OBJECTIVE: Synaptic vesicle mobilization and neurite outgrowth regulation molecules were examined in modulation of effects of methylphenidate (MPH) in Spontaneous Hypertensive Rats (SHRs), a model for attention-deficit hyperactivity disorder (ADHD). METHODS: We compared the changes in the protein expression level of Cyclin dependent kinase 5 (Cdk5) and molecular substrates of Cdk5; tropomyosin receptor kinase B (TrkB), syntaxin 1A (STX1A) and synaptosomal-associated protein 25 (SNAP25). Comparisons were made in prefrontal cortex of vehicle (distilled water i.p. for 7 days)-treated SHRs, vehicle-treated Wistar Kyoto Rats (WKYs) and MPH (2 mg/kg i.p. for 7 days) treated SHRs. RESULTS: The Cdk5 level of vehicle-treated SHRs was significantly decreased compared to the Cdk5 level of vehicle-treated WKY rats, but was restored to the expression level of vehicle-treated WKYs in MPH-treated SHR. The ratio of p25/p35 was significantly decreased in MPH-treated SHR compared to vehicle-treated SHR. Moreover, TrkB, STX1A and SNAP25 of vehicle-treated SHRs were significantly decreased compared to vehicle-treated WKY rats, but were restored to the expression level of vehicle-treated WKYs in MPH-treated SHR. CONCLUSION: The results show that Cdk5, TrkB, STX1A, and SNAP25 were involved in the modulation of MPH effects in prefrontal cortex of SHRs and play important role in treatment of ADHD.
Animals ; Cyclin-Dependent Kinase 5 ; Methylphenidate ; Neurites ; Phosphotransferases ; Prefrontal Cortex ; Rats ; Rats, Inbred WKY ; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins ; Synaptic Vesicles ; Synaptosomal-Associated Protein 25 ; Syntaxin 1 ; Tropomyosin ; Water

PURPOSE: Vesicle-associated membrane protein 8 (VAMP8) is a soluble N-ethylmaleimide-sensitive factor receptor protein that participates in autophagy by directly regulating autophagosome membrane fusion and has been reported to be involved in tumor progression. Nevertheless, the expression and prognostic value of VAMP8 in breast cancer (BC) remain unknown. This study aimed to evaluate the clinical significance and biological function of VAMP8 in BC. METHODS: A total of 112 BC samples and 30 normal mammary gland samples were collected. The expression of VAMP8 was assessed in both BC tissues and normal mammary gland tissues via a two-step immunohistochemical detection method. RESULTS: The expression of VAMP8 in BC tissues was significantly higher than that in normal breast tissues. Furthermore, increased VAMP8 expression was significantly correlated with tumor size (p=0.007), lymph node metastasis (p=0.024) and recurrence (p=0.001). Patients with high VAMP8 expression had significantly lower cumulative recurrence-free survival and overall survival (p < 0.001 for both) than patients with low VAMP8 expression. In multivariate logistic regression and Cox regression analyses, lymph node metastasis and VAMP8 expression were independent prognostic factors for BC. CONCLUSION: VAMP8 is significantly upregulated in human BC tissues and can thus be a practical and potentially effective surrogate marker for survival in BC patients.
Autophagy ; Biomarkers ; Breast Neoplasms* ; Breast* ; Humans ; Logistic Models ; Lymph Nodes ; Mammary Glands, Human ; Membrane Fusion ; Methods ; N-Ethylmaleimide-Sensitive Proteins ; Neoplasm Metastasis ; Prognosis ; R-SNARE Proteins* ; Recurrence

