Purpose: The purpose of this study was to determine the effects of different intensity of aerobic exercise for four weeks on cardiovascular risk factors, reactive oxygen, and antioxidant enzymes in old mice. Methods: Eighteen male C57BL/6 mice age 18 months were randomly classified into the control group (n=6), the moderate intensity exercise group (n=6), and the low intensity exercise group (n=6). The training groups performed the aerobic exercise twice daily for 20 minutes, five days weekly for four weeks. Data were analyzed using descriptive statistics, analysis of variance (ANOVA), the Chi-square test, and the Tukey’s test with the SPSSWIN 21.0 program. Results: In this study, among the risk factors of cardiovascular disease, blood sugar (BS) (p=.023) and total cholesterol (TC) (p=.001) were significantly different between the moderate intensity exercise group and the control group. Additionally, there were significant differences in the reactive oxygen malondialdehyde (MDA) (p=.001), the antioxidant enzymes superoxide dismutase (SOD) (p<.001) and glutathione reductase (GR) (p=.015) between the moderate intensity exercise group and the control group. Conclusion This finding suggests that moderate intensity aerobic exercise promotes the activity of antioxidant enzymes and lowers cardiovascular risk factors in older mice.

PURPOSE: The purpose of this study was to determine the effect of dehydroepiandrosterone (DHEA) on recovery of muscle atrophy induced by Parkinson's disease. METHODS: The rat model was established by direct injection of 6-hydroxydopamine (6-OHDA, 20 microg) into the left striatum using stereotaxic surgery. Rats were divided into two groups; the Parkinson's disease group with vehicle treatment (Vehicle; n=12) or DHEA treatment group (DHEA; n=22). DHEA or vehicle was administrated intraperitoneally daily at a dose of 0.34 mmol/kg for 21 days. At 22-days after DHEA treatment, soleus, plantaris, and striatum were dissected. RESULTS: The DHEA group showed significant increase (p<.01) in the number of tyrosine hydroxylase (TH) positive neurons in the lesioned side substantia nigra compared to the vehicle group. Weights and Type I fiber cross-sectional areas of the contralateral soleus of the DHEA group were significantly greater than those of the vehicle group (p=.02, p=.00). Moreover, extracellular signal-regulated kinase (ERK) phosphorylation significantly decreased in the lesioned striatum, but was recovered with DHEA and also in the contralateral soleus muscle, Akt and ERK phosphorylation recovered significantly and the expression level of myosin heavy chain also recovered by DHEA treatment. CONCLUSION: Our results suggest that DHEA treatment recovers Parkinson's disease induced contralateral soleus muscle atrophy through Akt and ERK phosphorylation.
Animals ; Corpus Striatum/drug effects/metabolism ; Dehydroepiandrosterone/*pharmacology/therapeutic use ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Male ; Muscle Fibers, Slow-Twitch/drug effects ; Muscle, Skeletal/drug effects/metabolism ; Muscular Atrophy/drug therapy/*etiology/*pathology ; Myosins/metabolism ; Neurons/drug effects/enzymology ; Oxidopamine/toxicity ; Parkinson Disease, Secondary/*chemically induced/*complications ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Rats, Sprague-Dawley ; Tyrosine 3-Monooxygenase/metabolism

This study was to determine the effect of exercise on the recovery of dopaminergic neuron loss and muscle atrophy in 6-OHDA-induced hemi Parkinson's disease model. Exercise was loaded twice per day for 30 minutes each time, at 5 days after 6-OHDA lesioning and continued for 16 days using a treadmill. Exercise significantly increased the number of tyrosine hydroxylase positive neuron in the lesioned substantia nigra and the expression level of tyrosine hydroxylase in the striatum compared with the control group. To examine which signaling pathways may be involved in the exercise, the phosphorylation of GSK3beta and ERK were observed in the striatum. In the control group, basal level of GSK3beta phosphorylation was less than in both striatum, but exercise increased it. ERK phosphorylation decreased in the lesioned striatum, but exercise recovered it. These findings suggest that exercise inactivates GSK3beta by phosphorylation which may be involved in the neuroprotective effect of exercise on the 6-OHDA-induced cell death. In the exercise group, weight, and Type I and II fiber cross-sectional area of the contralateral soleus significantly recovered and expression of myosin heavy chain and Akt and ERK phosphorylation significantly increased by exercise. These results suggest that exercise recovers Parkinson's disease induced dopaminergic neuron loss and contralateral soleus muscle atrophy.
Animals ; Atrophy ; Cell Death ; Dopaminergic Neurons ; Glycogen Synthase Kinase 3 ; Muscle, Skeletal ; Muscles ; Muscular Atrophy ; Myosin Heavy Chains ; Neurons ; Neuroprotective Agents ; Oxidopamine ; Parkinson Disease ; Phosphorylation ; Rats ; Substantia Nigra ; Tyrosine 3-Monooxygenase

