PubMed Health⌕ Search

Biomedical subjects

Jun Chen

Publications and source records attributed to Jun Chen.

At least 199 records · Page 11Linked to original sources
PubMed2004

PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain.

Glucocorticoid receptors (GRs) are extensively studied members of the steroid hormone receptor superfamily that regulate the transcription rates of numerous genes. Notwithstanding, the role of each GR amino acid in the various steps of transactivation is still unknown. A recent report shows that linear DNA has the same capacity as super-helical plasmid DNA for gene expression in transient transfection assays. Based on this observation, we describe a high-throughput assay to analyze a large set of alanine point mutations that are introduced by two rounds of PCR. The PCR products are then directly transfected into cells. This PCR expression mutagenesis (PEM) technique is used to identify several new residues of the GR ligand binding domain that influence ligand binding and/or transactivation. PEM thus provides a quick method for screening large quantities of mutant proteins. In combination with automation, PEM provides a more rapid and efficient tool for probing the role of each amino acid in the biological functions of a given protein.

Animals↗
PubMed2004

6-Hydroxydopamine induces dopaminergic cell degeneration via a caspase-9-mediated apoptotic pathway that is attenuated by caspase-9dn expression.

This study showed that primary dopaminergic neurons or the dopaminergic cell line MN9D, when exposed to 15 min of the parkinsonian toxin 6-hydroxydopamine (6-OHDA) in the range of 30-100 microM, underwent delayed degeneration and exhibited hallmarks of apoptosis. These results, along with the absence of any increase in lactate dehydrogenase (LDH) release from the degenerated cells, imply that apoptosis was the dominant mode of cell death. Moreover, a distinct elevation in the measured cellular activities of caspase-9 and -3 but not of caspase-8 points to the caspase-9/caspase-3 cascade as the predominant apoptotic pathway in the degeneration of dopaminergic neurons and MN9D cells. In addition, the presence of caspase-9 or -3 peptide inhibitors but not of caspase-8 inhibitor attenuated cell death significantly, supporting the notion that only the intrinsic apoptotic pathway is utilized to achieve cell death. Finally, overexpression of a mutant caspase-9 with dominant negative phenotype (caspase-9dn) in MN9D cells and primary dopaminergic neurons via the adenovirus and adenoassociated virus gene delivery system, respectively, conferred marked increases in tolerance to the toxicity of 6-OHDA. These results point to the intrinsic caspase-9/caspase-3 cascade as the predominant signaling pathway underlying dopaminergic cell death induced by 6-OHDA and suggest that gene delivery of caspase-9dn can attenuate this pathway and its degenerative consequences.

Animals↗
PubMed2004

Inhibitory effects of spinal propofol on the responses of spinal dorsal horn neurons in normal rats.

Spinal dorsal horn neurons play an important role in the processing of sensory information and are also targets of modulation by both endogenous and exogenous drugs. Propofol is an intravenous anesthetic and whether it has direct modulatory actions on sensory neuronal responses of the spinal cord dorsal horn has not been well studied. In the present study, a single dose (0.5 micromol) of propofol dissolved in dimethyl sulfoxide (DMSO) was directly applied onto the dorsal surface of the spinal cord and its effect was evaluated in 25 wide-dynamic-range (WDR) neurons and 10 low-threshold mechanoreceptive (LTM) neurons by using extracellular single unit recording technique in sodium pentobarbital anesthetized rats. Compared with the DMSO treatment, propofol produced a significant inhibition of WDR neuronal activity evoked by both noxious heat (45, 47, 49 or 53 degrees C, 15 s) and mechanical (pinch, 10 s) stimuli applied to their cutaneous receptive fields (cRF) on the ipsilateral hind paw skin. To investigate whether propofol exerts a modulatory effect on non-nociceptive afferent-mediated activity, the responses of WDR or LTM neurons to non-noxious brush and pressure were also evaluated. The non-noxious mechanically-evoked responses of both WDR and LTM neurons were significantly suppressed by propofol. The present results indicate that propofol has direct actions on the dorsal horn neurons of the spinal cord in rats. However, since both non-nociceptive and nociceptive afferent-mediated activity can be suppressed, the spinal effects of propofol are not likely to be specifically associated with anti-nociception.

Anesthetics, Intravenous↗
PubMed2004

Mechanism responsible for initiating carbon nanotube vacuum breakdown.