Reactive oxygen species (ROS) and nitrogen species (RNS) are implicated in cellular signaling processes and as a cause of oxidative stress. Recent studies indicate that ROS and RNS are important signaling molecules involved in nociceptive transmission. Xanthine oxidase (XO) system is a well-known system for superoxide anions (O2(.-)) generation, and sodium nitroprusside (SNP) is a representative nitric oxide (NO) donor. Patch clamp recording in spinal slices was used to investigate the role of O2(.-) and NO on substantia gelatinosa (SG) neuronal excitability. Application of xanthine and xanthine oxidase (X/XO) compound induced membrane depolarization. Low concentration SNP (10 microM) induced depolarization of the membrane, whereas high concentration SNP (1 mM) evoked membrane hyperpolarization. These responses were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger). Addition of thapsigargin to an external calcium free solution for blocking synaptic transmission, led to significantly decreased X/XO-induced responses. Additionally, X/XO and SNP-induced responses were unchanged in the presence of intracellular applied PBN, indicative of the involvement of presynaptic action. Inclusion of GDP-beta-S or suramin (G protein inhibitors) in the patch pipette decreased SNP-induced responses, whereas it failed to decrease X/XO-induced responses. Pretreatment with n-ethylmaleimide (NEM; thiol-alkylating agent) decreased the effects of SNP, suggesting that these responses were mediated by direct oxidation of channel protein, whereas X/XO-induced responses were unchanged. These data suggested that ROS and RNS play distinct roles in the regulation of the membrane excitability of SG neurons related to the pain transmission.
Animals ; Calcium ; Ethylmaleimide ; Humans ; Membranes ; Neurons* ; Nitric Oxide ; Nitrogen* ; Nitroprusside ; Oxidative Stress ; Rats* ; Reactive Oxygen Species* ; Substantia Gelatinosa* ; Superoxides ; Suramin ; Synaptic Transmission ; Thapsigargin ; Tissue Donors ; Xanthine ; Xanthine Oxidase

Stellate ganglion blockade (SGB) protects patients from focal cerebral ischemic injury, and transection of the cervical sympathetic trunk (TCST) in a rat model can mimic SGB in humans. The purpose of this study was to investigate the mechanisms underlying the neuroprotective effects of TCST on neuronal damage in the hippocampus in a rat model of middle cerebral artery occlusion (MCAO) in an attempt to elucidate the neuroprotective effects of SGB. The modified method of Zea Longa was used to establish the permanent MCAO model. Male Wistar rats were randomly divided into three groups: sham-operated group, MCAO group, and TCST group. The animals in TCST group were sacrificed 48 h after TCST which was performed after the establishment of the MCAO model. Proteins were extracted from the ipsilateral hippocampus and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE) and peptide mass fingerprinting (PMF). The levels of N-ethylmaleimide-sensitive factor (NSF) were measured as well. The results showed that 11 types of proteins were identified by 2D-DIGE. The expressions of eight proteins were changed both in the sham-operated and TCST groups, and the expressions of the other three proteins were changed in all three groups. Moreover, the expression of NSF was higher in the TCST group than in the MCAO group but lower in the MCAO group than in sham-operated group. The ratio of NSF expression between the MCAO group and shamoperated group was -1.37 (P<0.05), whereas that between the TCST group and MCAO group was 1.35 (P<0.05). Our results imply that TCST increases the expression of NSF in the hippocampus of adult rats with focal cerebral ischemia, which may contribute to the protection of the injured brain. Our study provides a theoretical basis for the therapeutic application of SGB to patients with permanent cerebral ischemia.
Animals ; Brain Injuries ; genetics ; metabolism ; pathology ; Brain Ischemia ; genetics ; metabolism ; pathology ; Gene Expression Regulation ; Hippocampus ; metabolism ; pathology ; Male ; N-Ethylmaleimide-Sensitive Proteins ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Stellate Ganglion ; metabolism ; pathology ; Transfection

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 microm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 microM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I h) in DRG neurons of rats.
Animals ; Diagnosis-Related Groups ; Ethylmaleimide ; Fires ; Ganglia, Spinal ; Membranes ; Neuralgia ; Neurons ; Pertussis Toxin ; Rats ; Spinal Nerve Roots

K+-Cl--cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A2 (PLA2)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K+ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K+ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA2 (iPLA2), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF3) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA2 (cPLA2) and the secretory PLA2 (sPLA2), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF3 and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA2-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.
5,8,11,14-Eicosatetraynoic Acid ; Acetophenones ; Apoptosis ; Arachidonic Acid ; Arachidonic Acids ; Cytosol ; Ethylmaleimide ; Hep G2 Cells ; Hepatoblastoma ; Humans ; Indomethacin ; Lipoxygenase ; Naphthalenes ; Nordihydroguaiaretic Acid ; Phospholipases A2 ; Prostaglandin-Endoperoxide Synthases ; Pyrones ; Reactive Oxygen Species ; Signal Transduction

BACKGROUNDQacA, a main exporter mediating the multidrug-resistance of Staphylococcus aureus to a variety of antiseptics and disinfectants, possesses a topology of 14 alpha-helical transmembrane segments (TMS). Our study aimed to determine the importance and topology of amino acid residues in and flanking the cytoplasmic end of TMS5.

