Correcting signal biases and detecting regulatory elements in STARR-seq data.

High-throughput reporter assays such as self-transcribing active regulatory region sequencing (STARR-seq) have made it possible to measure regulatory element activity across the entire human genome at once. The resulting data, however, present substantial analytical challenges. Here, we identify technical biases that explain most of the variance in STARR-seq data. We then develop a statistical model to correct those biases and to improve detection of regulatory elements. This approach substantially improves precision and recall over current methods, improves detection of both activating and repressive regulatory elements, and controls for false discoveries despite strong local correlations in signal.

The Effects of 1-O-Acetylbritannilactone Isolated from Inula britannica Flowers on Human Neutrophil Elastase and Inflammation of RAW 264.7 Cells and Zebrafish Larvae.

During a search for natural inflammatory inhibitors, 1-O-acetylbritannilactone (ABL), a sesquiterpene lactone, was isolated from the flowers of Inula britannica. ABL significantly inhibited human neutrophil elastase (HNE) with a half-maximal inhibitory concentration (IC50) of 3.2 ± 0.3 µM, thus did so more effectively than the positive control material (epigallocatechin gallate) (IC50 7.2 ± 0.5 µM). An enzyme kinetic study was performed. ABL noncompetitively inhibited HNE with an inhibition constant Ki of 2.4 µM. ABL inhibited lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by RAW 264.7 cells in a dose-dependent manner, as well as the protein-level expression of inducible nitric oxide synthase and cyclooxygenase-2. The anti-inflammatory effect of ABL was confirmed using a transgenic Tg(mpx:EGFP) zebrafish larval model. The exposure of the larvae to ABL inhibited neutrophil recruitment to the site of injury after tail fin amputation.

CaV α2δ Autoimmune Encephalitis: A Novel Antibody and its Characteristics.

OBJECTIVE: Discovery of a novel antibody would enable diagnosis and early treatment of autoimmune encephalitis. The aim was to discover a novel antibody targeting a synaptic receptor and characterize the pathogenic mechanism. METHOD: We screened for unknown antibodies in serum and cerebrospinal fluid samples from autoimmune encephalitis patients. Samples with reactivity to rat brain sections and no reactivity to conventional antibody tests underwent further processing for antibody discovery, using immunoprecipitation to primary neuronal cells, mass-spectrometry analysis, an antigen-binding assay on an antigen-overexpressing cell line, and an electrophysiological assay with cultured hippocampal neurons. RESULTS: Two patients had a novel antibody against CaV α2δ (voltage-gated calcium channel alpha-2/delta subunit). The patient samples stained neuropils of the hippocampus, basal ganglia, and cortex in rat brain sections and bound to a CaV α2δ-overexpressing cell line. Knockdown of CaV α2δ expression in cultured neurons turned off the immunoreactivity of the antibody from the patients to the neurons. The patients were associated with preceding meningitis or neuroendocrine carcinoma and responded to immunotherapy. In cultured neurons, the antibody reduced neurotransmitter release from presynaptic nerve terminals by interfering with tight coupling of calcium channels and exocytosis. INTERPRETATION: Here, we discovered a novel autoimmune encephalitis associated with anti-CaV α2δ antibody. Further analysis of the antibody in autoimmune encephalitis might promote early diagnosis and treatment. ANN NEUROL 2021;89:740-752.

Herbicidal characteristics and structural identification of a potential active compound produced by Streptomyces sp. KRA18-249.

