Reduction of gastrointestinal motility by unilateral thyroparathyroidectomy plus subdiaphragmatic vagotomy in rats.

AIM: To investigate whether the combined methods of unilateral thyroparathyroidectomy (TPX) and subdiaphragmatic vagotomy (VAX) can be adapted for rats and used as a reliable method to produce a rat model of long-term reduction of gastrointestinal (GI) motor function. METHODS: Male Sprague-Dawley rats were randomly divided into 3 groups, normal, sham-operated and unilateral TPX plus VAX. The TPX plus VAX rats received VAX 7 d after application of TPX, and dietary intake and fecal output were then measured daily for 1 wk. After completion of the experiments, gastric emptying and small bowel transit were measured in vivo, and the contractile responses of colonic strips to excitatory and inhibitory neurotransmitters were estimated using isometric force transducers in vitro. RESULTS: In comparison with normal and sham-operated rats, rats which received unilateral TPX plus VAX showed a significant decrease in body weight and in fecal pellet number and weight throughout the entire week. Application of TPX plus VAX to rats markedly delayed gastric emptying and small bowel transit. In TPX plus VAX rats, the longitudinal muscles of the proximal colon showed a significant reduction in contractile responses to acetylcholine (5 × 10(-6) mol/L), and a dramatic attenuation of contractile responses was also observed in both the longitudinal and circular muscles of the distal colon. However, the spontaneous contractility of the colonic strips from TPX plus VAX rats was not significantly affected by treatment with N-nitro-L-arginine-methyl ester (0.1 mol/L). CONCLUSION: The results indicate that unilateral TPX plus VAX reduced the motor function of the GI tract in rats, and the reduced gut motility is likely mediated, at least in part, by inhibition of the excitatory neurotransmitter system.

Regulatory roles of ganglioside GQ1b in neuronal cell differentiation of mouse embryonic stem cells.

Gangliosides play an important role in neuronal differentiation processes. The regulation of ganglioside levels is related to the induction of neuronal cell differentiation. In this study, the ST8Sia5 gene was transfected into mESCs and then differentiated into neuronal cells. Interestingly, ST8Sia5 gene transfected mESCs expressed GQ1b by HPTLC and immunofluorescence analysis. To investigate the effects of GQ1b over-expression in neurogenesis, neuronal cells were differentiated from GQ1b expressing mESCs in the presence of retinoic acid. In GQ1b expressing mESCs, increased EBs formation was observed. After 4 days, EBs were co-localized with GQ1b and nestin, and GFAP. Moreover, GQ1b co-localized with MAP-2 expressing cells in GQ1b expressing mESCs in 7-day-old EBs. Furthermore, GQ1b expressing mESCs increased the ERK1/2 MAP kinase pathway. These results suggest that the ST8Sia5 gene increases ganglioside GQ1b and improves neuronal differentiation via the ERK1/2 MAP kinase pathway.

Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts.

In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus-containing short hairpin (sh)RNA to knockdown ST3 ß-galactoside α-2, 3-sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72 h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin-resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 mitogen-activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.

Signalling pathways responsible for the methylisogermabullone-induced contraction of ileal longitudinal muscles.

OBJECTIVES: We have previously reported that methylisogermabullone (MIGB) stimulates small bowel motility through activation of acetylcholinergic receptors. This study investigated the cellular signalling pathways implicated in the regulation of ileal contractility by MIGB. METHODS: The ileal longitudinal muscles prepared from rats were treated with MIGB isolated from radish roots, and muscle contractility and protein expression were measured by force transducer and Western blot, respectively. KEY FINDINGS: MIGB at 30 µm induced a sustained phasic contraction of ileal longitudinal muscles. Acetylcholine (ACh, 0.5 µm) and MIGB stimulated translocation of protein kinase C (PKC) to cell membrane of ileal longitudinal muscles, and these stimulatory effects were remarkably attenuated by atropine (0.5 µm). ACh and MIGB induced phosphorylation of ERK 1/2 and p38 MAPKs in ileal longitudinal muscles, and they also phosphorylated the caldesmon and 20-kDa regulatory light chain of myosin (MLC(20) ). Additionally, PD-98058 (10 µm), a selective ERK 1/2 MAPK inhibitor, and SB-203580 (10 µm), a selective p38 MAPK inhibitor, significantly reduced the MIGB-induced contraction of ileal longitudinal muscles. CONCLUSIONS: The muscarinic receptor activated by MIGB translocates the PKC to cell membrane which phosphorylates the ERK 1/2 and p38 MAPKs, resulting in subsequent phosphorylation of caldesmon and MLC(20) . These cellular events likely converge on the contraction of ileal longitudinal muscles in rats.

