0.5 microM) or 4-diphenyllacetoxy-N(2-chloriethyl)-piperidine (4-DAMP, 0.5 microM) dramatically inhibited the acetylcholine (ACh, 0.1 microM)- and magnolol (3 microM)-induced longitudinal muscle contractions, but they were not affected by methoctramine (0.5 microM) and hexamethonium (0.5 microM). Ondansetron (0.1 microM) and GR113808 (2 microM) significantly reduced the tone of ileum longitudinal muscle contractions stimulated by 5-HT (10 microM), but not the amplitude. Magnolol (3 microM)-induced ileum longitudinal muscle contractions, both tone and amplitude, were significantly blocked by GR113808, but not by ondansetron. Taken together, magnolol differently regulates the spontaneous GI motility according to the region of GI tracts and orientation of smooth muscles, and magnolol-induced regulation of smooth muscle contractions in rat GI strips is likely to be mediated, at least in part, by activation of ACh and 5-HT receptors, possibly the M(3) and/or 5-HT(4) receptors.

Hexane extract of Poncirus trifoliata (L.) Raf. stimulates the motility of rat distal colon

AIM OF THE STUDY Poncirus trifoliata (L.) Raf. (Rutaceae, PT) has been commonly used for treating gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of colonic motility have not been clarified. This study investigated the regulatory effects of PT on the colonic motility. MATERIALS AND METHODS Immature fruits of PT were sequentially partitioned with MeOH, n-hexane, CHCl(3), EtOAc, n-BuOH and H(2)O, and the effects of PT extracts on the contractility of colonic strips and colonic luminal transit in rats were measured in vitro and in vivo, respectively. RESULTS Among six different extracts, only hexane extract of PT (PTHE) dose-dependently increased the low frequency contraction of longitudinal muscle in distal colonic strips, and the ED(50) value was revealed to be 0.71 microg/ml. The contractile patterns induced by PTHE were remarkably different from those caused by acetylcholine (ACh) and serotonin (5-HT). The stimulatory effects of PTHE on the whole distal colonic strips were more prominent than on the mucosa/submucosa-denuded segments. The M(2) receptor-preferring, methoctramine (0.5 microM), and M(3) receptor-preferring antagonist, 4-DAMP (0.5 microM) significantly blocked the PTHE (1 microg/ml)-induced contraction of distal colon longitudinal muscles, whereas the 5-HT receptor antagonists (1.0 microM, alone or in combination) selective for 5-HT(3) (ondansetron), 5-HT(4) (GR113808) and 5-HT(1, 2, 5-7) (methysergide) receptors did not change the PTHE (1 microg/ml)-induced contractility of distal colon longitudinal muscles. SNAP (0.1mM), a NO donor, enhanced the stimulatory effects of PTHE on the longitudinal muscle of distal colon, but l-NAME (0.1mM), a NO synthesis inhibitor, had no effects. PTHE (10-100mg/kg) caused a dose-dependent increase of colonic luminal transit. CONCLUSIONS Collectively, these findings suggest that PTHE specifically acts on the longitudinal muscle of distal colon in rats, and these stimulatory effects are likely mediated, at least, by activation of acetylcholinergic M(2) and M(3) receptors.

Chronic Administration of Monosodium Glutamate under Chronic Variable Stress Impaired Hypothalamic-Pituitary-Adrenal Axis Function in Rats

The hypothalamic-pituitary-adrenal (HPA) axis is the primary endocrine system to respond to stress. The HPA axis may be affected by increased level of corticotrophin-releasing factors under chronic stress and by chronic administration of monosodium glutamate (MSG). The purpose of this study was to investigate whether chronic MSG administration aggravates chronic variable stress (CVS)-induced behavioral and hormonal changes. Twenty-four adult male Sprague-Dawley rats, weighing 200~220 g, were divided into 4 groups as follows: water administration (CON), MSG (3 g/kg) administration (MSG), CVS, and CVS with MSG (3 g/kg) administration (CVS+MSG). In addition, for the purpose of comparing the effect on plasma corticosterone levels between chronic stress and daily care or acute stress, 2 groups were added at the end of the experiment; the 2 new groups were as follows: naïve mice (n=7) and mice exposed to restraint stress for 2 h just before decapitation (A-Str, n=7). In an open field test performed after the experiment, the CVS+MSG group significant decrease in activity. The increase in relative adrenal weights in the CVS and CVS+MSG group was significantly greater than those in the CON and/or MSG groups. In spite of the increase in the relative adrenal weight, there was a significant decrease in the plasma corticosterone levels in the CVS+MSG group as compared to all other groups, except the naïve group. These results suggest that impaired HPA axis function as well as the decrease in the behavioral activity in adult rats can be induced by chronic MSG administration under CVS rather than CVS alone.