The KRA18-249 strain, isolated from a natural recreational forest near Jeongseon, Gangwon-do, when applied to plants induced signs of wilting within 24 h, leading to plant death. The isolated actinomycete was identified as Streptomyces gardneri based on 16S rRNA gene homogeneity analysis. The culture filtrate was solvent fractionated to obtain the active substance, and the active compound 249-Y1 was isolated from the purified fractions via a herbicide activity test using Digitaria ciliaris. NMR and ESI-MS analyses revealed that the molecular formula of 249-Y1 is C20H16O6 [MW = 352.0947] and is an anthraquinone (rubiginone D2) produce by polyketide synthetase system. The active compound 249-Y1 showed strong (100%) herbicidal activity against several weeds at 500 µg mL-1 concentration. Twisting symptoms began to appear within 24 h of treatment and intensified over time. The KRA18-249 strain produced the herbicidal compound under specific culture conditions, that is, at 200 rpm, 35 °C, for eight days at an initial pH of 10. We also found that 249-Y1 inhibited chlorophyll, but was not a radical generator. Overall, the secondary metabolite 249-Y1, produced by KRA18-249, can be used as a new biological agent for weed control.

Evaluation of a Rapid and Simple Method for Assessing Retinal Vessel Structures in Adult Zebrafish.

The evaluation of retinal vascular structures is important for analyzing various ophthalmic diseases. Conventional trypsin digestion was used for separating retinal vasculatures in mouse, rat, and other animal models; however, the trypsin method alone is technically difficult to perform and has not been reported in zebrafish to date. In this study, we introduced a rapid and convenient method that allows the investigation of fine vessel structures at a cellular level in the relatively intact retinal vasculature of adult zebrafish. Using an anti-ZO-1 antibody, tight junction structures in retinal vessels were examined in detail and several different cell types constituting blood vessels in arterial and capillary areas were identified. In addition, using cell type-specific antibodies, we identified smooth muscle cells, blood cells, and endothelial cells in the retinal vasculature. Finally, using the hyperglycemic model, we observed the dilation of retinal vessels, the downregulation of tight junction proteins, and the reduction in smooth muscle cells. Based on these results, we provide a rapid and convenient method for the study of retinal vasculature disease in the zebrafish animal model.

Bayesian estimation of genetic regulatory effects in high-throughput reporter assays.

MOTIVATION: High-throughput reporter assays dramatically improve our ability to assign function to noncoding genetic variants, by measuring allelic effects on gene expression in the controlled setting of a reporter gene. Unlike genetic association tests, such assays are not confounded by linkage disequilibrium when loci are independently assayed. These methods can thus improve the identification of causal disease mutations. While work continues on improving experimental aspects of these assays, less effort has gone into developing methods for assessing the statistical significance of assay results, particularly in the case of rare variants captured from patient DNA. RESULTS: We describe a Bayesian hierarchical model, called Bayesian Inference of Regulatory Differences, which integrates prior information and explicitly accounts for variability between experimental replicates. The model produces substantially more accurate predictions than existing methods when allele frequencies are low, which is of clear advantage in the search for disease-causing variants in DNA captured from patient cohorts. Using the model, we demonstrate a clear tradeoff between variant sequencing coverage and numbers of biological replicates, and we show that the use of additional biological replicates decreases variance in estimates of effect size, due to the properties of the Poisson-binomial distribution. We also provide a power and sample size calculator, which facilitates decision making in experimental design parameters. AVAILABILITY AND IMPLEMENTATION: The software is freely available from www.geneprediction.org/bird. The experimental design web tool can be accessed at http://67.159.92.22:8080. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of Jakyakgamcho-Tang Extracts on H2O2-Induced C2C12 Myoblasts.

