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.

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.

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.

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.

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.

Amino acid transport system L is differently expressed in human normal oral keratinocytes and human oral cancer cells.

Previously, we reported the expression and function of system L amino acid transporter in KB human oral epidermoid carcinoma cells. In the present study, therefore, we investigated the expression and function of system L amino acid transporter in human normal oral keratinocytes (HNOK) and compared the expressions and functions of system L amino acid transporters in HNOK and KB cells. The HNOK expressed L-type amino acid transporter 1 (LAT1) and L-type amino acid transporter 2 (LAT2) with their subunit 4F2hc in the plasma membrane but the expression of LAT1 was very weak, which is in contrast to the KB cells expressing LAT1 but not LAT2 with the 4F2hc in the plasma membrane. The [14C] L-leucine uptake by HNOK, as well as KB cells, was inhibited by the system L selective inhibitor BCH. The majority of [14C] L-leucine uptake was, therefore, mainly mediated by LAT2 in the HNOK and by LAT1 in the KB cells. These results suggest that the transport of neutral amino acids including several essential amino acids into the HNOK and KB cells are mainly mediated by LAT2 and LAT1, respectively. The specific inhibition of LAT1 in oral cancer cells could be a new rationale for anti-cancer therapy.

Differential expression patterns of gangliosides in the ischemic cerebral cortex produced by middle cerebral artery occlusion.

Neuronal damage subsequent to transient cerebral ischemia is a multifactorial process involving several overlapping mechanisms. Gangliosides, sialic acid-conjugated glycosphingolipids, reduce the severity of acute brain damage in vitro. However their in vivo effects on the cerebral cortex damaged by ischemic infarct are unknown. To assess the possible protective role of gangliosides we examined their expression in the cerebral cortex damaged by ischemic infarct in the rat. Ischemia was induced by middle cerebral artery (MCA) occlusion, and the resulting damage was observed by staining with 2, 3, 5-triphenylterazolium chloride (TTC). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GM3 and GM1 increased in the damaged cerebral cortex, and immunofluorescence microscopy also revealed a significant change in expression of GM1. In addition, in situ hybridization demonstrated an increase in the mRNA for ganglioside GM3 synthase. These results suggest that gangliosides GM1 and GM3 may be synthesized in vivo to protect the cerebral cortex from ischemic damage.

Ganglioside GM3 inhibits the high glucose- and TGF-beta1-induced proliferation of rat glomerular mesangial cells.

Abrupt proliferation of glomerular mesangial cells (GMCs) is a common feature in the early stage of diabetic glomerulopathy, and ganglioside GM3 (NeuAcalpha3Galbeta4Glcbeta1Cer) is thought to regulate the proliferation of many cell types. Recently, we have reported ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats []. This study examined whether modulation of cellular ganglioside GM3 could regulate the high glucose- and transforming growth factor-beta1 (TGF-beta1)-induced proliferation of GMCs. To pharmacologically modulate the cellular ganglioside GM3, GMCs originated from rat kidneys were cultured with exogenous ganglioside GM3 or d-threo-PDMP, an inhibitor of ganglioside synthesis, in the RPMI 1640 media containing normal (5.6 mM, NG) or high (25 mM, HG) glucose. HG, TGF-beta1 (10 ng/ml) and d-threo-PDMP (20 microM) significantly stimulated the mesangial cell proliferation, whereas these increments were remarkable attenuated by exogenous ganglioside mixture (0.1-0.2 mg/ml) or GM3 (20-100 microM) in a dose-dependent manner. The mesangial cell proliferation caused by HG, TGF-beta1 and d-threo-PDMP was closely correlated with decreases in both cellular sialic acid contents and ganglioside GM3 synthase activity. Based upon the mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted, at least, of five different components of gangliosides, mainly ganglioside GM3. HG, TGF-beta1 and d-threo-PDMP induced a significant reduction of ganglioside expression with apparent changes in the composition of ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 expression reduced to about 35-54% of control. These results provide a pathophysiological link between mesangial cell proliferation and ganglioside GM3 expression, indicating that exogenously added ganglioside GM3 inhibits the high-ambient glucose- and TGF-beta1-induced proliferation of cultured GMCs.

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.

Modulation of cell proliferation and hypertrophy by gangliosides in cultured human glomerular mesangial cells.