Methylisogermabullone isolated from radish roots stimulates small bowel motility via activation of acetylcholinergic receptors

We have previously reported that extract of radish roots exhibits an increase in gastrointestinal motility through the activation of muscarinic acetylcholine (ACh) receptors. Based on the stimulatory activity‐guided fractionation on rat ileal segments, this study isolated methylisogermabullone (MIGB, C23H31O5NS, MW 433) from methanol extracts of radish roots. MIGB caused a significant increase of the isolated rat ileal contraction in a concentration‐dependent manner (23–693 μm), and the pattern of MIGB‐induced ileal contraction was different in the time course to that produced by ACh. The EC50 value of MIGB, to produce 50% maximum ileal contraction, was estimated to be 45.5 μm. MIGB (230 μm)‐induced ileal contractions were enhanced by pretreatment of segments with ACh (0.1 μm). Ileal contractions produced by MIGB (230 μm) or ACh (0.1 μm) at submaximal concentration were partially inhibited by pretreatment of hexamethonium (0.1 mm), a ganglionic blocker, whereas they were almost completely abolished by atropine (10 μm). Oral administration of MIGB to mice stimulated the small intestinal transit of charcoal in a dose‐dependent manner (10–100 mg kg−1), and MIGB (100 mg kg−1)‐induced stimulation of small intestinal transit was significantly attenuated by co‐administration of atropine (50 mg kg−1). Taken together, these results demonstrate that MIGB isolated from radish roots stimulates the small bowel motility through the activation of ACh receptors. These findings suggest that MIGB may become a potential regulatory agent for therapeutic intervention in dysfunction of gastrointestinal motility.

Pharmacological mechanism responsible for the Atractylodes japonica-induced distal colonic contraction in rats.

BACKGROUND AND AIM Atractylodes japonica Koidz (Compositae) has been commonly used to treat the gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of GI motility have not been clarified yet. METHODS Atractylodes japonica was sequentially partitioned with MeOH, n-hexane, CHCl(3), EtOAc and n-BuOH saturated with H(2)O, and the effects of Atractylodes japonica extracts on the spontaneous contractility of GI muscle strips prepared from rats were measured. RESULTS Among five different fractionations, EtOAc extracts of Atractylodes japonica (AJEA) dose-dependently increased the low frequency contraction of distal colon longitudinal muscles (DCLM), and the ED(50) values were revealed to be 1.71×10(-9) g/ml. Among GI tracts, a prominent contractile response to AJEA was observed only in the DCLM. The contractile patterns produced by AJEA remarkably differed from those caused by acetylcholine and 5-HT. 4-DAMP and methoctramine at 0.5 μM significantly blocked the AJEA (1.0 μg/ml)-induced contraction of DCLM, but ondansetron, GR113808 and methysergide at 1.0 μM in combination did not change the AJEA-induced DCLM contractions. Acetylethylcholine mustard (5.0 μM) significantly diminished the AJEA-induced DCLM contractions, whereas p-chlorophenyl alanine (1.0 μM) did not affect the stimulatory effects of AJEA on the DCLM contractions. CONCLUSION The present results suggest that AJEA may specifically act on the DCLM among GI smooth muscles, and AJEA-induced DCLM contraction is likely mediated, at least, by activation of ChAT and acetylcholinergic muscarinic receptors.