We report a physical mechanism responsible for initiating a vacuum breakdown process of a single carbon nanotube (CNT) during field emission. A quasidynamic method has been developed to simulate the breakdown process and calculate the critical field, critical emission current density and critical temperature beyond which thermal runaway occurs before the CNT temperature reaches its melting point. This model is in good agreement with experiments carried out with a single CNT on a silicon microtip.

Journal Article↗
PubMed2004

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling.

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.

Animals↗
PubMed2004

Cloning of a novel Apaf-1-interacting protein: a potent suppressor of apoptosis and ischemic neuronal cell death.

Cytochrome c-initiated activation of apoptotic protease activating factor-1 (Apaf-1) is a key step in the mitochondrial-signaling pathway for the activation of death-executing caspases in apoptosis. This signaling pathway has been implicated in the pathophysiology of various neurological disorders, including ischemic brain injury. In this study, we have cloned a novel rat gene product, designated as Apaf-1-interacting protein (AIP), which functions as a dominant-negative inhibitor of the Apaf-1-caspase-9 pathway. AIP is constitutively expressed in the brain, but at substantially lower levels than Apaf-1 and caspase-9. AIP can directly bind to Apaf-1 in vitro through its N-terminal caspase-recruiting domain, and this protein interaction was increased in cells undergoing apoptosis. Cytosolic extracts from cells overexpressing AIP were highly resistant to cytochrome c- dATP-induced activation of caspase-9 and caspase-3. Gene transfection of AIP into cell lines, including the neuronal-differentiated PC12 cells, potently suppressed apoptosis induced by various pro-apoptotic stimuli. To further investigate the functional role of AIP in primary neurons and in the brain, an adeno-associated virus (AAV) vector carrying the AIP cDNA was constructed. AAV-mediated overexpression of AIP in primary cortical- hippocampal neurons markedly reduced cell death and caspase-3 activation triggered by protein kinase C inhibition, DNA damage, or oxygen- glucose deprivation. Moreover, intracerebral infusion of the AAV vector resulted in robust AIP expression in the hippocampus and significantly promoted CA1 neuronal survival after transient global cerebral ischemia. These results suggest that molecular targeting of the Apaf-1-caspase-9 signaling pathway may be a feasible neuroprotective strategy to enhance the endogenous threshold for caspase activation and prevent neuronal loss in stroke and related disorders.

Animals↗
PubMed2004

D1 dopamine receptor mediates dopamine-induced cytotoxicity via the ERK signal cascade.

Postsynaptic striatal neurodegeneration occurs through unknown mechanisms, but it is linked to high extracellular levels of synaptic dopamine. Dopamine-mediated cytotoxicity of striatal neurons occurs through two distinct pathways: autoxidation and the D1 dopamine receptor-linked signaling pathway. Here we investigated the mitogen-activated protein kinase (MAPK) signaling pathways activated upon the acute stimulation of D1 dopamine receptors. In SK-N-MC neuroblastoma cells, endogenously expressing D1 dopamine receptors, dopamine caused activation of phosphorylated (p-)ERK1/2 and of the stress-signaling kinases, p-JNK and p-p38 MAPK, in a time- and dose-dependent manner. Selective stimulation of D1 receptors with the agonist SKF R-38393 caused p-ERK1/2, but not p-JNK or p-p38 MAPK activation, in a manner sensitive to the receptor-selective antagonist SCH 23390, protein kinase A inhibition (KT5720), and MEK1/2 inhibition (U0126 or PD98059). Activation of ERK by D1 dopamine receptors resulted in oxidative stress and cytotoxicity. In cells transfected with a catalytically defective mutant of MEK1, the upstream ERK-specific kinase, both dopamine- and SKF R-38393-mediated cytotoxicity was markedly attenuated, confirming the participation of the ERK signaling pathway. Cell fractionation studies showed that only a small amount of p-ERK1/2 was translocated to the nucleus, with the majority retained in the cytoplasm. From coimmunoprecipitation studies, p-ERK was found to form stable heterotrimeric complexes with the D1 dopamine receptor and beta-arrestin2. In cells transfected with the dominant negative mutant of beta-arrestin2, the formation of such complexes was substantially inhibited. These data provide novel mechanistic insights into the role of ERK in the cytotoxicity mediated upon activation of the D1 dopamine receptor.

Cell Line, Tumor↗
PubMed2004

Differential actions of intrathecal nociceptin on persistent spontaneous nociception, hyperalgesia and inflammation produced by subcutaneous bee venom injection in conscious rats.