Glomerular mesangial cells (GMCs) in diverse renal diseases undergo cell proliferation and/or hypertrophy, and gangliosides have been reported to play an important role in modulating cell structure and function. This study compared the effects of transforming growth factor-beta1 (TGF-beta1) and the effects of the application of exogenous gangliosides on GMCs and investigated whether the application of exogenous gangliosides regulated cellular proliferation and hypertrophy. Human GMCs were cultured with exogenous gangliosides and TGF-beta1 in a media containing 10% fetal bovine serum and in a media without the fetal bovine serum. Exogenous gangliosides biphasically changed the proliferation of human GMCs (0.1-1.0 mg/mL). A low concentration (0.1 mg/mL) of gangliosides mainly increased the number of human GMCs, whereas cellular proliferation was significantly reduced by raising the concentration of exogenous gangliosides. TGF-beta1 greatly reduced the number of human GMCs in a concentration-dependent manner (1-10 ng/mL). Serum deprivation accelerated the gangliosides- and TGF-beta1-induced inhibition of mesangial cell proliferation to a greater extent. Gangliosides (1.0 mg/ mL) and TGF-beta1 (10 ng/mL) both caused a significant increase in the incorporation of [3H]leucine per cell in the serum-deprived condition, whereas it was completely reversed in serum-supplemented condition. Similar results to the [3H]leucine incorporation were also observed in the changes in cell size measured by flow cytometric analysis. These results show that exogenous gangliosides modulate cell proliferation and hypertrophy in cultured human GMCs, and these cellular responses were regulated differently based on whether the media contained serum or not. Results from the present study raise new possibilities about the potential involvement of gangliosides in the development of mesangial cell proliferation and hypertrophy.

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.

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.

Involvement of staphylococcal protein A and cytoskeletal actin in Staphylococcus aureus invasion of cultured human oral epithelial cells.

Following the coincidental discovery that beta-actin isolated from renal epithelial cells was precipitated by staphylococcal protein A (SPA), the possibility that SPA and cytoskeletal actin filaments may be involved in Staphylococcus aureus infection of epithelial cells was considered. Therefore, to clarify the potential role of SPA and actin filaments in S. aureus infection, the invasion efficiency of S. aureus was determined quantitatively by measuring the number of cfu of viable organisms recovered from cultured KB cells. S. aureus invasion was found to be time dependent (0-60 min) and increased linearly when increasing numbers of bacteria were added (10(4)-10(6) cfu/ml). However, significant variation in the level of invasion was noted in protein A-deficient S. aureus Wood 46. Cytochalasin B inhibited the invasion efficiency of S. aureus in a dose-dependent manner. The present study suggests that interaction of staphylococcal protein A and cytoskeletal actin filaments is involved in the S. aureus invasion of cultured KB cells, and this process may contribute, in part, to the intracellular movement, cell-to-cell spread and dissemination of S. aureus within human oral epithelial cells in vivo.

Involvement of rat organic anion transporter 3 (rOAT3) in cephaloridine-induced nephrotoxicity: in comparison with rOAT1.

This study was performed to elucidate the possible involvement of organic anion transporter 3 (OAT3) in cephaloridine (CER)-induced nephrotoxicity and compare the substrate specificity between rOAT3 and rat OAT1 (rOAT1) for various cephalosporin antibiotics, using proximal tubule cells stably expressing rOAT3 (S2 rOAT3) and rOAT1 (S2 rOAT1). S2 rOAT3 exhibited a CER uptake and a higher susceptibility to CER cytotoxicity than did mock, which was recovered by probenecid. Various cephalosporin antibiotics significantly inhibited both estrone sulfate uptake in S2 rOAT3 and para-aminohippuric acid uptake in S2 rOAT1. The Ki values of CER, cefoperazone, cephalothin and cefazolin for rOAT3- and rOAT1-mediated organic anion transport ranged from 0.048 to 1.14 mM and from 0.48 to 1.32 mM, respectively. These results suggest that rOAT3, at least in part, mediates CER uptake and CER-induced nephrotoxicity as rOAT1. There was some difference of affinity between rOAT3 and rOAT1 for cephalosporin antibiotics.

Decreases of ganglioside GM3 in streptozotocin-induced diabetic glomeruli of rats.

Ganglioside GM(3) (NeuAcalpha3Galbeta4Glcbeta1Cer) is known to regulate the proliferation of many cell types and to maintain the charge-selective filtration barrier of glomeruli. Based on these, this study examined whether altered expression of ganglioside GM(3) was pathologically related with glomerular hypertrophy and proteinuria occurring in diabetic human and rat kidneys. Diabetic rats were produced by intraperitoneal injection of streptozotocin (80 mg/kg, I.P.). At 15 days after the induction of diabetes, glomerular volume and fibrotic matrix were dramatically elevated, whereas glomerular sialic acid contents were significantly reduced compared with control. Based upon mobility on high-performance thin-layer chromatography (HPTLC) and reactivity to anti-GM(3) monoclonal antibody, normal glomeruli showed a complex ganglioside pattern that consisted of six different components of gangliosides, mainly GM(3), and diabetes caused a severe reduction of these gangliosides with apparent changes in the composition of major ganglioside GM(3). Semi-quantitative analysis by HPTLC showed that ganglioside GM(3) was reduced to 57% of control in diabetic glomeruli. A prominent immunofluorescence microscopy showed a dramatic disappearance of GM(3) expression in diabetic glomeruli. These results indicate that diabetic glomeruli can be characterized by decreases of glomerular sialic acid content and ganglioside GM(3) expression, which may cause loss of charge-selective filtration barrier in renal glomeruli. These changes may be account, at least in part, for the development of glomerular hypertrophy and proteinuria seen in the early stage of diabetic glomerulopathy.

