Esculentoside H inhibits colon cancer cell migration and growth through suppression of MMP-9 gene expression via NF-kB signaling pathway.

A water-soluble saponin, Esculentoside H (EsH), 3-O-(O-ß-d-glucopyranosyl-(1→4)-ß-d-xylopyranosyl)-28-ß-d-glucopyranosylphytolaccagenin has been isolated and purified from the root extract of perennial plant Phytolacca esculenta. EsH is known to be an anticancer compound, having a capacity for TNF-α release. However, the effects of EsH on migration and growth in tumor cells have not yet been reported. In the current study, the suppressive effects of EsH on phorbol 12-myristate 13-acetate (PMA)-induced cell migration were examined in murine colon cancer CT26 cells and human colon cancer HCT116 cells. Interestingly, the transwell assay and wound healing show that EsH suppresses the PMA-induced migration and growth potential of HCT116 and CT26 colon cancer cells, respectively. EsH dose-dependently suppressed matrix metalloproteinases-9 (MMP-9) expression that was upregulated upon PMA treatment in messenger RNA levels and protein secretion. Since the expression of MMP-9 is correlated with nuclear factor-κB (NF-κB) signaling, it has been examined whether EsH inhibits PMA-induced IκB phosphorylation that leads to the suppression of NK-κB nuclear translocation. EsH repressed the phosphorylation level of JNK, but not extracellular signal-regulated kinase and p38 signaling when the cells were treated with PMA. Overall, these results demonstrated that EsH could suppress cancer migration through blockage of the JNK1/2 and NF-κB signaling-mediated MMP-9 expression.

Ganglioside GM3 suppresses lipopolysaccharide-induced inflammatory responses in rAW 264.7 macrophage cells through NF-κB, AP-1, and MAPKs signaling.

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase-2 (COX-2) protein and mRNA levels in lipopolysaccharide (LPS)-activated RAW 264.7 cells in a dose-dependent manner. Moreover, GM3 inhibited the expression and release of pro-inflammatory cytokines of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS-induced nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein (AP)-1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen-activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS-activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS-induced inflammatory response in RAW 264.7 macrophages by suppression of NF-κB, AP-1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.

Monosialyl Ganglioside GM3 Decreases Apolipoprotein B-100 Secretion in Liver Cells.

Some sialic acid-containing glycolipids are known to regulate development of atherosclerosis with accumulated plasma apolipoprotein B-100 (Apo-B)-containing lipoproteins, because Apo-B as an atherogenic apolipoprotein is assembled mainly in VLDL and LDL. Previously, we have elucidated that disialyl GD3 promotes the microsomal triglyceride transfer protein (MTP) gene expression and secretion of triglyceride (TG)-assembled ApoB, claiming the GD3 role in ApoB lipoprotein secretion in liver cells. In the synthetic pathway of gangliosides, GD3 is synthesized by addition of a sialic acid residue to GM3. Thus, there should be some regulatory links between GM3 and GD3. In this study, exogenous and endogenous monosialyl GM3 has been examined how GM3 plays a role in ApoB secretion in Chang liver cells in a view point of MTP and ApoB degradation in the same cells. The level of GM3 ganglioside in the GM3 synthase gene-transfected cells was increased in the cell extract, but not in the medium. In addition, GM3 synthase gene-transfected cells showed a diminished secretion of TG-enriched ApoB with a lower content of TG in the medium. Exogenous GM3 treatment for 24 h exerted a dose dependent inhibitory effect on ApoB secretion together with TG, while a liver-specific albumin was unchanged, indicating that GM3 effect is limited to ApoB secretion. GM3 decreased the mRNA level of MTP gene, too. ApoB protein assembly dysregulated by GM3 indicates the impaired ApoB secretion is caused by a proteasome-dependent pathway. Treatment with small interfering RNAs (siRNAs) decreased ApoB secretion, but GM3-specific antibody did not. These results indicate that plasma membrane associated GM3 inhibits ApoB secretion, lowers development of atherosclerosis by decreasing the secretion of TG-enriched ApoB containing lipoproteins, suggesting that GM3 is an inhibitor of ApoB and TG secretion in liver cells. J. Cell. Biochem. 118: 2168-2181, 2017. © 2017 Wiley Periodicals, Inc.