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.

Acetylene compound isolated from Atractylodes japonica stimulates the contractility of rat distal colon via inhibiting the nitrergic-purinergic relaxation.

AIM OF THE STUDY: Our previous research has showed that rhizome of Atractylodes japonica Koidz (Compositae) exhibits an increase in the spontaneous contractility of distal colon in rats. The aims of this study are to identify the phytochemical(s), which stimulate(s) the colonic contractility, contained in Atractylodes japonica and to evaluate the pharmacological mechanism responsible for the colonic muscle contraction. MATERIALS AND METHODS: Based on the stimulatory activity-guided fractionation on the isometric contraction of rat distal colonic strips, atractylodiol (ATD) and diacetyl-atractylodiol (DATD) were isolated from the CHCl(3) fractions of Atractylodes japonica. RESULTS: ATD and DATD dose-dependently increased both tension and amplitude of distal colon longitudinal muscle (DCLM), but they stimulated only amplitude in the distal colon circular muscle. The ED(50) values of ATD and DATD to stimulate the amplitude of DCLM were revealed as 9.1×10(-9)M and 1.8×10(-8)M, respectively. l-NAME (0.1mM) significantly increased the ADT (1µM)-induced contraction of DCLM, whereas SNAP (0.1mM) markedly reduced the stimulatory effects of ATD on DCLM contractility. The combined effects of SNAP and atropine (0.5µM) on the ATD-induced contraction of DCLM were similar to the inhibitory effects of SNAP alone. Suramin (0.1mM) significantly enhanced the increase of ATD-induced DCLM contraction, whereas ADPßS (0.1mM) markedly abolished the stimulatory effects of ATD on the spontaneous contractility of DCLM. CONCLUSIONS: The present results demonstrate that acetylene compounds, ATD and DATD, are the effective phytochemical of Atractylodes japonica to stimulate the motility of distal colon in rats, and ATD possibly enhances the spontaneous contractility of distal colon through inhibiting the mechanism of nitrergic-purinergic relaxation.

Skin lesions associated with Demodex sp. in a llama (Lama glama). [corrected]

A 2.5-yr-old female llama (Lama glama) [corrected] with skin lesions was presented to the Animal Health Center in Seoul Grand Park Zoo, Korea. Mites of the genus Demodex in the absence of other mites or fungi were identified from the lesions by skin scrapings. The bodies were elongated, tapered, and 200-280 1m in length; four pairs of stumpy legs were present at the front of the body, and the striated opisthosoma constituted about half of the body length. Histologic examination of the skin biopsy showed typical folliculitis, hyperkeratinization of epidermis, and infiltration of inflammatory cells, consisting mainly of eosinophils and monocytes, in dermis. Although mites were undetected in the dilated hair follicles, the histologic features are consistent with descriptions of infestation by Demodex spp. This is believed to be the first case of skin lesions associated with Demodex sp. in L. glama. [corrected] Incidental findings were previously described in a different species of llamas (Lama glama) without any recognized symptoms. Treatment with amitraz (0.025%) eliminated the mites and resolved the clinical signs.

Estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized rats.

This study examined the estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized (OVX) rats. The components of silkworm pupae were extracted in distilled water at room temperature for 6 hours. The ovaries of six-week old female rats were then bilaterally removed. One week after OVX, the animals were treated with 200, 400 or 600 mg/kg/day of silkworm pupae extracts. The body weights of the OVX rats increased remarkably compared to the control rats, however their relative uterus weights to body weights decreased significantly. Treatment with the aqueous extracts of silkworm pupae dramatically improved the decreased uterus weights of OVX rats, with the highest increase observed in treatment with 200 mg/kg/day of the aqueous extracts. Additionally, treatment with aqueous extracts (200 mg/kg/day) of silkworm pupae significantly elevated the serum 17beta-estradiol contents of OVX rats when compared to the control animals. To examine the toxic effects of silkworm pupae on the hepatic functions of OVX rats, the levels of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were measured. The serum GOT and GPT levels did not change in response to the administration of aqueous extracts (200, 400 and 600 mg/kg/day) for 4-weeks. Taken together, these results suggest that the aqueous extracts of silkworm pupae may have estrogenic activity, which suggests that silkworm pupae may be useful in the prevention and/or treatment of menopausal disorders caused by deficiencies in female sexual hormones, including estrogen.