Mechanism for the negative regulation of cell proliferation by ganglioside GM3 in high glucose-treated glomerular mesangial cells.

We recently identified ganglioside GM3 as a modulator of glomerular hypertrophy in streptozotocin-induced diabetic rats (Life Sci., 72: 1997-2006, 2003). This study examined whether alteration of ganglioside GM3 expression could modulate the high glucose-induced proliferation of glomerular mesangial cells (GMCs). GMCs isolated from rat kidneys were cultured under normal (5.6 mM) or high (25 mM) glucose condition for 24-72 hrs. Cell proliferation was predominantly stimulated when GMCs were cultured with high glucose as well as 20 microM of d-threo-PDMP, an inhibitor of ganglioside biosynthesis, for 24 hrs, whereas raising ambient glucose significantly reduced the mesangial sialic acid contents. Based upon mobility on high-performance thin-layer chromatography (HPTLC), GMCs showed a complex pattern of ganglioside expression that consisted of three major components of gangliosides, mainly GM3. High glucose induced a significant reduction of ganglioside expression with apparent changes in the composition of major ganglioside GM3, and semi-quantitative analysis by HPTLC showed that ganglioside GM3 was reduced to 62% of GMCs cultured under normal glucose condition. A prominent immunofluorescence microscopy using anti-GM3 monoclonal antibody also showed a dramatic disappearance of immunoreactivity in high glucose-treated GMCs. Moreover, high glucose significantly lowered the Km values of GM3 synthase (16 microM vs. 49 microM), but did not change the Vmax. These results provide the pathophysiological relationship between the high glucose-induced proliferation of GMCs and the decreased expression of ganglioside GM3, indicating a mechanism for the negative regulation of mesangial proliferation by ganglioside GM3. This mechanism may play an important role in the development of diabetic glomerulopathy.

Antitumor activity of Soamsan, a traditional Korean medicine, via suppressing angiogenesis and growth factor transcription.

Antiangiogenic and antitumor activities of Soamsan known as an anticancer remedy in traditional Korean medicine were examined. In contrast to the normal branching of vascular vessels in chorioallantoic membrane (CAM), blood vessels in CAM treated with Soamsan (50 microg per egg) were run parallel to each other with less branching. Oral administration of Soamsan (20 mg/kg per day) for 4 weeks significantly inhibited the rat corneal neovascularization induced by suture, and the length of blood vessels in Soamsan-treated rat cornea was conspicuously low compared to control. When HT1080 cells, human fibrosarcoma, were treated with 2.18 mg/ml of Soamsan up to 24 h, mRNA transcription of VEGF, TGF-beta and bFGF genes was dramatically reduced in a time-dependent manner. Soamsan showed a prolongation of life span and a reduction of tumor volume in CT-26 cell (colon adenocarcinoma)-bearing mice. These results suggest that antitumor activity of Soamsan may be mediated, at least in part, by antiangiogenic mechanism.

Anti-angiogenic activities of Cnidium officinale Makino and Tabanus bovinus.

This study investigated the anti-angiogenic activities of Cnidium officinale Makino and Tabanus bovinus by using cultured glomerular capillary endothelial cells (GECs), chorioallantoic membrane (CAM) and rat cornea. Treatment of GECs with several concentrations (5-50 microg/ml) of C. officinale Makino and T. bovinus extracts for 24 h inhibited angiotensin II (10(-8) M)-induced increases of [3H]thymidine uptake and cell numbers in a concentration-dependent manner. The extent of inhibitory rate of [3H]thymidine incorporation by C. officinale Makino and T. bovinus at 50 microg/ml was a similar to that by 10(-5) M of retinoic acid. Herbal extracts also conspicuously inhibited the neovascularization. In contrast to the normal branching of vascular vessels, blood vessel patterns in CAMs treated with extracts (50 microg per egg) of C. officinale Makino and T. bovinus were ran parallel to each other without much branching. Moreover, oral administration of herbal extracts (20 mg/kg per day) for 4 weeks significantly inhibited the rat corneal neovascularization induced by suture, and the length of blood vessels in herbal medicine-treated rat cornea was conspicuously lower than that in control animals. A similar inhibitory effect to these was also observed in the rat cornea treated with thalidomide (200 mg/kg per day). These findings indicate that the anti-angiogenic properties of C. officinale Makino and T. bovinus may be one of the pharmacological mechanisms underlying the anti-tumor and anti-metastatic activities of herbal extracts tested in this study.