Exogenous and Endogeneous Disialosyl Ganglioside GD1b Induces Apoptosis of MCF-7 Human Breast Cancer Cells.

Gangliosides have been known to play a role in the regulation of apoptosis in cancer cells. This study has employed disialyl-ganglioside GD1b to apoptosis in human breast cancer MCF-7 cells using exogenous treatment of the cells with GD1b and endogenous expression of GD1b in MCF-7 cells. First, apoptosis in MCF-7 cells was observed after treatment of GD1b. Treatment of MCF-7 cells with GD1b reduced cell growth rates in a dose and time dependent manner during GD1b treatment, as determined by XTT assay. Among the various gangliosides, GD1b specifically induced apoptosis of the MCF-7 cells. Flow cytometry and immunofluorescence assays showed that GD1b specifically induces apoptosis in the MCF-7 cells with Annexin V binding for apoptotic actions in early stage and propidium iodide (PI) staining the nucleus of the MCF-7 cells. Treatment of MCF-7 cells with GD1b activated apoptotic molecules such as processed forms of caspase-8, -7 and PARP (Poly(ADP-ribose) polymerase), without any change in the expression of mitochondria-mediated apoptosis molecules such as Bax and Bcl-2. Second, to investigate the effect of endogenously produced GD1b on the regulation of cell function, UDP-gal: ß1,3-galactosyltransferase-2 (GD1b synthase, Gal-T2) gene has been transfected into the MCF-7 cells. Using the GD1b synthase-transfectants, apoptosis-related signal proteins linked to phenotype changes were examined. Similar to the exogenous GD1b treatment, the cell growth of the GD1b synthase gene-transfectants was significantly suppressed compared with the vector-transfectant cell lines and transfection activated the apoptotic molecules such as processed forms of caspase-8, -7 and PARP, but not the levels of expression of Bax and Bcl-2. GD1b-induced apoptosis was blocked by caspase inhibitor, Z-VAD. Therefore, taken together, it was concluded that GD1b could play an important role in the regulation of breast cancer apoptosis.

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.

Ganglioside GM3 participates in the TGF-ß1-induced epithelial-mesenchymal transition of human lens epithelial cells.

TGF-ß (transforming growth factor-ß)-induced EMT (epithelial-mesenchymal transition) induces the proliferation and migration of the HLE (human lens epithelial) cells. Ganglioside GM3, simple sialic-acid-containing glycosphingolipids on mammalian cell membranes, regulates various pathological phenomena such as insulin resistance and tumour progression. However, the relationship between ganglioside GM3 and TGF-ß-induced EMT in the HLE B-3 cells is poorly understood. In the present study we demonstrated that ganglioside GM3 was involved in TGF-ß1-induced EMT in HLE B-3 cells. Our results indicated that the expression of ganglioside GM3 and GM3 synthase mRNA were significantly increased in TGF-ß1-induced HLE B-3 cells. Reporter gene analysis also demonstrated that transcriptional activation of the GM3 synthase gene was regulated by Sp1 (specificity protein 1) in HLE B-3 cells upon TGF-ß1 stimulation. Interestingly, the inhibition of ganglioside GM3 expression by d-PDMP [d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol] and GM3 synthase shRNA (short hairpin RNA) resulted significantly in the suppression of cell migration and EMT-related signalling in HLE B-3 cells stimulated by TGF-ß. Furthermore, exogenous treatment of ganglioside GM3 rescued the expression of EMT molecules and cell migration suppressed by the depletion of ganglioside GM3 in TGF-ß1-induced HLE B-3 cells. We also found that ganglioside GM3 interacted with TGFßRs (TGF-ß receptors) in TGF-ß1-induced HLE B-3 cells. Taken together, these results suggest that ganglioside GM3 induced by TGF-ß1 regulates EMT by potential interaction with TGFßRs.

Implementation of Guideline-Based HBV Reactivation Management in Patients with Chronic HBV Infections of HBsAg or Resolved HBV Infection Undergoing Immunosuppressive Therapy.