Oxidative stress is a major contributor to muscle aging and loss of muscle tissue. Jakyakgamcho-tang (JGT) has been used in traditional Eastern medicine to treat muscle pain. Here, we compared the total phenolic and flavonoid contents in 30% ethanol and water extracts of JGT and tested the preventive effects against oxidative stress (hydrogen peroxide)-induced cell death in murine C2C12 skeletal muscle cells. The total phenolic content and total flavonoid content in 30% ethanol extracts of JGT were higher than those of water extracts of JGT. Ethanol extracts of JGT (JGT-E) had stronger antioxidant activities of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2'-diphenyl-1-picrylhydrazyl-scavenging activity (DPPH) than water extracts of JGT (JGT-W). JGT-E contained 19-53% (1.8 to 4.9-fold) more active compounds (i.e., albiflorin, liquiritin, pentagalloylglucose, isoliquiritin apioside, isoliquiritin, liquiritigenin, and glycyrrhizin) than JGT-W. The ethanol extracts of JGT inhibited hydrogen peroxide-induced cell death and intracellular reactive oxygen species generation more effectively than the water extract of JGT in a dose-dependent manner. For the first time, these results suggest that ethanol extract of JGT is relatively more efficacious at protecting against oxidative stress-induced muscle cell death.

Osteomeles schwerinae Extract and Its Major Compounds Inhibit Methylglyoxal-Induced Apoptosis in Human Retinal Pigment Epithelial Cells.

The accumulation and formation of advanced glycation end products (AGEs) are related to diabetes and age-related disease. Osteomeles schwerinae C. K. Schneid. (Rosaceae, OSSC) is used traditionally for the treatment of various diseases in Asia. Previous studies have shown that OSSC elicits preventive effects in an in vivo model of diabetes. This study was to evaluate the antiapoptotic effects of dried leaves and twigs of OSSC extract and its major compounds in ARPE-19 cells-spontaneously arising human retinal pigment epithelial cells-under diabetic conditions. To examine the effects of an OSSC extract and its active compounds (acetylvitexin, hyperoside and quercitrin) on apoptosis in methylglyoxal (MG, the active precursor in the formation of AGEs)-treated ARPE-19 cells and the mechanism by which these effects occur, apoptosis was measured using flow cytometry analysis. Protein expression levels of phospho-p53 (p-p53), Bax and Bcl-2 were determined by western blot analyses. The OSSC extract inhibited apoptosis in MG-treated ARPE-19 cells in a dose-dependent manner. The major compounds also reduced the rate of apoptosis. Both the extract and major compounds also inhibited the expression of p-p53 and Bax and increased the levels of Bcl-2 that had been previously reduced by MG treatment. The OSSC extract (0.1 µg/mL) and its major compounds (0.01 µM) attenuated apoptosis in ARPE-19 cells under toxic diabetic conditions by downregulating of expression of p-p53 and Bax. OSSC may serve as an alternative therapy to retard the development of diabetic retinopathy.

Modeling hypercholesterolemia and vascular lipid accumulation in LDL receptor mutant zebrafish.

Elevated plasma LDL cholesterol is the dominant risk factor for the development of atherosclerosis and cardiovascular disease. Deficiency in the LDL receptor (LDLR) is a major cause of familial hypercholesterolemia in humans, and the LDLR knockout mouse is a major animal model of atherosclerosis. Here we report the generation and characterization of an ldlr mutant zebrafish as a new animal model to study hypercholesterolemia and vascular lipid accumulation, an early event in the development of human atherosclerosis. The ldlr mutant zebrafish were characterized by activated SREBP-2 pathway and developed moderate hypercholesterolemia when fed a normal diet. However, a short-term, 5-day feeding of ldlr mutant larvae with a high-cholesterol diet (HCD) resulted in exacerbated hypercholesterolemia and accumulation of vascular lipid deposits. Lomitapide, an inhibitor of apoB lipoprotein secretion, but not the antioxidant probucol, significantly reduced accumulation of vascular lipid deposits in HCD-fed ldlr mutant larvae. Furthermore, ldlr mutants were defective in hepatic clearance of lipopolysaccharides, resulting in reduced survival. Taken together, our data suggest that the ldlr knockout zebra-fish is a versatile model for studying the function of the LDL receptor, hypercholesterolemia, and related vascular pathology in the context of early atherosclerosis.

Ordinal response generalized difference in differences with varying categories: The health effect of a disability program in Korea.