Nociceptin is an endogenous ligand for the opioid receptor-like 1 (ORL1) receptor. The present study was designed to investigate spinal actions of nociceptin on the spontaneous nociception, hyperalgesia and inflammation induced by subcutaneous bee venom injection. Subcutaneous injection of bee venom into one hindpaw of conscious rat produced a persistent spontaneous nociception followed by a long-lasting primary heat and mechanical hyperalgesia as well as local inflammation. Compared with the pre-saline group, pretreatment with intrathecal injection of three doses (3, 10 and 30 nmol) of nociceptin produced significant suppression on the spontaneous paw flinching reflex. The inhibitory rates were 37+/-7, 43+/-6 and 57+/-11%, respectively, which were enhanced with an increase in the concentration of nociceptin. The inhibitory action of nociceptin was completely antagonized by a new selective ORL1 receptor antagonist CompB (30 nmol). Pre-treatment with 10 nmol nociceptin prior to bee venom produced no significant effect on the occurrence of primary heat and mechanical hyperalgesia, nor did post-treatment with the same dose again 3 h after bee venom injection. Additionally, pre-treatment with nociceptin had no effect on the bee venom-induced increase in paw thickness and volume and the plasma protein extravasation. These results indicate that intrathecal nociceptin has no effect on primary heat and mechanical hyperalgesia as well as inflammation, but has dose-related anti-nociceptive effect on the bee venom-induced persistent spontaneous nociception via activation of spinal ORL1 receptor.

Analgesics↗
PubMed2004

[Effects of etomidate on local synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord].

By using blind spinal slice whole-cell patch-clamp technique, we observed the influence of etomidate (ET) on synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord. Male adult Sprague-Dawley rats (7~8 weeks old) were anaesthetized with urethane (1.2 g/kg, i.p.), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1~S3) was removed and placed in preoxygenated Krebs solution at 1~3 degrees C. After cutting all of the ventral and dorsal roots, the pia-arachnoid membrane was removed. The spinal cord was mounted on a vibrating microslicer and then a 500 microm thick transverse slice was cut. The slice was placed on a nylon mesh in the recording chamber, and then perfused at a rate of 15~20 ml/min with Krebs solution saturated with 95% O2 and 5% CO2, and maintained at 36+/-1 degrees C. Substantia gelatinosa neurons were identified by their location. Under a binocular microscope and with transmitted illumination, the substantia gelatinosa was clearly discernible as a relatively translucent band across the dorsal horn. The resistance of patch clamp electrodes was 8~12 Msigma. Signals were gained by using an Axopatch 200B amplifier with low-passfiltered at 5 kHz, and digitized at 333 kHz with an A/D converter. The results are as follows. (1) To see whether or not ET has any effects on the local miniature excitatory postsynaptic currents (mEPSC), the holding potential was set up at -70 mV. Under such a condition extracellular superfusion was made with 1 micromol/L TTX for 2 min first, which was followed by consistent application of 500 micromol/L ET and 1 micromol/L TTX for 1 min. It was shown that ET did not influence the decay time, frequency and amplitude of mEPSC, when compared to the control. (2) To see whether or not ET has any effects on the local miniature inhibitory postsynaptic currents (mIPSC) mediated by GABA(A) receptor, the holding potential was set up at 0 mV. Under this condition extracellular superfusion was made with 1 micromol/L TTX and 1 micromol/L strychnine, an antagonist of glycine receptor, for 2 min, and then with consistent application of 50 micromol/L ET, 1 micromol/L TTX and 1 micromol/L strychnine for 1 min. ET prolonged the decay time of GABAergic mIPSC by 45.57+/-12.46% (P<0.05), but did not influence the frequency and amplitude of GABAergic mIPSC, when compared with the control. (3) To see whether or not ET has any effects on the local mIPSC mediated by glycine receptor, the holding potential was also set up at 0 mV, and under this condition extracellular superfusion was made with 1 mmol/L TTX and 10 mmol/L bicuculline, an antagonist also set up at 0 mV, and under this condition extracellular superfusion was made with 1 micromol/L TTX and 10 micromol/L bicuculline, an antagonist of GABA(A) receptor, for 2 min, and then with consistent application of 50 micromol/L ET, 1 micromol/L TTX and 10 micromol/L bicuculline for 1 min. ET had no effects on decay time, frequency and amplitude of glycinergic mIPSC. The above-mentioned results show that ET plays anesthetic or analgesic roles by modulating the decay time of GABAergic mIPSC, i.e. by prolonging the mean open time of GABA(A) receptors, however, ET has no direct effect on local excitatory synaptic transmission in substantia gelatinosa neurons of the adult rat spinal cord.