INTRODUCTION: Although patients with HBV have a risk of reactivation after immunosuppressive therapy (IST), the status of their risk management is unclear in Japan. This study aims to describe the proportion of patients who received preventive management of HBV reactivation during ISTs in patients with chronic HBV infection of HBsAg or resolved HBV infection. METHOD: A retrospective cohort study was conducted using the JMDC Japanese claims database from April 2011 to June 2021. Patients with HBV infections of HbsAg who received ISTs or patients who had resolved HBV infections who received ISTs were identified from the database and evaluated for appropriate management to prevent HBV reactivation. RESULTS: In total, 6242 eligible patients were identified. The proportions of patients with appropriate HBV reactivation management, stratified by the HBV reactivation risk level of IST, was 43.1% (276/641) for high-risk, 40.2% (223/555) for intermediate-risk and 14.9% (741/4965) for low-risk patients. When the evaluation period for the outcome calculation was shortened from 360 to 180 days, the proportion for high risk increased to 52.7%. The odds ratios of large hospitals for receiving appropriate management were 2.16 (95% CI 1.12-4.44) in the high-risk, 4.63 (95% CI 2.34-10.25) in the intermediate-risk and 3.60 (95% CI 3.07-4.24) in the low-risk patients. CONCLUSION: HBV reactivation management was tailored according to the reactivation risk associated with IST. However, adherence to HBV reactivation management guidelines was sub-optimal, even among high-risk patients. This is especially the case for ensuring smaller-sized medical institutions, highlighting the need for further educational activities.

The study assesses the implementation of guideline-based management of hepatitis B virus reactivation during immunosuppressive therapy in Japan. The appropriate management of hepatitis B virus treatment involves prophylactic nucleos(t)ide analog (NUC) therapy and regular monitoring of hepatitis B virus DNA. This study aims to assess the extent to which these management practices are implemented in a clinical setting in Japan using a retrospective cohort study using the Japanese Medical Claims Database. The analysis identified 6242 eligible patients and identified whether they received appropriate management to prevent hepatitis B virus reactivation based on the level of risk associated with their immunosuppressive therapy. Based on the guidelines, the proportions of patients receiving appropriate reactivation management were 43.1% for high-risk, 40.2% for intermediate-risk and 14.9% for low-risk immunosuppressive therapy patients. Shortening the evaluation period from 360 to 180 days showed an increase in the proportion of high-risk patients to 52.7%, which indicated the potential challenge for continued monitoring after immunosuppressive therapy administration. The study shows that large hospitals present higher odds of patients receiving appropriate management. Overall, adherence to hepatitis B virus reactivation management guidelines was suboptimal, especially in smaller medical institutions, emphasizing the need for additional educational activities.

Comorbidities and the use of comedications among patients with chronic hepatitis C in Korea: A nationwide cross-sectional study.

BACKGROUND/AIMS: Chronic hepatitis C (CHC) is the second leading cause of liver-related mortality and is more prevalent in the elderly population in Korea. Decisions to initiate treatment and selection of proper antiviral agents may be challenging among elderly patients due to relevant comorbidities, comedications, and drug-drug interaction (DDI). It may be helpful to understand the current demographic status and comorbidities of CHC patients in the country. METHODS: Patients aged ≥ 18 years and diagnosed with CHC (KCD-7 code B18.2) were extracted from the Korean Health Insurance Review & Assessment Service database in 2018. Data on comorbidities and comedications were assessed and potential DDIs were analyzed. RESULTS: A total of 50,476 patients with CHC, with a mean age of 60.3 years and 46.7% male patients were identified. The proportion of patients with cirrhosis, hepatocellular carcinoma, and liver transplantation was 6.0%, 4.1%, and 0.3%, respectively and 37.2% of patients were more than 65 years of age. The three most common comorbidities were diseases of the digestive system (83.7%), respiratory system (58.2%), and musculoskeletal system and connective tissue (57.6%). The three most common comedications were analgesics (91.6%), gastrointestinal agents (85%), and antibacterials (80.3%). Lipid-lowering agents and anticonvulsants were prescribed in 28.5% and 14.8% of patients. Rate of potential DDI for contraindication was 2.2%, 13.1%, and 15.6% with sofosbuvir/velpatasvir, ledipasvir/sofosbuvir, and glecaprevir/pibrentasvir. CONCLUSION: With the increasing age of patients with CHC, comorbidity, comedication, and potential DDI should be considered when choosing antivirals in Korea. Sofosbuvir-based regimens showed favorable DDI profiles among Korean patients.

Efficacy and safety of sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir for hepatitis C in Korea: a Phase 3b study.

