Biomedical subjects

Zhen Yan

Publications and source records attributed to Zhen Yan.

70 records · Page 4Linked to original sources

PubMed2003

Impaired modulation of GABAergic transmission by muscarinic receptors in a mouse transgenic model of Alzheimer's disease.

It has long been recognized that muscarinic acetylcholine receptors (mAChRs) are crucial for the control of cognitive processes, and drugs that activate mAChRs are helpful in ameliorating cognitive deficits of Alzheimer's disease (AD). On the other hand, GABAergic transmission in prefrontal cortex (PFC) plays a key role in "working memory" via controlling the timing of neuronal activity during cognitive operations. To test whether the muscarinic and gamma-aminobutyric acid (GABA) system are interconnected in normal cognition and dementia, we examined the muscarinic regulation of GABAergic transmission in PFC of an animal model of AD. Transgenic mice overexpressing a mutant gene for beta-amyloid precursor protein (APP) show behavioral and histopathological abnormalities resembling AD and, therefore, were used as an AD model. Application of the mAChR agonist carbachol significantly increased the spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude in PFC pyramidal neurons from wild-type animals. In contrast, carbachol failed to increase the sIPSC amplitude in APP transgenic mice, whereas the carbachol-induced increase of the sIPSC frequency was not significantly changed in these mutants. Similar results were obtained in rat PFC slices pretreated with the beta-amyloid peptide (Abeta). Inhibiting protein kinase C (PKC) blocked the carbachol enhancement of sIPSC amplitudes, implicating the PKC dependence of this mAChR effect. In APP transgenic mice, carbachol failed to activate PKC despite the apparently normal expression of mAChRs. These results show that the muscarinic regulation of GABA transmission is impaired in the AD model, probably due to the Abeta-mediated interference of mAChR activation of PKC.

Action Potentials↗

PubMed2003

Activation of muscarinic receptors inhibits beta-amyloid peptide-induced signaling in cortical slices.

Deposition of fibrillar aggregates of the beta-amyloid peptide (Abeta) is a key pathologic feature during the early stage of Alzheimer's disease. The initial neuronal responses to Abeta in cortical circuits and the regulation of Abeta-induced signaling remain unclear. In this study, we found that exposure of cortical slices to Abeta(1-42) or Abeta(25-35) induced a marked increase in the activation of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII), two enzymes critically involved in a variety of cellular functions. Activation of M1 muscarinic receptors, but not nicotinic receptors, significantly inhibited the Abeta activation of PKC and CaMKII. Increasing inhibitory transmission mimicked the M1 effect on Abeta, whereas blocking GABA(A) receptors eliminated the M1 action. Moreover, electrophysiological evidence shows that application of Abeta to cortical slices induced action potential firing and enhanced excitatory postsynaptic currents, whereas muscarinic agonists potently increased inhibitory postsynaptic currents. These results suggest that Abeta activates PKC and CaMKII through enhancing excitatory activity in glutamatergic synaptic networks. Activation of M1 receptors inhibits Abeta signaling by enhancing the counteracting GABA(ergic) inhibitory transmission. Thus the muscarinic reversal of the Abeta-induced biochemical and physiological changes provides a potential mechanism for the treatment of Alzheimer's disease with cholinergic enhancers.

Alzheimer Disease↗

PubMed2003

Conditional expression of SV40 T-antigen in mouse cardiomyocytes facilitates an inducible switch from proliferation to differentiation.

Studies of cardiac muscle gene expression and signaling have been hampered by the lack of immortalized cardiomyocyte cell lines capable of proliferation and irreversible withdrawal from the cell cycle. With the goal of creating such cell lines, we generated transgenic mice using cardiac-specific cis-regulatory elements from the mouse Nkx2.5 gene to drive the expression of a simian virus 40 large T-antigen (TAg) gene flanked by sites for recombination by Cre recombinase. These transgenic mice developed tumors within the ventricular myocardium. Cells isolated from these tumors expressed cardiac markers and proliferated rapidly during serial passage in culture, without apparent senescence. However, they were unable to exit the cell cycle and failed to exhibit morphological features of terminal differentiation. Introduction of Cre recombinase to these cardiac cell lines by adenoviral delivery resulted in the elimination of TAg expression, accompanied by rapid cessation of cell division, and increase in cell size without an apparent induction of cellular differentiation. Incubation of cells lacking TAg in serum-deficient media with various pharmacological agents (norepinephrine, phenylephrine, or bone morphogenetic protein-2/4) or constitutively active calcium/calmodulin-dependent protein kinase I and/or calcineurin led to the formation of sarcomeres and up-regulation of cardiac genes involved in excitation-contraction coupling. The combination of TAg expression under the control of an early cardiac promoter and Cre-mediated recombination allowed us to derive an immortal cell line from the ventricular myocardium that could be controllably withdrawn from the cell cycle. The conditional expression of TAg in this manner permits propagation and regulated growth termination of cell types that are otherwise unable to be maintained in cell culture and may have applications for cardiac repair technologies.