Anesthetics, Intravenous↗
PubMed2004

Joint analysis of two microarray gene-expression data sets to select lung adenocarcinoma marker genes.

BACKGROUND: Due to the high cost and low reproducibility of many microarray experiments, it is not surprising to find a limited number of patient samples in each study, and very few common identified marker genes among different studies involving patients with the same disease. Therefore, it is of great interest and challenge to merge data sets from multiple studies to increase the sample size, which may in turn increase the power of statistical inferences. In this study, we combined two lung cancer studies using microarray GeneChip, employed two gene shaving methods and a two-step survival test to identify genes with expression patterns that can distinguish diseased from normal samples, and to indicate patient survival, respectively. RESULTS: In addition to common data transformation and normalization procedures, we applied a distribution transformation method to integrate the two data sets. Gene shaving (GS) methods based on Random Forests (RF) and Fisher's Linear Discrimination (FLD) were then applied separately to the joint data set for cancer gene selection. The two methods discovered 13 and 10 marker genes (5 in common), respectively, with expression patterns differentiating diseased from normal samples. Among these marker genes, 8 and 7 were found to be cancer-related in other published reports. Furthermore, based on these marker genes, the classifiers we built from one data set predicted the other data set with more than 98% accuracy. Using the univariate Cox proportional hazard regression model, the expression patterns of 36 genes were found to be significantly correlated with patient survival (p < 0.05). Twenty-six of these 36 genes were reported as survival-related genes from the literature, including 7 known tumor-suppressor genes and 9 oncogenes. Additional principal component regression analysis further reduced the gene list from 36 to 16. CONCLUSION: This study provided a valuable method of integrating microarray data sets with different origins, and new methods of selecting a minimum number of marker genes to aid in cancer diagnosis. After careful data integration, the classification method developed from one data set can be applied to the other with high prediction accuracy.

Adenocarcinoma↗
PubMed2004

Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia.

Astragalus membranaceus is a herbal medicine that has been used clinically in stroke patients in China for decades, but its potential neuroprotective effect against ischemic brain injury has not been experimentally tested. In this study, we investigated the effect of Astragaloside IV, a purified extract from Astragalus membranaceus, in a murine model of focal cerebral ischemia/reperfusion produced by transient (1.5 h) middle cerebral artery occlusion. As determined at 72 h after ischemia, post-ischemic treatment of Astragaloside IV (20 or 40 mg/kg) markedly and significantly (P < 0.03 vs. vehicle-treated animals) reduced infarct volume. Astragaloside IV treatment also decreased the levels of malondialdehyde, an indicator of lipid peroxidation, and increased the levels of the antioxidant enzymes glutathione peroxidase and superoxide dismutase in ischemic tissues. The results presented here provide the first evidence of a neuroprotective effect of Astragaloside IV in the model of ischemic brain injury. We suggest that the anti-infarction effect by Astragaloside IV may be derived at least in part from its antioxidant properties.

Animals↗
PubMed2004

[Endoscopic thyroidectomy with 150 cases].

OBJECTIVE: To discuss the method, the advantages and disadvantages of endoscopic thyroidectomy. METHODS: Endoscopic thyroidectomy via areola of breasts approach was performed in 150 patients, including 41 cases of thyroid adenoma, 64 cases of nodular goiter, 40 cases of Graves' disease, and 5 cases of thyroid carcinoma. RESULTS: The endoscopic thyroidectomy was successfully carried out in 144 cases, including tumor dissection in 32 cases, one lobe partial thyroidectomy in 54 cases, two lobe partial thyroidectomy in 19 cases, subtotal thyroidectomy in 37 cases of Graves' disease, and radical thyroidectomy in 2 cases of thyroid carcinoma. The operative time length ranged from 50 to 270 min (mean 80 min). There were no complications such as damage to recurrent laryngeal nerve or parathyroid glands. Postoperative hospital stay ranged from 3 to 7 days (mean 4 days). The post-operative following-up for 1 approximately 13 months indicated that all the patients were satisfied with the cosmetic results and the same curative effects as conventional surgery were obtained. However, the operations were converted into open surgery in 6 cases. CONCLUSIONS: Endoscopic thyroidectomy is a safe and effective method of thyroid surgery. Since all the minimal incisions are on concealed parts of the body, the obvious cosmetic effect of this method is guaranteed.