We consider the use of difference in differences (DD) to evaluate the effect of an activity assistance program on the health of severely disabled Koreans. There are, however, 2 problems in the data: the number of response categories for self-assessed health changed over the waves of the repeated cross-section survey and the "parallel untreated response path" assumption for DD is suspect. We show how to overcome these problems by renormalizing parameters and applying "generalized difference in differences (GDD)." We find a significantly positive effect of the program with DD, but not with GDD. Our solutions should prove useful in applications in which one or other of these problems arise.

Halomonas salicampi sp. nov., a halotolerant and alkalitolerant bacterium isolated from a saltern soil.

A Gram-stain-negative, halotolerant and alkalitolerant bacterium, designated strain BH103T, was isolated from saltern soil in Gomso, Korea. Cells of strain BH103T were strictly aerobic, motile, straight rods and grew at pH 7.0-10.8 (optimum, pH 8.5), at 10-55 °C (optimum, 28 °C) and at salinities of 0-23 % (w/v) NaCl (optimum, 14 % NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain BH103T belongs to the genus Halomonas, showing highest sequence similarity to Halomonas boliviensis LC1T (97.7 %), Halomonas neptunia Eplume1T (97.7 %), Halomonas variabilis IIIT (97.7 %), Halomonas alkaliantarctica CRSST (97.7 %), Halomonas olivaria TYRC17T (97.5 %), Halomonas titanicae BH1T (97.2 %) and Halomonas sulfidaeris Esulfide1T (96.2 %). The predominant ubiquinone was Q-9. The major fatty acids were C18 : 1ω7c, C16 : 1ω7c and/or iso-C15 : 0 2-OH, C16 : 0 and C12 : 0 3-OH. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, aminophospholipid and an unknown phospholipid. The DNA G+C content of this novel isolate was 54.7 mol%. DNA-DNA relatedness between strain BH103T and H. boliviensis KACC 16615T, H. neptunia KCTC 2888T, H. variabilis KCTC 2889T, H. alkaliantarctica KCTC 22844T, H. olivaria DSM 19074T, H. titanicae JCM 16411T and H. sulfidaeris DSM 15722T was 45, 41, 39, 32, 38, 45 and 35 %, respectively. On the basis of polyphasic analysis from this study, strain BH103T represents a novel species of the genus Halomonas, for which the name Halomonas salicampi sp. nov. is proposed. The type strain is BH103T ( = KACC 17609T = NBRC 109914T = NCAIM B 02528T).

Pontibacillus salicampi sp. nov., a moderately halophilic bacterium isolated from saltern soil.

A Gram-stain-positive, moderately halophilic bacterium, designated BH043(T), was isolated from saltern soil of Gomso in Korea. Cells were motile rods, producing ellipsoidal endospores at a terminal position in swollen sporangia. Strain BH043(T) was strictly aerobic, grew at pH 6.0-10.0 (optimal growth at pH 7.5), at 10-55 °C (optimal growth at 30 °C) and at salinities of 1-20 % (w/v) NaCl, growing optimally with 7 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain BH043(T) belongs to the family Bacillaceae and was most closely related to the type strains of the five recognized species of the genus Pontibacillus, showing sequence similarity to Pontibacillus yanchengensis Y32(T) (97.5 % similarity), Pontibacillus marinus BH030004(T) (97.4 %), Pontibacillus chungwhensis BH030062(T) (97.0 %), Pontibacillus litoralis JSM 072002(T) (96.4 %) and Pontibacillus halophilus JSM 076056(T) (96.2 %). The major cellular fatty acids of strain BH043(T) were iso-C15 : 0 and anteiso-C15 : 0. The genomic DNA G+C content was 42.5 mol%. The major isoprenoid quinone was MK-7 and meso-diaminopimelic acid was present in the cell-wall peptidoglycan as the diagnostic diamino acid. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. DNA-DNA relatedness between strain BH043(T) and the type strains of other species of the genus Pontibacillus, P. yanchengensis CGMCC 1.10680(T) and P. marinus KCTC 3917(T) and P. chungwhensis KCTC 3890(T), was 35, 24 and 18 %, respectively. On the basis of polyphasic analysis from this study, strain BH043(T) represents a novel species of the genus Pontibacillus for which the name Pontibacillus salicampi sp. nov. is proposed. The type strain is BH043(T) ( = KACC 17607(T) = NBRC 109831(T) = NCAIM B.02529(T)).

