Protective effects of citrus based mixture drinks (CBMDs) on oxidative stress and restraint stress.

In the current study investigated the protective effects of citrus based mixture drinks (CBMDs) using oxidative stress in human dermal fibroblast (HDF) cells and restraint-stressed rats. The CBMDs contained citrus bioflavonoids including narirutin and hesperidin. The cell viability of HDF cells treated with H2O2 was observed at 53.9% but treated with CBMD-1 and CBMD-2 (500 µg/mL) on H2O2 exposed HDF cells significantly increased the relative cell viability at 65.0 and 72.2%, respectively. In the treadmill test, the time spent on the electrode plate in the restraint-stressed group was analyzed 24.1 s, but restraint-stressed rats with administered CBMDs (300 mg/kg) had significantly decreased the time at 2.4 (CBMD-1) and 4.7 (CBMD-2) s, respectively. In addition, number of touches the electrode plate in restraint-stressed group was observed at 42.4 ea, but, restraint-stressed rats with administered CBMD-1 and CBMD-2 (300 mg/kg) were significantly decreased at 7.0 and 10.2 ea, respectively.

Toxicological Evaluation of Brassica napus Extract Containing Vanadium, Nutricultured in Jeju Water.

This study evaluated the mutagenicity and acute toxicity of the juice extract of nutricultured Brassica napus containing vanadium (BECV). The BECV was prepared by nutriculture for 7 days in Jeju water containing vanadium. The mutagenic effects of BECV were investigated using the bacterial reverse mutation test, chromosome aberration test, and micronucleus test. Based on the results of the mutagenicity test, we propose that BECV is not a mutagenicity-inducing agent. In the acute oral toxicity study, male and female Sprague-Dawley rats were administered a single limiting dose of 0.014, 0.14, or 1.4 µg BECV/kg body weight; the rats were then observed for 7 days. No acute lethal effect was observed at the maximal dose of 1.4 µg BECV/kg body weight. In the subacute study, male and female rats were administered once daily, by oral gavage, a dose of 0.028, 0.14, and 0.7 µg/kg body weight of BECV for 28 days. No significant toxicity was observed not only hematological, biochemical, and pathological parameters but also the body and organ weights when compared to controls. The level of BECV with no observed adverse effects in male and female rats was 0.7 µg/kg body weight (concentration of vanadium in BECV) in the subacute toxicity study.

The Anticancer Effects of Garlic Extracts on Bladder Cancer Compared to Cisplatin: A Common Mechanism of Action via Centromere Protein M.

Although garlic induces apoptosis in cancer cells, it is unclear whether the effects are similar to those of cisplatin against bladder cancer (BC). Therefore, this study investigated whether garlic extracts and cisplatin show similar activity when used to treat BC. The effect of garlic on T24 BC cell line was examined in a BALB/C-nude mouse xenograft model and compared with that of cisplatin. Tissue microarray analysis and gene network analysis were performed to identify differences in gene expression by control tumors and tumors exposed to garlic extract or cisplatin. Investigation of gene expression based on tissues from 165 BC patients and normal controls was then performed to identify common targets of garlic and cisplatin. Tumor volume and tumor weight in cisplatin (0.05[Formula: see text]mg/kg)- and garlic-treated mice were significantly smaller than those in negative control mice. However, cisplatin-treated mice also showed a significant reduction in body weight. Microarray analysis of tumor tissue identified 515 common anticancer genes in the garlic and cisplatin groups ([Formula: see text]). Gene network analysis of 252 of these genes using the Cytoscape and ClueGo software packages mapped 17 genes and 9 gene ontologies to gene networks. BC (NMIBC and MIBC) patients with low expression of centromere protein M (CENPM) showed significantly better progression-free survival than those with high expression. Garlic extract shows anticancer activity in vivo similar to that of cisplatin, with no evident of side effects. Both appear to act by targeting protein-DNA complex assembly; in particular, expression of CENPM.

Garlic extract in bladder cancer prevention: Evidence from T24 bladder cancer cell xenograft model, tissue microarray, and gene network analysis.