BACKGROUND/AIMS: Despite the availability of direct-acting antivirals (DAAs) for chronic hepatitis C virus (HCV) infection in Korea, need remains for pangenotypic regimens that can be used in the presence of hepatic impairment, comorbidities, or prior treatment failure. We investigated the efficacy and safety of sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir for 12 weeks in HCV-infected Korean adults. METHODS: This Phase 3b, multicenter, open-label study included 2 cohorts. In Cohort 1, participants with HCV genotype 1 or 2 and who were treatment-naive or treatment-experienced with interferon-based treatments, received sofosbuvir-velpatasvir 400/100 mg/day. In Cohort 2, HCV genotype 1 infected individuals who previously received an NS5A inhibitor-containing regimen ≥ 4 weeks received sofosbuvir-velpatasvir-voxilaprevir 400/100/100 mg/day. Decompensated cirrhosis was an exclusion criterion. The primary endpoint was SVR12, defined as HCV RNA < 15 IU/mL 12 weeks following treatment. RESULTS: Of 53 participants receiving sofosbuvir-velpatasvir, 52 (98.1%) achieved SVR12. The single participant who did not achieve SVR12 experienced an asymptomatic Grade 3 ASL/ALT elevation on day 15 and discontinued treatment. The event resolved without intervention. All 33 participants (100%) treated with sofosbuvir-velpatasvir-voxilaprevir achieved SVR 12. Overall, sofosbuvir-velpatasvir and sofosbuvir-velpatasvir-voxilaprevir were safe and well tolerated. Three participants (5.6%) in Cohort 1 and 1 participant (3.0%) in Cohort 2 had serious adverse events, but none were considered treatment-related. No deaths or grade 4 laboratory abnormalities were reported. CONCLUSION: Treatment with sofosbuvir-velpatasvir or sofosbuvir-velpatasvir-voxilaprevir was safe and resulted in high SVR12 rates in Korean HCV patients.

Citreorosein inhibits production of proinflammatory cytokines by blocking mitogen activated protein kinases, nuclear factor-κB and activator protein-1 activation in mouse bone marrow-derived mast cells.

Citreorosein (CIT), an anthraquinone component of Polygoni cuspidati (P. cuspidati) radix, suppressed gene expression of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the molecular mechanisms underlying CIT inhibition of the pro-inflammatory cytokine production, its effects on the activation of both nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were assessed. CIT attenuated phosphorylation of the MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase and c-Jun NH(2)-terminal kinase (JNK). Furthermore, CIT strongly inhibited DNA binding activity of NF-κB through the inhibition of phosphorylation and degradation of inhibitor of kappaB (IκB) as well as activator protein-1 (AP)-1 through the reduction of phosphorylation of c-Jun. These results demonstrate that CIT inhibits proinflammatory cytokines production through the inhibition of both MAPKs and AKT-mediated IκB kinase (IKK) phosphorylation and subsequent inhibition of transcription factor NF-κB activation, thereby attenuating the production of proinflammatory cytokines.

Immunocytochemical localization of the AMPA glutamate receptor subtype GluR2/3 in the squid optic lobe.

As a major excitatory neurotransmitter in the cephalopod visual system, glutamate signaling is facilitated by ionotropic receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR). In cephalopods with large and well-developed brains, the optic lobes (OL) mainly process visual inputs and are involved in learning and memory. Although the presence of AMPAR in squid OL has been reported, the organization of specific AMPAR-containing neurons remains unknown. This study aimed to investigate the immunocytochemical localization of the AMPA glutamate receptor subtype 2/3-immunoreactive (GluR2/3-IR) neurons in the OL of Pacific flying squid (Tordarodes pacificus). Morphologically diverse GluR2/3-IR neurons were predominantly located in the tangential zone of the medulla. Medium-to-large GluR2/3-IR neurons were also detected. The distribution patterns and cell morphologies of calcium-binding protein (CBP)-IR neurons, specifically calbindin-D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-IR neurons, were similar to those of GluR2/3-IR neurons. However, two-color immunofluorescence revealed that GluR2/3-IR neurons did not colocalize with the CBP-IR neurons. Furthermore, the specific localizations and diverse types of GluR2/3-IR neurons that do not express CB, CR, or PV in squid OL were determined. These findings further contribute to the existing data on glutamatergic visual systems and provide new insights for understanding the visual processing mechanisms in cephalopods.