Animals↗

PubMed2003

Muscarinic potentiation of GABA(A) receptor currents is gated by insulin signaling in the prefrontal cortex.

Cholinergic neurotransmission and insulin signaling in cognitive areas, such as the prefrontal cortex (PFC), play a key role in regulating learning and memory. However, the cellular mechanisms by which this regulation occurs are unclear. Because GABAergic inhibition in the PFC controls the timing of neuronal activity during cognitive operations, we examined the potential regulation of GABA transmission by cholinergic and insulin signaling in PFC pyramidal neurons. Activation of muscarinic acetylcholine receptors (mAChRs) with carbachol produced an enhancement of GABA(A) receptor currents in acutely dissociated cells after a short treatment with insulin. Inhibiting phosphoinositide-3 kinase (PI3K), a downstream target of insulin signaling, eliminated this effect as well as the carbachol-induced enhancement of GABAergic miniature IPSC amplitudes in PFC slices. The muscarinic potentiation of GABA(A) currents was blocked by PKC inhibitors, broad-spectrum protein tyrosine kinase inhibitors, and specific inhibitors of the nonreceptor tyrosine kinase Src. Additionally, muscarinic receptors in PFC slices activated PKC and the focal adhesion kinase Pyk2 (a potential molecular link between PKC and Src) in a PI3K-dependent manner. Together, our results show that mAChR activation in PFC pyramidal neurons enhances GABA(A) receptor functions through a PKC-dependent, Src-mediated signaling cascade that is gated by an insulin/PI3K pathway. Given the significance of GABAergic transmission in regulating PFC functions, our results provide a novel mechanism for understanding the role of cholinergic systems and insulin signaling in learning and memory.

Animals↗

PubMed2003

Construction of a new tumour necrosis factor fusion-protein expression vector for high-level expression of heterologous genes in Escherichia coli.

We report the construction and application of a new fusion-protein expression plasmid (TNFHis) for Escherichia coli. The plasmid contains both P(R) and P(L) promoters and is optimized to allow a higher level of expression of mature coding sequences. It also contains a six-histidine tag for convenient purification as well as thrombin and hydroxylamine recognition sites for cleaving heterologous protein. The potential use of this expression vector is demonstrated by comparing the expression levels of human tumour necrosis factor (TNF), interferon, interleukin 11, colony-forming factor, osteoprotegrin and interleukin 2 in E. coli. Furthermore, all expressed TNF fusion proteins can be detected by anti-TNF alpha antibody or by specific antibodies and purified by Ni(2+)-nitrilotriacetate beads. The expressed TNF fusion proteins can be cleaved by hydroxylamine.

Escherichia coli↗

PubMed2003

Cell proliferation in the endolymphatic sac in situ after the rat Waldeyer ring equivalent immunostimulation.