Idiomarina halophila sp. nov., isolated from a solar saltern sediment.

A Gram-stain-negative, halophilic bacterium, designated strain BH195(T), was isolated from the sediment of the solar saltern pond located in Gomso, Republic of Korea. Strain BH195(T) was a strictly aerobic, non-motile rod, which grew at pH 3.5-10.5 (optimum, pH 7.5), at 4-55 °C (optimum, 30 °C) and at salinities of 0.5-11% (w/v) NaCl [optimum, 2-3% (w/v) NaCl]. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain BH195(T) belongs to the genus Idiomarina , showing the highest sequence similarity to Idiomarina salinarum ISL-52(T) (97.4%), Idiomarina homiensis PO-M2(T) (96.8%), Idiomarina aestuarii KYW314(T) (96.7%), and Idiomarina tainanensis PIN1(T) (96.7%). The major cellular fatty acids of strain BH195(T) were iso-C(11 : 0) 3-OH, iso-C(15 : 0) and iso-C(11 : 0). The DNA G+C content was 51.3 mol% and the major respiratory quinone was ubiquinone 8. The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unknown phospholipid. DNA-DNA relatedness between strain BH195(T) and I. salinarum KCTC 12971(T) was 33%. On the basis of this polyphasic analysis, strain BH195(T) represents a novel species of the genus Idiomarina for which the name Idiomarina halophila sp. nov. is proposed. The type strain is BH195(T) ( = KACC 17610(T) = NCAIM B 02544(T)).

Wnt/ß-catenin signalling maintains self-renewal and tumourigenicity of head and neck squamous cell carcinoma stem-like cells by activating Oct4.

Accumulating evidence suggests that a distinct subpopulation of cancer stem cells (CSCs) is responsible for tumour initiation and progression in head and neck squamous cell carcinoma (HNSCC). Wnt/ß-catenin signalling is essential for stem cell regulation and tumourigenesis, but its molecular mechanism in HNSCC CSCs remains unknown. We investigated whether Wnt/ß-catenin signalling regulates self-renewal and tumourigenicity of HNSCC stem-like cells in vitro and in vivo. Cytoplasmic/nuclear ß-catenin, a major effector of Wnt/ß-catenin signalling, was expressed in a subpopulation of tumour cells in primary HNSCC tissue but in none of normal head and neck tissues. Overexpression of ß-catenin increased proliferation of HNSCC cells and induced dedifferentiation of these cells to cells with stem-like features. Knockdown of ß-catenin in HNSCC stem-like cells blocked their self-renewal capacity, stemness-associated gene expression, chemoresistance, and in vivo tumourigenicity. Furthermore, ß-catenin directly regulates Oct4 transcription in HNSCC stem-like cells. In addition, the effect of shRNA-mediated repression of ß-catenin on CSC traits in HNSCC stem-like cells was reversed by overexpression of Oct4. In patients with HNSCC, higher levels of both cytoplasmic/nuclear ß-catenin and Oct4 correlated with the worst prognosis. These results suggest inhibition of Wnt/ß-catenin signalling as a novel therapeutic strategy for targeting HNSCC stem-like cells.

Lignans from the stems and leaves of Brandisia hancei and their effects on VEGF-induced vascular permeability and migration of HRECs and DLAV formation in zebrafish.