The Organization of Somatostatin-Immunoreactive Cells in the Visual Cortex of the Gerbil.

Somatostatin (SST) is widely expressed in the brain and plays various, vital roles involved in neuromodulation. The purpose of this study is to characterize the organization of SST neurons in the Mongolian gerbil visual cortex (VC) using immunocytochemistry, quantitative analysis, and confocal microscopy. As a diurnal animal, the Mongolian gerbil provides us with a different perspective to other commonly used nocturnal rodent models. In this study, SST neurons were located in all layers of the VC except in layer I; they were most common in layer V. Most SST neurons were multipolar round/oval or stellate cells. No pyramidal neurons were found. Moreover, 2-color immunofluorescence revealed that only 33.50%, 24.05%, 16.73%, 0%, and 64.57% of SST neurons contained gamma-aminobutyric acid, calbindin-D28K, calretinin, parvalbumin, and calcium/calmodulin-dependent protein kinase II, respectively. In contrast, neuropeptide Y and nitric oxide synthase were abundantly expressed, with 80.07% and 75.41% in SST neurons, respectively. Our immunocytochemical analyses of SST with D1 and D2 dopamine receptors and choline acetyltransferase, α7 and ß2 nicotinic acetylcholine receptors suggest that dopaminergic and cholinergic fibers contact some SST neurons. The results showed some distinguishable features of SST neurons and provided some insight into their afferent circuitry in the gerbil VC. These findings may support future studies investigating the role of SST neurons in visual processing.

Immunocytochemical localization of tyrosine hydroxylase in the visual cortex of the microbat, Rhinolophus ferrumequinum.

INTRODUCTION: In order to enhance our understanding of bat vision, we investigated tyrosine hydroxylase (TH)-immunoreactive (IR) fibers in the visual cortex of the microbat. MATERIAL AND METHODS: The study was conducted on 12 freshly-caught adult bats (Rhinolophus ferrumequinum, both sexes, weighing 15-20 g). We used standard immunocytochemistry and confocal microscopy. RESULTS: TH-IR fibers were distributed throughout all layers of the visual cortex, with the highest density in layer I. Two types of TH-IR fibers were observed: small and large varicose fibers. TH-IR cells were not found in the microbat visual cortex. The microbat substantia nigra and ventral tegmental areas, previously identified sources of TH-IR fibers in the mammalian visual cortex, all contained strongly labeled TH-IR cells. The average diameters of TH-IR cells in the substantia nigra and the ventral tegmental areas were 14.39 ± 0.13 µm (mean ± SEM) and 11.85 ± 0.13 µm, respectively. CONCLUSIONS: Our results suggest that the microbat has a well-constructed neurochemical organization of THIR fibers. This observation should provide fundamental insights into a better understanding of the nocturnal, echolocating bat visual system.

Association of Lung Function with Serum 25-Hydroxyvitamin D Level according to the Presence of Past Pulmonary Tuberculosis in Korean Adults.

BACKGROUND: Vitamin D deficiency is associated with an increased risk of pulmonary tuberculosis (PTB) infection and the treatment outcome. The aim of this study was to examine the relationship between the serum 25-hydroxyvitamin D (25[OH]D) level and lung function in Korean adults according to whether or not there is a history of PTB. METHODS: The data for subjects aged 19 years or older from the Korea National Health and Nutrition Examination Survey 2008-2012 who underwent spirometry, chest radiography, and serum 25(OH)D level measurement were analyzed. RESULTS: Evidence of past PTB infection was found in 1,482 (9.6%) of 15,516 subjects. The serum 25(OH)D level was lower in the group with past PTB than in the non-PTB group (P=0.013). Respiratory dysfunction was more common in the past PTB group than in the non-PTB group (restrictive pattern, 14.0% vs. 9.6%; obstructive pattern, 29.6% vs. 8.2%; both P<0.001). After adjusting for age, sex, height, and season, the mean difference in forced expiratory volume in 1 second (FEV1) between the highest and lowest quartiles of 25(OH)D was 100.2 mL (standard error=49.3 mL, P for trend=0.049) in the past PTB group and 34.7 mL (standard error=13.6 mL, P=0.009) in the nonPTB group. CONCLUSION: FEV1 tended to increase as the vitamin D quartile increased in both study groups. This relationship was more pronounced in subjects with a history of PTB. A higher serum 25(OH)D level might be beneficial in preserving lung function after PTB infection.