METHODSSite-directed mutagenesis was used to mutate 5 residues, including L146, A147, V148, W149 and S150, into cysteine. A minimum inhibitory concentration (MIC) and transport assay with or without N-ethylmaleimide (NEM) were performed to analyse the function of these mutants.

RESULTSAll of the mutants showed comparable protein expression levels. MIC analysis suggested that mutant W149C showed low resistance levels to the drugs, but the mutations at L146, A147, V148, and S150C had little or no effect on the resistance level. And the results of the fluorimetric transport assay were in agreement with those of MIC analysis, that is to say, W149C did not allow transport to the substrates to be tested, while the other mutants retained significant transport ability. The reaction of the different mutant proteins with Fluorescein-NEM revealed that the mutant L146C was highly reactive with NEM; the W149C and S150C mutants were moderately reactive; A147C was barely reactive and V148C showed no reactivity.

CONCLUSIONSThe study identified that residues W149 and S150 situated at the interface of the aqueous: lipid junction as functionally important residues, probably involved in the substrate binding and translocation of QacA.

Bacterial Proteins ; chemistry ; physiology ; Drug Resistance, Bacterial ; Ethylmaleimide ; pharmacology ; Indoles ; metabolism ; Membrane Transport Proteins ; chemistry ; physiology ; Structure-Activity Relationship

AIMTo observe protein expression changes of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and related regulatory protein in the hippocampus and amygdala in chronic immobilization stressed rat and Xiaoyaosan's regulatory effect.

METHODSRats were tied 3 h per day to establish immobilization stress condition and treatment with Xiaoyaosan. After 7 days and 21 days stress, the protein expression of AMPA receptor subunit (GluR2/3), N-ethylmaleimide sensitive factor (NSF) and protein interacting with C-kinase 1 (PICK1) in hippocampus and amygdala were detected by using Western blot techniques.

RESULTSThe expression of GluR2/3, NSF in dentate gyrus (DG) and amygdala were markedly attenuated (P < 0.05) and PICK1 in CA1 region were significantly increased (P < 0.05) in 7 d immobilization stressed rats while 7 days xiaoyaosan treatment showed an effective regulatory result to PICK1's changes. Under 21 days immobilization stressed condition, the expression of GluR2/3, NSF in CA1 region showed an increasing trend, and GluR2/3 showed a markedly increase (P < 0.01), but showed an significantly decreased trend in amygdala, Xiaoyaosan showed an effective result to such changes above (P < 0.05). The expression of PICK1 showed increasing trend in amygdala and xiaoyaosan could lower its expression (P < 0.05).

CONCLUSIONThere are different trends of the expression of AMPA receptor in repeat short-term stress versus chronic immobilization stress, and in hippocampal CA1 region versus amygdala. Xiaoyaosan has better regulation effect on the expression of AMPA receptors in the condition of chronic immobilization stress than those of repeat shortterm stress.

Amygdala ; metabolism ; Animals ; CA1 Region, Hippocampal ; metabolism ; Carrier Proteins ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Male ; N-Ethylmaleimide-Sensitive Proteins ; metabolism ; Nuclear Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; metabolism ; Restraint, Physical ; Stress, Psychological

OBJECTIVETo observe the changes of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit and related regulatory protein mRNA expression in the hippocampus and amygdala in immobilization stressed rats and effect of Xiaoyaosan (XYS) on them.

