Differential effect of ginsenoside metabolites on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Ginsenosides are major active ingredients of Panax ginseng. They have a number of pharmacological and physiological actions and are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is derived from protopanaxadiol (PD) ginsenosides, whereas M4 is derived from protopanaxatriol (PT) ginsenosides. Recent reports show that ginsenosides act as pro-drugs for these metabolites. In previous work we demonstrated that the ginsenoside Rg2 regulates human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity [Choi et al. (2003)]. In the present study, we investigated the effect of CK and M4 on the activity of the human 5-HT3A receptor channel. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. Treatment with CK or M4 had no effect on oocytes injected with 5-HT3A receptor cRNA. However pretreatment with M4 or CK followed by injection of 5-HT3A receptor cRNA led to reversible inhibition of the 5-HT-induced inward peak current (I(5-HT)). Half maximal inhibitory concentrations (IC50) of CK and M4 were 36.9 +/- 9.6 and 7.3 +/- 2.2 microM, respectively. Inhibition by M4 was non-competitive and voltage-independent. These results indicate that M4, a metabolite of PT ginsenosides, acts primarily on 5-HT3A receptors and further, that ginsenosides as well as ginsenoside metabolites can influence 5-HT3A receptor channel activity in Xenopus oocytes.     

Differential expression of the chitin synthase genes of Aspergillus nidulans, chsA, chsB, and chsC, in response to developmental status and environmental factors

To understand the role of the chitin synthase genes of Aspergillus nidulans, we analyzed the expression of chsA, chsB, and chsC both by Northern blotting and by a vital reporter system with sgfp encoding a modified version of green fluorescent protein, sGFP. chsA was expressed specifically during asexual differentiation, but not during either vegetative growth or sexual differentiation. The expression of chsB was ubiquitous throughout the fungal body and relatively independent of the change in developmental status of the cells. chsC was expressed moderately during sexual development as well as during the early phase of vegetative growth, but was expressed weakly in old vegetative mycelia and in asexual structures. Furthermore its expression was spatially differentiated, i.e., relatively strong in young cleistothecia and in mature ascospores, but negligible in Hülle cells. Osmostress caused by high concentrations (up to 1.2M) of KCl or NaCl stimulated the expression of chsA and chsC, but not that of chsB. Sodium acetate, especially at high concentration (3%), strongly enhanced the expression of all the three genes. Neither heat shock nor the sugar carbon sources tested (glucose, sucrose, or lactose) affected the expression of any of the three chitin synthase genes.     

Nano-scale proteomics approach using two-dimensional fibrin zymography combined with fluorescent SYPRO ruby dye

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID-TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).     

Determination of glimepiride in human plasma using semi-microbore high performance liquid chromatography with column-switching

A fully automated semi-microbore high performance liquid chromatographic (HPLC) method with column-switching using UV detection was developed for the determination of glimepiride from human plasma samples. Plasma sample (900 microl) was deproteinated and extracted with ethanol and acetonitrile. The extract (70 microl) was directly injected into a Capcell Pak MF Ph-1 pre-column where the primary separation occurred to remove proteins and retain drugs using a mixture of acetonitrile and 10mM phosphate buffer (pH 2.18) (20:80, v/v). The analytes were transferred from the pre-column to an intermediate column using a switching valve and then subsequently separated on an analytical column and monitored with UV detection at 228 nm. Glimepiride was eluted with retention time 34.9 min without interference of endogenous substance from plasma. The limit of quantification (LOQ) was 10 ng/ml for glimepiride. The calibration curves were linear over the concentration range of 10-400 ng/ml (r(2) = 0.9997). Moreover, inter- and intra-day precisions of the method were less than 15% and accuracies were higher than 99%. The developed method was successfully applied for the quantification of glimepiride in human plasma and was used to support a human pharmacokinetic study following a single oral administration of 2 mg glimepiride.     

Ischaemic preconditioning in the pig assessed by myocardial perfusion imaging and histochemistry

Ischaemic preconditioning (IP) is a strong endogenous infarct reducing stimulus which has not previously been evaluated with myocardial perfusion imaging using 99mTc-MIBI. Factors responsible for cellular MIBI uptake are affected by both IP and acute ischaemia (plasma membrane and mitochondrial membrane potential and oxidative metabolism). IP seems to involve mitochondrial K-ATP channels affecting mitochondrial membrane potential and thereby potentially MIBI uptake. The study evaluated the performance of MPI with MIBI as a tracer to characterise the extent that severely ischaemic compromised myocardium was salvaged by IP. In a closed chest model, an ischaemic preconditioned group (8 pigs) subjected to IP before introducing a 45 min period of catheter based coronary occlusion was compared with a control group (9 pigs). Area at risk'(AAR), infarct size (IS) and IS relative to AAR was determined by MIBI SPECT and by a standard histochemical method. The results demonstrated that infarct size was significantly smaller in the IP group both relative to left ventricle (IS/LV) and to area at risk (IS/AAR). Both AAR/LV and IS/LV, however, were greater when measured by MPI than with histochemistry. There was no difference in the ratio between infarct size and area at risk (IS/AAR). In conclusion, MPI with MIBI is a reliable measurement of infarct reduction by ischaemic preconditioning. Myocardium affected by recent ischaemia is correctly distinguished as viable by MPI in early reperfusion, when compared to a standard histochemical technique.     

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

Microvirga splendida sp. nov., bacteria isolated from soil

Two bacterial strains, BT325^T and BT690, were isolated from soil samples collected in Korea. Both strains were Gram stain-negative, short rod-shaped, and formed light-pink colored colonies. The 16S rRNA sequence similarity of strains BT325^T and BT690 shared a sequence similarity of 99.7%. Both strains shared the highest 16S rRNA gene similarity of 98.6% with Microvirga arabica SV2184P^T, followed by Microvirga ossetica V5/3 M T (98.5% and 98.2%, respectively), Microvirga soli R491^T (98.3% and 98.2%, respectively), Microvirga aerilata (98.2% and 98.08%, respectively), Microvirga makkahensis (98.08% and 97.8%, respectively). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain BT325^T and BT690 were positioned in a distinct lineage within the family Methylobacteriaceae (order Rhizobiales, class Alphaproteobacteria). The genome size of strain BT325^T was 5,200,315 bp and the genomic DNA G?+?C content was 64.3 mol%. The sole respiratory quinone of strain BT325^T was Q-10 and the predominant cellular fatty acids were summed feature 3 (C_16:1 ω7c/C_16:1 ω6c) and summed feature 8 (C_18:1 ω7c/C_18:1 ω6c). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine. Polyphasic taxonomic analysis of biochemical, chemotaxonomic, and phylogenetic analyses suggested that strains BT325^T represents a novel bacterial species within the genus Microvirga, for which the name Microvirga splendida is proposed. The type strain of Microvirga splendida is BT325^T (=?KCTC 72406 ^T?=?NBRC 114847 ^T).

     

Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function.     

Lysosomal Trafficking of TGFBIp via Caveolae-Mediated Endocytosis

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin α_Vβ₃. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.     

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.