Propolis induces cell cycle arrest and apoptosis in human leukemic U937 cells through Bcl-2/Bax regulation.

We investigated mechanism(s) where propolis induces apoptosis in human leukemic U937 cells. Propolis inhibited the proliferation of U937 cells in a dose-dependent manner by inducing apoptosis and blocking cell cycle progression in the G2/M phase. Western blot analysis showed that propolis increases the expression of p21 and p27 proteins, and decreases the levels of cyclin B1, cyclin A, Cdk2 and Cdc2, thereby contributing to cell cycle arrest. DAPI staining assay revealed typical morphology features of apoptotic cells. Propolis-induced apoptosis was also confirmed by assays with annexin V-FITC, PI-labeling and DNA fragmentation assay. The increase in apoptosis level induced by propolis was associated with down-regulation of Bcl-2 and activation of caspase-3, but not with Bax. These results suggests that propolis-induced apoptosis is related to the selective activation of caspase-3 and induction of Bcl-2/Bax regulation.

Association between Picky Eating Behaviors and Nutritional Status in Early Childhood: Performance of a Picky Eating Behavior Questionnaire.

Picky eating behaviors are frequently observed in childhood, leading to concern that an unbalanced and inadequate diet will result in unfavorable growth outcomes. However, the association between picky eating behaviors and nutritional status has not been investigated in detail. This study was conducted to assess eating behaviors and growth of children aged 1-5 years from the Seoul Metropolitan area. Primary caregivers completed self-administered questionnaires and 3-day diet records. Differences in the nutrient intake and growth indices between picky and non-picky eaters were tested by analysis of covariance. Children "eating small amounts" consumed less energy and micronutrients (with the exception of calcium intake), but picky behaviors related to a "limited variety" resulted in a significant difference regarding nutrient density for some micronutrients. Children with the behavior of "eating small amounts" had a lower weight-for-age than that of non-picky eaters; especially, the older children with the behaviors of "eating small amounts" or "refusal of specific food groups" had lower height-for-age compared with non-picky eaters. These results suggest that specific picky eating behaviors are related to different nutrient intake and unfavorable growth patterns in early childhood. Thus, exploration of potential interventions according to specific aspects of picky eating and their efficacy is required.

Ascofuranone inhibits lipopolysaccharide-induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1ß in RAW 264.7 macrophages.

The natural fungal compound ascofuranone (5-chloro-3-[(2E,6E)-7-[(2S)-5,5-dimethyl-4-oxo-tetrahydrofuran-2-yl]-3-methyl-octa-2,6-dienyl]-2,4-dihydroxy-6-methyl-benzaldehyde, MW 420.93) (AF) isolated from Ascochyta viciae has been known to promote cell cycle arrest and inhibit invasion of tumor cells. We have previously studied a structurally similar compound ascochlorin (ASC; MW 404.93) with regard to its anti-inflammatory activity in LPS- stimulated RAW 264.7 macrophages. In order to examine the relationship between the anti-inflammatory activities and the molecular differences between AF and ASC, the activity of AF is herein studied, because ASC has a unique trimethyl oxocyclohexyl structure, while AF has a unique dimethyl-oxo-tetrahydrofuran structure. AF dose-dependently inhibited the production of NO and iNOS and the COX-2 mRNA and protein levels in RAW 264.7 cells. In addition, AF suppressed mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and IL-1ß, as assessed by RT-PCR. AF (30-50 µg/ml) treatment clearly inhibited the nuclear translocation of NF-κB, AP-1 (p-c-Jun) from the cytosolic space. Phosphorylation of IκB, which functions to maintain the activity of NF-κB, was decreased by AF treatment. Moreover, AF suppressed the binding of NF-κB (p65). Inhibition of IkBa phosphorylation and degradation inhibits nuclear translocation of p65. Immunofluorescence confocal microscopy analysis also revealed that translocation of NF-κB and AP-1 (p-c-Jun) was decreased upon AF treatment. AF specifically decreased the expression level of p-ERK, but not the expression level of p-p38 or p-JNK. Given these results, we suggest that AF suppresses the inflammatory response by targeting p-ERK. This indicates that AF is a negative regulator of LPS-stimulated nuclear translocation of NF-κB and AP-1 (p-c-Jun) in RAW 264.7 macrophages, and specifically it targets p-ERK. Therefore, AF and ASC exert their effects in different ways, most probably because their structural differences allow for specific recognition and inhibition of their target MAPKs. Our results further suggest that AF could be a natural bioactive compound useful for treating inflammation-mediated pathological diseases.