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer (BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165 BC patients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20 mg/kg (p<0.05), 200 mg/kg, and 1000 mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36 genes and 37 gene ontologies were mapped to gene networks. Protein kinase A (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity in vivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

Protein Expression Level of Skin Wrinkle-Related Factors in Hairless Mice Fed Hyaluronic Acid.

The aim of this study was to evaluate the wrinkle improving effect of hyaluronic acid intakes. Wrinkles were induced by exposing the skin of hairless mice to ultraviolet B (UVB) irradiation for 14 weeks. Hyaluronic acid was administered to the mice for 14 weeks including 4 weeks before experiments. Skin tissue was assayed by enzyme-linked immunosorbent assay to determine protein expression of wrinkle-related markers. The group supplemented with high concentrations of hyaluronic acid appeared significantly better than control group for collagen, matrix metalloproteinase 1, interleukin (IL)-1ß, and IL-6 assay. Transforming growth factor-ß1 (TGF-ß1) and hyaluronic acid synthase 2 (HAS-2) were not shown to be significantly different. In conclusion, hyaluronic acid administration regulated expression levels of proteins associated with skin integrity, and improved the wrinkle level in skin subjected to UVB irradiation.

HSP70-1 is required for interleukin-5-induced angiogenic responses through eNOS pathway.

We report a pivotal role for IL-5 as an angiogenic activator. IL-5 increased proliferation, migration and colony tube formation in HUVECs associated with the phosphorylation of ERK and AKT/eNOS, and promoted microvessel sprouting from an angiogenesis animal model. The angiogenic effects were confirmed in IL-5-deficient mice and addition of IL-5 antibody. HSP70-1 was identified via expression profiling following IL-5 stimulation. A siRNA knockdown of HSP70-1 suppressed angiogenic responses and eNOS phosphorylation induced by IL-5. HSP70-1 overexpression enhanced IL-5-induced angiogenic responses. In addition, IL-5-induced neo-vascular formation was verified in both HSP70-1 knockout and HSP70-1 transgenic mice. Furthermore, transcription factor AP-1 was a main factor in IL-5-induced HSP70-1 in response to ERK and AKT signaling pathway. Angiogenic responses induced by VEGF had no effect in either HSP70-1 siRNA in vitro or HSP70-1 knockout mice. IL-5-induced angiogenic responses depended on the binding of IL-5Rα. Our data demonstrate that binding of IL-5 to IL-5Rα receptors enhances angiogenic responses by stimulating the expression of HSP70-1 via the eNOS signaling pathway.

Reduced immunoreactivities of B-type natriuretic peptide in pulmonary arterial hypertension rats after ranolazine treatment.

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by sustained increase in the pulmonary arterial pressure and excessive thickening and remodeling of the distal small pulmonary arteries. During disease progression, structural remodeling of the right ventricular (RV) impairs pump function, creates pro-arrhythmic substrates and triggers for arrhythmias. Notably, RV failure and lethal arrhythmias are major contributors to cardiac death in PAH that are not directly addressed by currently available therapies. Ranolazine (RAN) is an anti-anginal, anti-ischemic drug that has cardioprotective effects of heart dysfunction. RAN also has anti-arrhythmic effects due to inhibition of the late sodium current in cardiomyocytes. Therefore, we hypothesized that RAN could reduce the mal-adaptive structural remodeling of the RV, and prevent triggered ventricular arrhythmias in the monocrotaline-induced rat model of PAH. RAN reduced ventricular hypertrophy, reduced levels of B-type natriuretic peptide, and decreased the expression of fibrosis. In addition, RAN prevented cardiovascular death in rat model of PAH. These results support the notion that RAN can improve the functional properties of the RV, highlighting its potential benefits in the setting of heart impairment.

An ethanol extract of Ramulus mori improves blood circulation by inhibiting platelet aggregation.