TGF-β1-induced PINCH-1-ILK-α-parvin complex formation regulates mesangial cell proliferation and hypertrophy

TGF-β1-induced glomerular mesangial cell (GMC) injury is a prominent characteristic of renal pathology in several kidney diseases, and a ternary protein complex consisting of PINCH-1, integrin-linked kinase (ILK) and α-parvin plays a pivotal role in the regulation of cell behavior such as cell proliferation and hypertrophy. We report here that PINCH-1-ILK-α-parvin (PIP) complex regulates the TGF-β1-induced cell proliferation and hypertrophy in cultured rat GMCs. When GMCs were treated with TGF-β1 for 1, 2 and 3 days, the PIP complex formation was up-regulated after 1 day, but it was down-regulated on day 2. Cell numbers were significantly elevated on day 2, but dramatically decreased on day 3. In contrast, a significant increase in cellular protein contents was observed 3 days after TGF-β1-treatment. TGF-β1 induced early increase of caspase-3 activity. In GMCs incubated with TGF-β1 for 2 days, cytosolic expression of p27(Kip1) was dramatically reduced, but its nuclear expression was remarkably elevated. A significantly decreased expression of phospho-Akt (Ser 473) was observed in the cells treated with TGF-β1 for 1 day. TGF-β1 induced early increase of phospho-p27(Kip1) (Thr 157) expression with subsequent decrease, and similar responses to TGF-β1 were observed in the p38 phosphorylation (Thr 180/Thr 182). Taken together, TGF-β1 differently regulates the PIP complex formation of GMCs in an incubation period-dependant fashion. The TGF-β1-induced up- and down-regulation of the PIP complex formation likely contributes to the pleiotropic effects of TGF-β1 on mesangial cell proliferation and hypertrophy through cellular localization of p27(Kip1) and alteration of Akt and p38 phosphorylation. TGF-β1-induced alteration of the PIP complex formation may be importantly implicated in the development and progression of glomerular failure shown in several kidney diseases.

Fatty diets retarded the propulsive function of and attenuated motility in the gastrointestinal tract of rats

Digestive functions are considered to be alterable by the ingestion of fatty diets. This study aimed to investigate the hypothesis that dietary fats may exert site-specific effects on the propulsive functions of the gastrointestinal (GI) tract. After male Wistar rats were fed either low-fat diet or high-fat diet (HFD) for 8 weeks, the propulsive function of the luminal contents of the entire GI tract was simultaneously examined in vivo. In comparison with a low-fat diet, an HFD significantly increased the body weight gains but significantly decreased the diet and caloric intakes, fecal weights, and fecal pellet numbers. Gastric emptying in the HFD-fed rats tended to be delayed, but this was not significant. High-fat diet feeding significantly slowed the small bowel transit times, and the luminal residuals emptied from the gastric antrum were largely accumulated in the proximal parts of the small intestine. An HFD also significantly prolonged the colonic transit times. In conclusion, fatty diets retarded the propulsive function of the entire GI tract, and the delayed gastroduodenal transit of fatty diets may act as a primary causal factor for producing the attenuated motile function of the GI tract in rats.

Pathophysiological implication of ganglioside GM3 in early mouse embryonic development through apoptosis.

Apoptosis may occur in early embryos where the execution of essential developmental events has failed, and gangliosides, sialic acid-conjugated glycosphingolipids, are proposed to regulate cell differentiation and growth. To evaluate the regulatory roles of ganglioside GM3 in early embryonic development, this study examined its expressional patterns in apoptotic cells during early embryonic development in mice. Pre-implanted embryos were obtained byin vitro fertilization, which were treated at the 4-cell stage with three the apoptosis inducers, actinomycin D, camptothecin and cycloheximide, for 15 h. All three inducers significantly increased the percentage of apoptotic cells, as measured using a TUNEL method, but remarkably reduced the total cell numbers. The numbers of morula and blastocyst stages were significantly decreased by treatment of the embryos with the three apoptosis inducers compared with the control, with a similar result also observed in the number of blastomeres. Staining of early embryos with Hoechst 33342 revealed a significant percentage of apoptotic nuclei. Prominent immunofluorescence microscopy revealed a significant difference in the ganglioside GM3 expression in apoptotic embryos compared with the control, and RT-PCR also demonstrated a dramatic increase in ganglioside GM3 synthase mRNA in the apoptotic embryos. These results suggest that ganglioside GM3 may be pathophysiologically implicated in the regulation of early embryonic development through an apoptotic mechanism.