Disialyl GD2 ganglioside suppresses ICAM-1-mediated invasiveness in human breast cancer MDA-MB231 cells.

The disialoganglioside GD3 has been considered to be involved in tumor progression or suppression in various tumor cells. However, the significance of the biological functions of GD3 in breast cancer cells is still controversial. This prompted us to study the possible relationship(s) between GD3 expression and the metastatic potential of a breast cancer MDA-MB231 cells as an estrogen receptor negative (ER-) type. The human GD3 synthase cDNA was transfected into MDA-MB231 cells, and G-418 bulk selection was used to select cells stably overexpressing the GD3 synthase. In vitro invasion potentials of the GD3 synthase over-expressing cells (pc3-GD3s) were significantly suppressed when compared with control cells. Expression of intercellular adhesion molecule-1 (ICAM-1; CD54) was down-regulated in the pc3-GD3s cells and the decrease in ICAM-I expression is directly related to the decrease in invasiveness of the pc3-GD3s cells. Another type of ER negative SK-BR3 cells exhibited the similar level of ICAM-1 expression as MDA-MB231 cells, while the ER positive MCF-7 cells (ER+) showed the increased expression level of ICAM-1. Then, we investigated signaling pathways known to control ICAM-1 expression. No difference was observed in the phosphorylation of ERK and p38 between the pc3-GD3s and control cells (pc3), but the activation of AKT was inhibited in pc3-GD3s, and not in the control (pc3). In addition, the composition of total gangliosides was changed between control (pc3) and pc3-GD3s cells, as confirmed by HPTLC. The pc3-GD3s cells had an accumulation of the GD2 instead of the GD3. RT-PCR results showed that not only GD3 synthase, but also GM2/GD2 synthase (ß4-GalNc T) expression was increased in pc3-GD3s cells. Overexpression of GD3 synthase suppresses the invasive potential of human breast cancer MDA-MB-231 cells through down-regulation of ICAM-1 and the crucial pathway to allow the apoptotic effect has been attributed to accumulation of the GD2 ganglioside. ER has been linked to the ICAM-1 expression with GD3 to GD2 conversion in human breast cancer cells. This is the first finding of the endogenous sialyltransferase functions in tumor cells.

Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells.

The disialic acid-containing glycosphingolipid GD3 recruited membrane transglutaminase 2 (TG2) as a signaling molecule for erythroid differentiation in human chronic myelogenous leukemia (CML) K562 cells. The α1-adrenergic receptor (α1-AR)/TG2-mediated signaling pathway regulated GD3 functions, including gene expression and production, to differentiate CML K562 cells into erythroid lineage cells. Epinephrine, an AR agonist, increased membrane recruitment as well as GTP-photoaffinity of TG2, inducing GD3 synthase gene expression. Epinephrine activated PI3K/Akt signaling and GTPase downstream of TG2 activated Akt. The coupling of TG2 and GD3 production was specifically suppressed by prazosin (α1-AR antagonist), but not by propranolol (ß-AR antagonist) or rauwolscine (α2-AR antagonist), indicating α1-AR specificity. Small interfering RNA (siRNA) experiment results indicated that the α1-AR/TG2-mediated signaling pathway activated PKCs α and δ to induce GD3 synthase gene expression. Transcription factors CREB, AP-1, and NF-κB regulated GD3 synthase gene expression during α1-AR-induced differentiation in CML K562 cells. In addition, GD3 synthase gene expression was upregulated in TG2-transfected cells via α1-AR with expression of erythroid lineage markers and benzidine-positive staining. α1-AR/TG2 signaling pathway-directed GD3 production is a crucial step in erythroid differentiation of K562 cells and GD3 interacts with α1-AR/TG2, inducing GD3/α1-AR/TG2-mediated erythroid differentiation. These results suggest that GD3, which acts as a membrane mediator of erythroid differentiation in CML cells, provides a therapeutic avenue for leukemia treatment.

Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR.

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90kDa consistent with the 90,470Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70-75°C. The enzyme possessed 3'→5' exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.