Inappropriate platelet aggregation can cause blood coagulation and thrombosis. In this study, the effect of an ethanol extract of Ramulus mori (ERM) on blood circulation was investigated. The antithrombotic activity of ERM on rat carotid arterial thrombosis was evaluated in vivo, and the effect of ERM on platelet aggregation and blood coagulation time was evaluated ex vivo. To evaluate the safety of ERM, its cytotoxicity to platelets and its effect on tail bleeding time were assessed; ERM was not toxic to rat platelets and did not prolong bleeding time. Moreover, administering ERM to rats had a significant preventive effect on carotid arterial thrombosis in vivo, and significantly inhibited adenosine diphosphate- and collagen-induced platelet aggregation ex vivo, whereas it did not prolong coagulation periods, such as prothrombin time and activated partial thromboplastin time. The results suggest that ERM is effective in improving blood circulation via antiplatelet activity rather than anticoagulation activity.

Antioxidant and anti-ageing activities of citrus-based juice mixture.

The production of excessive reactive oxygen species by exposure to oxidative stress and solar radiation are primary factors in skin damage. We examined the effects of a citrus-based juice mixture and its bioactive compounds on antioxidant and anti-ageing activities in human dermal fibroblasts and hairless mice via the regulation of antioxidant enzymes and the mitogen-activated protein kinase pathway. The citrus-based juice mixture reduced H2O2-induced cell damage and intracellular reactive oxygen species production in human dermal fibroblasts. Citrus-based juice mixture pretreatment suppressed the activation of the H2O2-mediated mitogen-activated protein kinase pathway by activating the expression of activator protein 1 and matrix metalloproteinases. Moreover, it increased the expression levels of antioxidant enzymes such as glutathione reductase, catalase and manganese superoxide dismutase. In addition, oral administration of the citrus-based juice mixture decreased skin thickness and wrinkle formation and increased collagen content on an ultraviolet light B-exposed hairless mouse. These results indicate that the citrus-based juice mixture is a potentially healthy beverage for the prevention of oxidative stress-induced premature skin ageing.

Therapeutic Effects of Umbilical Cord Blood Derived Mesenchymal Stem Cell-Conditioned Medium on Pulmonary Arterial Hypertension in Rats.

BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) may have multiple therapeutic applications for cell based therapy including the treatment of pulmonary artery hypertension (PAH). As low survival rates and potential tumorigenicity of implanted cells could undermine the mesenchymal stem cell (MSC) cell-based therapy, we chose to investigate the use of conditioned medium (CM) from a culture of MSC cells as a feasible alternative. METHODS: CM was prepared by culturing hUCB-MSCs in three-dimensional spheroids. In a rat model of PAH induced by monocrotaline, we infused CM or the control unconditioned culture media via the tail-vein of 6-week-old Sprague-Dawley rats. RESULTS: Compared with the control unconditioned media, CM infusion reduced the ventricular pressure, the right ventricle/(left ventricle+interventricular septum) ratio, and maintained respiratory function in the treated animals. Also, the number of interleukin 1α (IL-1α), chemokine (C-C motif) ligand 5 (CCL5), and tissue inhibitor of metalloproteinase 1 (TIMP-1)-positive cells increased in lung samples and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique (TUNEL)-positive cells decreased significantly in the CM treated animals. CONCLUSIONS: From our in vivo data in the rat model, the observed decreases in the TUNEL staining suggest a potential therapeutic benefit of the CM in ameliorating PAH-mediated lung tissue damage. Increased IL-1α, CCL5, and TIMP-1 levels may play important roles in this regard.

Microarray analysis after umbilical cord blood derived mesenchymal stem cells injection in monocrotaline-induced pulmonary artery hypertension rats.

Pulmonary arterial hypertension (PAH) is associated with structural alterations of lung vasculature. PAH is still a devastating disease needing an aggressive therapeutic approach. Despite the therapeutic potential of human umbilical cord mesenchymal stem cells (MSCs), the molecular parameters to define the stemness remain largely unknown. Using high-density oligonucleotide microarrays, the differential gene expression profiles between a fraction of mononuclear cells of human umbilical cord blood (UCB) and its MSC subpopulation were obtained. Of particular interest was a subset of 46 genes preferentially expressed at 7-fold or higher in the group treated with human UCB-MSCs. This subset contained numerous genes involved in the inflammatory response, immune response, lipid metabolism, cell adhesion, cell migration, cell differentiation, apoptosis, cell growth, transport, cell proliferation, transcription, and signal transduction. Our results provide a foundation for a more reproducible and reliable quality control using genotypic analysis for the definition of human UCB-MSCs. Therefore, our results will provide a basis for studies on molecular mechanisms controlling the core properties of human MSCs.