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.

5-hydroxytryptophan acts on the mitogen-activated protein kinase extracellular-signal regulated protein kinase pathway to modulate cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 cells.

In this study, we evaluated the effect of 5-hydroxytryptophan on anti-inflammatory and analgesic activity in RAW 264.7 cells. Cells were treated with different concentrations of 5-hydroxytryptophan for either 1 h or for 24 h. The anti-inflammatory effect was then analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting and reverse transcription polymerase chain reaction (RT-PCR). In addition, the analgesic activity was evaluated by measuring the acetic acid-induced writhing response. We found that 5-hydroxytryptophan significantly reduced the acetic acid-induced writhing response. Moreover we evaluated the effects of 5-hydroxytryptophan on the release of several inflammatory mediators including nitric oxide (NO) and interleukin-6 (IL-6). Our results demonstrated that 5-hydroxytryptophan inhibited the lipopolysaccharide (LPS)-induced expression of NO and IL-6. Furthermore, we found that 5-hydroxytryptophan played a role in LPS induced inducible nitric oxide synthase (iNOS), cyclo oxygenase-2 (COX-2) and extracellular-signal regulated protein kinase (ERK) activation. Taken together, these results indicate that 5-hydroxytryptophan has the potential for use in the treatment of inflammatory disease and as an analgesic.

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.

Different stimulatory effects of methylisogermabullone on the spontaneous contractility of rat gastrointestinal segments.

Using rat gastrointestinal (GI) strips, this study investigated the stimulatory effects of methylisogermabullone (MIGB) purified from radish on the spontaneous contractility of GI smooth muscles and pharmacological mechanisms involved in the MIGB-induced GI contraction. MIGB at 30 microM differently regulated the tone and amplitude of spontaneous GI contractility according to the region (fundus through distal colon) and orientation (longitudinal and circular) of smooth muscles: a significant increase in both tone and amplitude of spontaneous contraction in the ileum longitudinal and distal colon circular muscles and in amplitude only in the fundus, jejunum and distal colon longitudinal muscles. Pretreatment of ileum longitudinal muscles with atropine (0.5 microM) or 4-DAMP (0.5 microM) significantly inhibited the acetylcholine (ACh, 1 microM)- and MIGB (30 microM)-stimulated contraction, and methoctramine (0.5 microM) also obviously reduced the tone and amplitude increased by ACh and MIGB, respectively. In the presence of methysergide (1 microM), pretreatment of ileum longitudinal muscles with both ondansetron (0.1 microM) and GR113808 (0.1 microM) significantly inhibited the contraction stimulated by 5-HT (10 microM), but not by MIGB. Taken together, it is concluded that MIGB differently regulates the spontaneous contractility (tone and/or amplitude) of GI segments according to the region of gut and orientation of smooth muscles, and these contractile responses of GI tracts to MIGB are likely mediated, at least, by activation of acetylcholinergic M2 and M3 receptors.

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors.

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.

TGF-beta1-induced PINCH-1-ILK-alpha-parvin complex formation regulates mesangial cell proliferation and hypertrophy.