In our continuing search for novel antiangiogenic agents, a new lignan glycoside, (7R,8R)-1-(4-O-ß-d-glucopyranosyl-3-methoxyphenyl)-2-{2-methoxy-4-[1-(E)-propene-3-ol]-phenoxyl}-propane-1,3-diol (1), along with three known lignans (2-4), were isolated from the 80% EtOH extract of Brandisia hancei stems and leaves. These isolates (1-4) were subjected to an in vitro bioassay to evaluate their effects on vascular endothelial growth factor (VEGF)-induced vascular permeability and migration of human retinal endothelial cells (HRECs). Of the compounds tested, compound 1 resulted in the greatest reduction in VEGF-induced vascular permeability by about 31.5% at 10 µM compared to the VEGF-treated control. In the migration assay, compounds 1 and 2 significantly decreased VEGF-induced HREC migration. Furthermore, zebrafish embryos treated with compounds 1 and 2 showed mild reductions of dorsal longitudinal anastomotic vessel (DLAV) formation.

Difference in differences for stayers with a time-varying qualification: health expenditure elasticity of the elderly.

In difference in differences, a treatment is applied only to a qualified group at some time point. The qualification may be time-constant as in gender, or time-varying as in residential location. When the qualification is time-varying, there appear four groups: the newly qualified (in-movers), the already qualified (in-stayers), the newly disqualified (out-movers), and the already disqualified (out-stayers). A change in qualification may affect the response variable of interest even when the treatment effect is zero, which is an 'untreated moving effect'. Also, when the treatment effect is not zero, it may be different across the four groups. The conventional difference in differences fails to remove untreated moving effects and ignores the possible treatment effect heterogeneity across the groups. This paper shows how to account for untreated moving effects and proposes 'the effect on in-stayers' as the main effect of interest. Our proposal can be implemented with least squares estimator for panel models or with nonparametric methods. An empirical analysis is provided using Korean data for the effects of the basic elder pension on health-care expenditure.

Culture optimization of Streptomyces sp. KRA16-334 for increased yield of new herbicide 334-W4.

This study aimed to isolate actinomycetes that exhibit strong herbicidal activity, identify compounds active against weeds, and researching methods to improve the production of these compounds through culture optimization to establish a foundation for the development of environmentally friendly bioherbicides. 334-W4, one of the herbicidal active substances isolated from the culture broth of Streptomyces sp. KRA16-334, exhibited herbicidal activity against various weeds. The molecular formula of 334-W4 was determined to be C16H26N2O6, based on ESI-MS (m/z) and 1H and 13C NMR spectral data. It had molecular weight 365.1689 [M+Na] and 343.1869 [M+H], indicating the presence of the epoxy-ß-aminoketone moiety based on HMBC correlations. Additionally, selective culture was possible depending on the addition of trifluoroacetic acid (TFA) during culture with GSS medium. Experiments confirmed that exposure of the KRA16-334 strain to UV irradiation (254 nm, height 17 cm) for 45 seconds improved the yield of the active substance (334-W4) by over 200%. As a result of examining yields of active materials of four mutants selected through optimization of culture conditions such as temperature, agitation, and initial pH, the yield of one mutant 0723-8 was 264.7 ± 12.82 mg/L, which was 2.8-fold higher than that of wild-type KRA16-334 at 92.8 ± 5.48 mg/L.

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose.

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.

Polygonum cuspidatum inhibits pancreatic lipase activity and adipogenesis via attenuation of lipid accumulation.