METHODSImmobilization model rats were established by binding for 3 h per day and intervened with XYS, the expression of AMPA receptor subunit (GluR1-4), N-ethylmaleimide sensitive factor (NSF) and protein interacting with C-kinase 1 (PICK1) mRNA expression in model rats' CA1 and CA3 regions of hippocampus, dentate gyrus and amygdala were detected on day 7 and day 21 after modeling.

RESULTSOn day 7, expression of GluR1 mRNA was significantly decreased in CA1 region (P < 0.05) and increased in CA3 region and amygdala (all P < 0.05); expression of GluR2 and GluR3 mRNA in amygdala (all P < 0.05) and GluR4 mRNA in CA1 region (P < 0.01) significantly increased, but the expression of NSF and PICK1 mRNA in amygdala only showed an increasing trend. XYS showed effective regulation on GluR4 mRNA in CA1 region (P < 0.01) and GluR1-3 mRNA expression (P < 0.05, P < 0.01) in amygdala. On day 21, the expression of GluR4 mRNA in CA1 region (P < 0.05) and GluR2 mRNA in dentate gyrus (P < 0.05) markedly lowered and expression of GluR1 mRNA in amygdala increased (P < 0.01); XYS significantly regulated the expression of GluR1 and GluR4 mRNA in CA1 region (all P < 0.05).

CONCLUSIONRepeated stress in a short time shows effect on expression of AMPA receptor subunit mRNA stronger than chronic stress. The regulation of XYS to AMPA receptor subunit mRNA expression were obvious in hippocampal CA1 region and amygdala.

Amygdala ; drug effects ; metabolism ; Animals ; Carrier Proteins ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression ; drug effects ; Hippocampus ; drug effects ; metabolism ; Male ; N-Ethylmaleimide-Sensitive Proteins ; genetics ; Nuclear Proteins ; genetics ; Protein Subunits ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, AMPA ; genetics ; Restraint, Physical ; Reverse Transcriptase Polymerase Chain Reaction ; Stress, Psychological ; physiopathology

Peripheral insulin resistance in obese/type II diabetes animals results from an impairment of insulin-stimulated glucose uptake into skeletal muscle. Insulin stimulate the translocation of GLUT4 from intracellular location to the plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) is implicated in mediation of fusion of GLUT4-containing vesicle with the plasma membrane. Present study investigated regulatory effects of Rhodiola sachalinensis administration and exercise training on the expression of GLUT4 protein and SNAREs protein in skeletal muscles of obese Zucker rats. Experimental animals were randomly assigned into one of five groups ; lean control (LN), obese control (OB), exercise-treated (EXE), Rhodiola sachalinensis-treated (Rho), combine of Rho & EXE (Rho-EXE). All animals of exercise training (EXE, Rho-EXE) performed treadmill running for 8 weeks, and animals of Rho groups (Rho, Rho-EXE) were dosed daily by gastric gavage during the same period. After experiment, blood were taken for analyses of glucose, insulin, and lipids levels. Mitochondrial oxidative enzyme (citrate synthase, CS ; beta-hydroxyacyl-CoA dehydrogenase, beta-HAD) activity were analysed. Skeletal muscles were dissected out for analyses of proteins (GLUT4, VAMP2, syntaxin4, SNAP23). Results are as follows. Exercise and/or Rhodiola sachalinensis administration significantly reduced body weight and improved blood lipids (TG, FFA), and increased insulin sensitivity. Endurance exercise significantly increased the activity of mitochondrial enzymes and the expression of GLUT4 protein, however, administration of Rhodiola sachalinensis did not affect them. The effect of exercise and/or Rhodiola sachalinensis administration on the expression of SNARE proteins was unclear. Our study suggested that improvement insulin sensitivity by exercise and/or Rhodiola sachalinensis administration in obese Zucker rats is independent of expression of SNARE proteins.
Animals ; Body Weight ; Cell Membrane ; Glucose Transporter Type 4 ; Glucose* ; Insulin Resistance* ; Insulin* ; Muscle, Skeletal* ; Negotiating ; Obesity ; Oxidoreductases ; Rats* ; Rats, Zucker ; Rhodiola* ; Running ; SNARE Proteins ; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins ; Vesicle-Associated Membrane Protein 2