Adolescent↗
PubMed2004

Liquid chromatographic method for the determination of rizatriptan in human plasma.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the determination of rizatriptan in human plasma. Following a single-step liquid-liquid extraction with methyl tertiarybutyl ether, the analytes were separated using a mobile phase consisting of 0.05% (v/v) triethylamine in water (adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v). Fluorescence detection was performed at an excitation wavelength of 225nm and an emission wavelength of 360nm. The linearity for rizatriptan was within the concentration range of 0.5-50ng/ml. The intra- and inter-day precisions of the method were not more than 8.0%. The lower limit of quantification (LLOQ) was 0.5ng/ml for rizatriptan. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.

Calibration↗
PubMed2004

High-performance liquid chromatographic analysis of zolmitriptan in human plasma using fluorescence detection.

A simple, rapid and sensitive high-performance liquid chromatographic (HPLC) method has been developed to quantify zolmitriptan in plasma using an isocratic system with fluorescence detection. The method included a single-step liquid-liquid extraction with methyl tertiary butyl ether. HPLC separation was carried out by reversed phase chromatography with a mobile phase composed of 0.05% (v/v) triethylamine in water(adjusting to pH 2.75 with 85% phosphoric acid) and acetonitrile (92:8, v/v), pumped at flow rate of 1.5 ml/min. Fluorescence detection was performed at 225 nm (excitation) and 360 nm (emission). The calibration curve for zolmitriptan was linear from 0.2 to 40 ng/ml. The validation method yielded good results regarding linearity, precision, accuracy, specificity and recoveries. The values of the limit of detection (LOD) and limit of quantification (LOQ) were 20 and 40 pg, respectively. The method was sensitive, simple and repeatable enough to be used in pharmacokinetic studies.

Adolescent↗
PubMed2004

Ag nanowires coated with Ag/Pd alloy sheaths and their use as substrates for reversible absorption and desorption of hydrogen.

When the surfaces of Ag nanowires were coated with thin sheaths of Pd/Ag alloys, they exhibited hydrogen absorption/desorption behaviors and capacities similar to those of pure Pd powders or nanotubes. The stronger mechanical strengths of these cable-like nanostructures also allowed them to undergo more absorption/desorption cycles with no morphological changes. These nanostructures can be used as a good model system to study the interaction between hydrogen and metal alloys with relatively low concentrations of Pd (<10%) with respect to structural, thermodynamic, and kinetic features.

Journal Article↗
PubMed2004

Disposition of flavonoids via enteric recycling: enzyme-transporter coupling affects metabolism of biochanin A and formononetin and excretion of their phase II conjugates.

The purpose of this study was to continue our effort to determine how enzyme-transporter coupling affect disposition of flavonoids. The rat intestinal perfusion and Caco-2 cell models were used together with relevant microsomes. In perfusion model, isoflavone (i.e., formononetin and biochanin A) absorption and subsequent excretion of its metabolites were always site-dependent. Maximal amounts of intestinal and biliary conjugates excreted per 30 min were 31 and 51 nmol for formononetin, more than that for pure biochanin A (12 and 20 nmol). When a standardized red clover extract (biochanin A/formononetin = 10:7) was used, the results indicated that more metabolites of biochanin A than formononetin were found in the perfusate (36.9 versus 22.8 nmol) and bile (78 versus 51 nmol). In metabolism studies, rat intestinal and liver microsomes always glucuronidated biochanin A faster (p < 0.05) than formononetin, whereas intestinal microsomes glucuronidated both isoflavones faster (p < 0.05) than liver microsomes. However, rapid metabolism in the microsomes did not translate into more efficient excretion in either the rat perfusion model as shown previously or in the Caco-2 model. In the Caco-2 model, both isoflavones were rapidly absorbed, efficiently conjugated, and the conjugates excreted apically and basolaterally. More formononetin conjugates were excreted than biochanin A when used alone, but much more biochanin A conjugates were found when using the isoflavone mixture. In conclusion, efficiency of enzyme-transporter coupling controls the amounts of metabolites excreted by the intestine and liver and determines the relative contribution of enteric and enterohepatic recycling to the in vivo disposition of isoflavones.

Absorption↗