Amphetamine, a synthetic psychostimulant, is transported by the dopamine transporter (DAT) to the cytosol and increases the exchange of extracellular amphetamine by intracellular dopamine. Recently, we reported that the phosphorylation levels of ezrin-radixin-moesin (ERM) proteins are regulated by psychostimulant drugs in the nucleus accumbens, a brain area important for drug addiction. However, the significance of ERM proteins phosphorylation in response to drugs of abuse has not been fully investigated. In this study, using PC12 cells as an in vitro cell model, we showed that amphetamine increases ERM proteins phosphorylation and protein kinase C (PKC) beta inhibitor, but not extracellular signal-regulated kinase (ERK) or phosphatidylinositol 3-kinases (PI3K) inhibitors, abolished this effect. Further, we observed that DAT inhibitor suppressed amphetamine-induced ERM proteins phosphorylation in PC12 cells. These results suggest that PKCbeta-induced DAT regulation may be involved in amphetmaine-induced ERM proteins phosphorylation.
Amphetamine ; Animals ; Brain ; Cytosol ; Dopamine ; Dopamine Plasma Membrane Transport Proteins ; Nucleus Accumbens ; PC12 Cells ; Phosphatidylinositol 3-Kinases ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Proteins ; Street Drugs ; Substance-Related Disorders

Matrix metalloproteinase 2 (MMP2) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in metastatic cancer. Although there have been various studies on the MMP2 gene, further studies of regulatory factors are required to achieve inhibition of MMP2 enzyme activities. MicroRNAs (miRNAs) play key roles in tumor metastasis. However, the specific functions of miRNAs in metastasis are unclear. In this study, we assessed the function of the microRNA-29 family (miR-29s) in HT1080 human fibrosarcoma cells and examined the regulatory mechanisms of these miRNAs on MMP2 activation. Using miRanda, TargetScan, and PicTar databases, miR-29s were identified as candidate miRNAs targeting MMP2. Gain-of-function studies showed that overexpression of miR-29s could inhibit the invasion of HT1080 cells, suggesting their tumor-suppressive roles in HT1080 cells. In addition, dual luciferase reporter assays indicated that miR-29s could inhibit the expression of the luciferase gene containing the 3'-untranslated region of MMP2 mRNA. Ectopic expression of miR-29s down-regulated the expression of MMP2. Moreover, ectopic expression of miR-29s reduced MMP2 enzyme activity. These results suggested that miR-29s could decrease the invasiveness of HT1080 cells by modulating MMP2 signaling. Taken together, our results demonstrated that miR-29s may serve as therapeutic targets to control tumor metastasis.
Ectopic Gene Expression ; Fibrosarcoma ; Humans ; Humans ; Luciferases ; Matrix Metalloproteinase 2 ; MicroRNAs ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.
Blotting, Western ; Cell Proliferation ; Cyclic AMP/metabolism ; Cyclic AMP Response Element-Binding Protein/genetics/metabolism ; Flowers/*chemistry ; Gas Chromatography-Mass Spectrometry ; Humans ; Intramolecular Oxidoreductases/genetics/metabolism ; Lotus/*chemistry ; Melanins/*biosynthesis ; Melanocytes/*drug effects/metabolism ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Monophenol Monooxygenase/genetics/metabolism ; Phosphorylation ; Plant Oils/*pharmacology ; RNA, Messenger/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/cytology/drug effects/metabolism

Acer mono is known to contain bioactive substances that exhibit beneficial effects in osteoporosis, gastric ulcers, hepatic damage, and pathologic angiogenesis. The current study aimed to investigate the effects of Acer mono extract on the invasive activities and cell-cycle progression of human fibrosarcoma cells. Cytotoxicity of Acer mono extract was assessed by MTT assay, in-vitro invasiveness of HT1080 fibrosarcoma cells was measured using matrigel assay, expression of invasion- and cell-cycle-related proteins was analyzed by western blot analysis, and that of E2F target genes was quantified using qRT-PCR. Acer mono extract did not show distinct cytotoxicity in the experimental concentrations used. Invasiveness of HT1080 fibrosarcoma cells and expression of cyclin D1 and CDK4 in them were significantly reduced in a dose-dependent manner after treatment with Acer mono extract. Acer mono extract showed inhibitory effects on the G1/S transition during cell-cycle progression; the active phosphorylated Rb protein level was decreased, and expression of E2F target genes was downregulated by the Acer mono extract. Our data collectively demonstrated that Acer mono extract exerts inhibitory effects on the invasiveness and cell-cycle progression of HT1080 human fibrosarcoma cells.