BACKGROUND: Obesity causes metabolic disease and is a serious health problem around the world. Polygonum cuspidatum (POCU1b) has been used clinically for the treatment of constipation, gallstones, hepatitis, and inflammation in East Asian countries. The principal aim of this study was to investigate for the first time whether the extract of Polygonum cuspidatum (POCU) biologically affects adipogenesis in 3 T3-L1 preadipocytes. METHODS: Fractions (n-hexan, ethyl acetate, n-butanol, and water) of POCU ethanol extract were evaluated in vitro for their inhibitory activities on pancreatic lipase. Of the fractions, the n-butanol of POCU ethanol extract (POCU1b) was examined anti-obesity activity in 3 T3-L1 preadipocytes. To examine the inhibitory effect of POCU1b on adipogenesis, 3 T3-L1 preadipocytes were treated every the other day with POCU1b at various concentrations (0 ~ 25 µg/mL) for twelve days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation. The expression of mRNA and proteins associated lipid accumulation was measured using RT-PCR and Western blotting analysis. We also examined the effect of POCU1b on level of phosphorylated AMP-activated protein kinase (pAMPK) in 3 T3-L1 preadipocytes with POCU1b at various concentrations during adipocyte differentiation. RESULTS: POCU1b exhibited the most pronounced inhibitory effects on pancreatic lipase activity. We found that POCU1b inhibited adipocyte differentiation in 3 T3-L1 preadipocytes in a dose-dependent manner, as evidenced by the reduced formation of lipid droplets and decreased glycerol-3-phosphate dehydrogenase (GPDH) activity. We also showed that the expression levels of adipocyte differentiation-related protein (ADRP) and perilipin (a protein that coats lipid droplets in adipocytes) were both reduced after POCU1b treatment. Peroxisome proliferator-activated receptor-gamma (PPAR-γ) and CCAAT/enhancer-binding protein-alpha (C/EBP-α) proteins, both major adipogenic transcription factors, were markedly reduced by POCU1b. Moreover, ADRP, perilipin, C/EBP-α, and PPAR-γ mRNA levels were also reduced by POCU1b. Levels of phosphorylated AMP-activated protein kinase (pAMPK) were elevated after POCU1b treatment (5, 10, and 25) in a dose-dependent manner. CONCLUSIONS: Taken together, these results suggest that the anti-obesity effects of POCU1b involve the inhibition of pancreatic lipase activity and adipogenesis via the down-regulation of lipid accumulation.

Effects of Allium victorialis leaf extracts and its single compounds on aldose reductase, advanced glycation end products and TGF-ß1 expression in mesangial cells.

BACKGROUND: Accumulating evidences suggest that aldose reductase (AR) inhibitors and advanced glycation end product (AGE) formation inhibitors may prevent chronic hyperglycemia-induced long-term complication in diabetes. Transforming growth factor-beta1 (TGF-ß1) plays an important role in the development of diabetic nephropathy. Allium species have been utilized in folk medicine throughout the world for the treatment of various physical disorders. However, the benefits of Allium victorialis (A. victorialis) against diabetic complications, especially nephropathy, have yet to be explored. In the present study, we investigated the protective effect of the compounds isolated from A. victorialis leaf on diabetic nephropathy. METHODS: In vitro AR activity, AGEs formation, and AGE-receptor for AGEs (RAGE) binding in human RAGE (hRAGE)-overexpressing cells were tested. High glucose-induced transforming growth factor-beta1 (TGF-ß1) expression was also examined in mouse kidney mesangial cells (MMCs) cultured under high glucose. RESULTS: Of the isolated eight compounds from A. victorialis leaf extracts tested, quercitrin exhibited the most pronounced inhibitory effects on AR activity (IC50 value of 0.17 µM) and AGEs formation (IC50 value of 4.20 µM). Furthermore, quercitrin disrupted AGE-RAGE binding in a concentration-dependent manner in hRAGE-overexpressing cells. Additionally, of the eight compounds tested, ferulic acid significantly reduced high glucose-induced TGF-ß1 expression and secretion in MMCs. CONCLUSIONS: Our results suggest that active compounds isolated from A. victorialis leaf exhibit inhibitory effects on AR activity in rat lenses and AGE formation. Further, ferulic acid reduces TGF-ß1 mRNA expression and secretion in MMCs under diabetic conditions. Thus, A. victorialis is a good candidate for the development of treatments for diabetic nephropathy.