Region-specific changes in the immunoreactivity of Atg9A in the central nervous system of SOD1(G93A) transgenic mice.

Autophagy is a eukaryotic self-degradation system that plays a pivotal role in the maintenance of cellular homeostasis. Atg9 is the only transmembrane Atg protein required for autophagosome formation. Although the subcellular localization of the Atg9A has been examined, little is known about its precise cell and tissue distribution. In the present study, we used G93A mutation in superoxide dismutase 1 [SOD1(G93A)] mutant transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of Atg9A immunoreactivity in the central nervous system of these mice. Atg9A-immunoreactivity was detected in the spinal cord, cerebral cortex, hippocampal formation, thalamus and cerebellum of symptomatic SOD1(G93A) transgenic mice. By contrast, no Atg9A-immunoreactivity were observed in any brain and spinal cord region of wtSOD1, pre-symptomatic and early symptomatic mice, and the number and staining intensity of Atg9A-positive cells did not differ in SOD1(G93A) mice between 8 and 13 weeks of age. These results provide evidence that Atg9A-immunoreactivity were found in the central nervous system of SOD1(G93A) transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for Atg9A and the functional implications require elucidation.

Age-related changes in the distribution of transient receptor potential vanilloid 4 channel (TRPV4) in the central nervous system of rats.

Transient receptor potential vanilloid type 4 (TRPV4) channels are expressed in the central nervous system, but their role in regulating the aging process under physiological and pathological conditions is still largely unknown. To identify age-related changes in the TRPV4 channel that contribute to the central nervous system, we investigated the distribution of TRPV4 in the brain and spinal cord regions of adult and aged rats. The expression of TRPV4 in the brain and spinal cord of adult and aged Sprague-Dawley rats was compared using immunohistochemistry performed with antibodies recognizing TRPV4 on free floating sections and western blotting analysis. TRPV4 immunoreactivity was significantly increased in the cerebral cortex, hippocampal formation, thalamus, basal nuclei, cerebellum and spinal cord of aged rats compared with adult control rats. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of aged rats. In addition, TRPV4 immunoreactivity was increased in the spinal cord, hippocampal formation, thalamus, basal nuclei and cerebellum of aged rats. This first demonstration of age-related increases in TRPV4 expression in the brain and spinal cord may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases. The exact regulatory mechanism and its functional significance require further elucidation.

Region-specific changes in the immunoreactivity of TRPV4 expression in the central nervous system of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis.

Transient receptor potential vanilloid 4 (TRPV4) is a broadly expressed Ca(2+)-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. It is activated by warm temperature, lipids downstream of arachidonic acid metabolism, hypoosmolarity, or mechanical stimulation. In the present study, we used SOD1(G93A) mutant transgenic mice as the animal model of amyotrophic lateral sclerosis (ALS) and investigated the changes of TRPV4 immunoreactivity in the central nervous system of these mice by immunohistochemical studies. An increased expression of TRPV4 was pronounced in the cerebral cortex, hippocampal formation, thalamus, cerebellum and spinal cord of symptomatic SOD1(G93A) transgenic mice. In the cerebral cortex, TRPV4 immunoreactivity was significantly increased in pyramidal cells of SOD1(G93A) transgenic mice. In the hippocampal formation, pyramidal cells of the CA1-3 areas and in the granule cells of the dentate gyrus demonstrated increased TRPV4 immunoreactivity. In addition, TRPV4 immunoreactivity was increased in the spinal cord, thalamus and cerebellum of the symptomatic SOD1(G93A) transgenic mice. This study, which showed increased TRPV4 in different brain and spinal cord regions of SOD1(G93A) transgenic mice, may provide clues to the understanding of many basic neuronal functions in ALS. These findings suggest a role for TRPV4 in the neuronal functions in ALS but the mechanisms and functional implications of increased TRPV4 require elucidation.

DNA aptamers against the receptor binding region of hemagglutinin prevent avian influenza viral infection.

The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular region (residues 101-257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells.

Biological activities of Fructus arctii fermented with the basidiomycete Grifola frondosa.