Acer mono is known to contain bioactive substances that exhibit beneficial effects in osteoporosis, gastric ulcers, hepatic damage, and pathologic angiogenesis. The current study aimed to investigate the effects of Acer mono extract on the invasive activities and cell-cycle progression of human fibrosarcoma cells. Cytotoxicity of Acer mono extract was assessed by MTT assay, in-vitro invasiveness of HT1080 fibrosarcoma cells was measured using matrigel assay, expression of invasion- and cell-cycle-related proteins was analyzed by western blot analysis, and that of E2F target genes was quantified using qRT-PCR. Acer mono extract did not show distinct cytotoxicity in the experimental concentrations used. Invasiveness of HT1080 fibrosarcoma cells and expression of cyclin D1 and CDK4 in them were significantly reduced in a dose-dependent manner after treatment with Acer mono extract. Acer mono extract showed inhibitory effects on the G1/S transition during cell-cycle progression; the active phosphorylated Rb protein level was decreased, and expression of E2F target genes was downregulated by the Acer mono extract. Our data collectively demonstrated that Acer mono extract exerts inhibitory effects on the invasiveness and cell-cycle progression of HT1080 human fibrosarcoma cells.

Cytochrome P450 3A4 (CYP3A4), is the dominant human liver hemoprotein enzyme localized in the endoplasmic reticulum (ER), and is responsible for the metabolism of more than 50% of clinically relevant drugs. While we were studying CYP3A4 expression and activity in human liver, we found that anti-CYP3A4 antibody cross-reacted with a lower band in liver cytoplasmic fraction. We assessed the activities of CYP3A4 and its truncated form in the microsomal and cytoplasmic fraction, respectively. In the cytoplasmic fraction, truncated CYP3A4 showed catalytic activity when reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b5. In order to determine which site was deleted in the truncated form in vitro, we transfected cells with N-terminal tagged or C-terminal tagged human CYP3A4 cDNA. The truncated CYP3A4 is the N-terminal deleted form and was present in the soluble cytoplasmic fraction. Our result shows, for the first time, that N-terminal truncated, catalytically active CYP3A4 is present principally in the cytoplasm of human liver cells.
Blotting, Western ; Catalysis ; Cell Line ; Cytochrome P-450 CYP3A/chemistry/*metabolism ; Cytoplasm/*enzymology ; Humans ; Microsomes, Liver/*enzymology

Glutamate induced rapid phosphorylation of moesin, one of ERM family proteins involved in the ligation of membrane to actin cytoskeleton, in rat hippocampal cells (JBC, 277:16576-16584, 2002). However, the identity of glutamate receptor has not been explored. Here we show that a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is responsible for glutamate-induced RhoA activation and phosphorylation of moesin. Glutamate induced phosphorylation at Thr-558 of moesin was still detectible upon chelation of Ca(2+), suggesting involvement of AMPA receptor instead of N-methyl D-Aspartate (NMDA) receptor in this phosphorylation of moesin. AMPA but not NMDA- induced moesin phosphorylation was independent of Ca(2+). Both AMPA and NMDA but not Kainate induced moesin phosphorylation at similar levels. However, the kinetics of phosphorylation varied greatly between AMPA and NMDA where AMPA treatment rapidly increased phosphomoesin, which reached a maximum at 10 min after treatment and returned to a basal level at 30 min. In contrast, NMDA-induced phosphorylation of moesin reached a maximum at 30 min after treatment and was remained at higher levels at 60 min. A possible involvement of RhoA and its downstream effector, Rho kinase in the AMPA receptor-triggered phosphorylation of moesin was also explored. The kinetics for the glutamate- induced membrane translocation of RhoA was similar to that of moesin phosphorylation induced by AMPA. Moreover, Y-27632, a specific Rho kinase inhibitor, completely blocked AMPA-induced moesin phosphorylation but had no effect on NMDA-induced moesin phosphorylation. These results suggest that glutamate-induced phosphorylation of moesin may be mediated through the AMPA receptor/RhoA/Rho kinase pathway.
Animals ; Calcium/metabolism ; Cell Line ; Excitatory Amino Acid Agonists/*metabolism ; Glutamic Acid/metabolism ; Kainic Acid/metabolism ; Microfilament Proteins/*metabolism ; N-Methylaspartate/*metabolism ; Phosphorylation ; Protein-Serine-Threonine Kinases/metabolism ; Rats ; Receptors, AMPA/metabolism ; Receptors, N-Methyl-D-Aspartate/metabolism ; Research Support, Non-U.S. Gov't ; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/*metabolism ; rhoA GTP-Binding Protein/*metabolism