TGF-beta1-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 alpha-parvin plays a pivotal role in the regulation of cell behavior such as cell proliferation and hypertrophy. We report here that PINCH-1-ILK-alpha-parvin (PIP) complex regulates the TGF-beta1-induced cell proliferation and hypertrophy in cultured rat GMCs. When GMCs were treated with TGF-beta1 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-beta1-treatment. TGF-beta1 induced early increase of caspase-3 activity. In GMCs incubated with TGF-beta1 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-beta1 for 1 day. TGF-beta1 induced early increase of phospho-p27(Kip1) (Thr 157) expression with subsequent decrease, and similar responses to TGF-beta1 were observed in the p38 phosphorylation (Thr 180/Thr 182). Taken together, TGF-beta1 differently regulates the PIP complex formation of GMCs in an incubation period-dependant fashion. The TGF-beta1-induced up- and down-regulation of the PIP complex formation likely contributes to the pleiotropic effects of TGF-beta1 on mesangial cell proliferation and hypertrophy through cellular localization of p27(Kip1) and alteration of Akt and p38 phosphorylation. TGF-beta1-induced alteration of the PIP complex formation may be importantly implicated in the development and progression of glomerular failure shown in several kidney diseases.

Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells.

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.

TGF-beta1 regulates the PINCH-1-integrin-linked kinase-alpha-parvin complex in glomerular cells.

Glomerular damage is a major cause of renal failure. Recent studies suggest that a ternary protein complex that consists of PINCH-1, integrin-linked kinase, and alpha-parvin, cytoplasmic components of cell-extracellular matrix adhesions, plays pivotal roles in regulation of glomerular cell behavior. It is reported here that TGF-beta1, a key factor in the progression of glomerular failure, regulates the PINCH-1-integrin-linked kinase-alpha-parvin (PIP) complex formation in glomerular podocytes and mesangial cells. Treatment of podocytes with TGF-beta1 inhibited the PIP complex formation. Forced disruption of the PIP complex in podocytes activated p38 mitogen-activated protein kinase and promoted apoptosis. Importantly, inhibition of p38 mitogen-activated protein kinase, either with a chemical p38 inhibitor (SB202190) or with a dominant negative form of p38alpha, alleviates podocyte apoptosis that is induced by the disruption of the PIP complex. In contrast to an inhibitory role in podocytes, TGF-beta1 promotes the PIP complex formation in mesangial cells. Thus, TGF-beta1 regulates the PIP complex in a cell type-dependent manner. Because the PIP complex promotes glomerular mesangial matrix deposition and protects podocytes from apoptosis, the TGF-beta1-induced up- and downregulation of the PIP complex likely contribute to the pleiotropic effects of TGF-beta1 on different glomerular cell types and hence the progression of glomerular failure.

Differential expression of gangliosides in the ovary and uterus of streptozotocin-induced and db/db diabetic mice.

Gangliosides are widely distributed in mammalian cells and play important roles in various functions such as cell differentiation and growth control. In addition, diabetes and obesity cause abnormal development of reproductive processes in a variety of species. However, the mechanisms underlying these effects, and how they are related, are not fully understood. This study examined whether the differential expression of gangliosides is implicated in the abnormal follicular development and uterine architecture of streptozotocin (STZ)-induced and db/db diabetic mice. Based upon the mobility on high-performance thin-layer chromatography, mouse ovary consisted of at least five different ganglioside components, mainly gangliosides GM3, GM1, GD1a and GT1b, and diabetic ovary exhibited a significant reduction in ganglioside expression with apparent changes in the major gangliosides. A prominent immunofluorescence microscopy showed a dramatic loss of ganglioside GD1a expression in the primary, secondary and Graafian follicles of STZ-induced and db/db diabetic mice. A significant decrease in ganglioside GD3 expression was also observed in the ovary of db/db mice. In the uterus of STZ-induced diabetic mice, expression of gangliosides GD1a and GT1b was obviously reduced, but gangliosides GM1, GM2 and GD3 expression was increased. In contrast, the uterus of db/db mice showed a significant increase in gangliosides GM1, GD1a and GD3 expression. Taken together, a complex pattern of ganglioside expression was seen in the ovary and uterus of normoglycemic ICR and db/+ mice, and the correspoding tissues in diabetic mice are characterized by appreciable changes of the major ganglioside expression. These results suggest that alterations in ganglioside expression caused by diabetes mellitus may be implicated in abnormal ovarian development and uterine structure.

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.

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 microM), 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 microM. MIGB (230 microM)-induced ileal contractions were enhanced by pretreatment of segments with ACh (0.1 microM). Ileal contractions produced by MIGB (230 microM) or ACh (0.1 microM) 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 microM). 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.

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 by in 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.