Fructus arctii extract containing phenolic glycosides was cultured with Grifola frondosa mycelia to produce ß-glucosidase and its biological activities were studied. This ß-glucosidase converted the glycosides (arctiin and caffeic acid derivatives) into aglycones (arctigenin and caffeic acid). Fermented Fructus arctii extract (G-FAE) with G. frondosa had antioxidant and 5-lipoxygenase inhibitory activities. The photoprotective potential of G-FAE was tested in human dermal fibroblasts (HDF) exposed to ultra-violet A (UVA). It was revealed that G-FAE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF. The treatment of UVA-irradiated HDF with G-FAE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. G-FAE also showed notable stimulation of collagen biosynthetic activity for fibroblasts. These diverse functionalities suggest that G-FAE could be a promising cosmetic ingredient.

Anti-inflammatory effects of an ethanol extract of Angelica gigas in a Carrageenan-air pouch inflammation model.

Anti-inflammatory effects of an ethanol extract of Angelica gigas (EAG; 50, 160, or 500 mg/kg) were investigated in a carrageenan-induced air pouch inflammation model. Injection of 1 ml of carrageenan (1%) into mouse air pouches markedly increased the exudate volume and exudate albumin concentration, which were significantly attenuated by oral pretreatment with EAG. EAG also markedly reduced carrageenan-induced infiltrations of neutrophils, monocytes, and lymphocytes, but did not influence eosinophils or basophils. Carrageenan dramatically increased levels of tumor necrosis factor-alpha and interleukin-6, which might be derived from the infiltrated cells. It also elevated nitric oxide, and slightly increased prostaglandin E(2). EAG pretreatment significantly lowered tumor necrosis factor-alpha and nitric oxide, but did not alter interleukin-6 or prostaglandin E(2) levels. These results indicate that EAG attenuates some inflammatory responses by blocking the tumor necrosis factor-alpha-nitric oxide pathway, and that EAG could be a promising anti-inflammatory drug candidate for inflammatory diseases.

Identification of the nuclear localization motif in the ETV6 (TEL) protein.

ETV6, or Translocation-Ets-Leukemia (TEL), is an ETS family transcriptional repressor that is essential for establishing hematopoiesis in neonatal bone marrow, and is frequently a target of chromosomal translocations in human cancer. ETV6 is predominantly a nuclear phosphoprotein that represses transcription by binding directly to the promoters of target genes. The nuclear localization mechanism of ETV6, however, is not well understood. In this report, we provide evidence that a nuclear localization signal (NLS) exists in the C-terminal region of ETV6. ETV6 proteins with mutations outside of amino acids 332-452 localize to the nucleus, whereas proteins with mutations within amino acids 332-452 remain in the cytoplasm. Furthermore, when a fragment of ETV6 comprised of amino acids 332-452 was fused to cytoplasmic beta-galactosidase protein, the fusion protein was able to enter the nucleus. These results strongly indicate that residues 332-452 mediate nuclear localization of ETV6.

Protein nanoarray on Prolinker surface constructed by atomic force microscopy dip-pen nanolithography for analysis of protein interaction.

Protein nanoarrays are addressable ensembles of nano-scale protein domain on solid surfaces. This method can serve as a useful platform for ultraminiaturized bioanalysis. In this study, we investigated single molecular nanopatterning and molecular interaction of proteins that were immobilized on Prolinker surface of gold-coated silicon wafer by using dip-pen nanolithography (DPN) method. Contact force and humidity were optimized at 0.01 nN and 80%, respectively. The domain features of protein nanoarrays were developed at the contact time of 5 s. The optimized conditions for the nanoarray process were applied to create protein nanoarray using integrin alpha(v)beta3 and angiogenin. Constructed protein nanoarrays using integrin alpha(v)beta3 have single molecular monolayer with regular domain shape (height 15 +/- 5 nm). The changed height value due to the single molecular interaction between integrin alpha(v)beta3 and vitronectin was approximately 30 +/- 5 nm on Prolinker surface as measured with atomic force microscopy tip. Taken together, these results suggest that protein nanoarray on Prolinker surface fabricated by well-controlled DPN process can be used to analyze single molecular interaction of protein.