OBJECTIVES/HYPOTHESIS: It has been recognized that immunological mechanisms could be involved in the pathogenesis of different inner ear disorders, such as progressive sensorineural hearing loss, Meniere's disease, and even sudden deafness. The endolymphatic sac acts as an immune control organ for the inner ear and has been considered as an effector site of the mucosa-associated lymphoid tissue. The purpose of the study was to determine the potentially immunological relationship between endolymphatic sac and Waldeyer ring equivalent, one of the most important affector organs in mucosa-associated lymphoid tissue. STUDY DESIGN: Animal model. METHODS: Thirty-six rats were employed. Two animals were killed for histological observation of Waldeyer ring equivalent, and another 34 animals were randomly divided into experimental and control groups and received bilateral intranasal immunizations with keyhole limpet hemocyanin or Freund adjuvant, respectively. The ears of immunized animals and control animals were examined for keyhole limpet hemocyanin-positive memory cells and immunoglobulin G-positive plasma cells with the technique of immunohistochemical analysis. The histopathological changes and cell proliferation in those ears were also assessed. RESULTS: There were paired and organized lymphoid tissues in the lateral wall of the first choana in the rat. Keyhole limpet hemocyanin-positive lymphocytes appeared within the endolymphatic sac at 3 days after the first anti-genetic stimulus of the Waldeyer ring equivalent. Endolymphatic hydrops in the cochlea, elevated amounts of immunocompetent cells, and increased activity of cell proliferation within the endolymphatic sac were also considered after four intranasal injections of keyhole limpet hemocyanin. CONCLUSION: Presumably, endolymphatic sac is supplied with immunocompetent cells from Waldeyer ring equivalent and has an ability of co-reaction with Waldeyer ring equivalent.

Animals↗

PubMed2003

[Induction of specific cytotoxic T lymphocytes and humoral immune response by MUC1 DNA vaccine in mice].

AIM: To observe the specific cytotoxic T lymphocyte and humoral immune response induced by MUC1 DNA inmunization in mice. METHODS: Female BALB/c mice were immunized intramuscularly with 100 microg pcDNA3.1-MUC1 3 times at intervals of 3 weeks. Three weeks after the last immunization, tumor challenge experiments were performed by inoculation of MUC1 positive breast cancer cell line EMT6. Both humoral immune response and CTL-specific cytotoxicity were detected by immunohistochemical staining and (51)Cr release assay, respectively. RESULTS: The cytotoxicity of MUC1-specific CTLs to EMT6 target cells showed that the cytotoxic effect was different at various ratio of effector cells to target cells. At ratios of 100:1, 50:1, 25:1 and 12.5:1, the specific lysis in MUC1cDNA group reached 54.1%, 39.8%, 26.4% and 20.1%, while two control groups 13.2%, 10%, 8.2%, 7.2% and 11.7%, 9.8%, 7.7%, 7.0%, respectively. The former was markedly higher than the two latters(P<0.01). MUC1 expression was exhibited to be positive in breast cancer tissue by immunohistochemical staining with BALB/c mouse sera against MUC1 cDNA. There was only 40% tumorigenic rate in MUC1 cDNA immunized group, while there 100% in pcDNA3.1(+) and NS control groups. There was a significant difference between MUC1 cDNA group and control groups (P<0.05). CONCLUSION: Specific CTLs and antibody prodution are elicited by MUC1 DNA inmunization. MUC1 cDNA inmunization partly inhibit the growth of implanted tumor in mice.

Animals↗

PubMed2002

Navigating the signalling network in mouse cardiac myocytes.

Cardiac myocytes have a complex network of signals that regulates their essential role in the rhythmic pumping of the heart. This network is an appealing model system in which to study the basic principles underlying cellular signalling mechanisms. Progress in this effort has come through the establishment of standardized myocyte isolation and culture procedures and characterization of important signalling responses.

Aging↗

PubMed2002

Highly coordinated gene regulation in mouse skeletal muscle regeneration.

Mammalian skeletal muscles are capable of regeneration after injury. Quiescent satellite cells are activated to reenter the cell cycle and to differentiate for repair, recapitulating features of myogenesis during embryonic development. To understand better the molecular mechanism involved in this process in vivo, we employed high density cDNA microarrays for gene expression profiling in mouse tibialis anterior muscles after a cardiotoxin injection. Among 16,267 gene elements surveyed, 3,532 elements showed at least a 2.5-fold change at one or more time points during a 14-day time course. Hierarchical cluster analysis and semiquantitative reverse transcription-PCR showed induction of genes important for cell cycle control and DNA replication during the early phase of muscle regeneration. Subsequently, genes for myogenic regulatory factors, a group of imprinted genes and genes with functions to inhibit cell cycle progression and promote myogenic differentiation, were induced when myogenic stem cells started to differentiate. Induction of a majority of these genes, including E2f1 and E2f2, was abolished in muscles lacking satellite cell activity after gamma radiation. Regeneration was severely compromised in E2f1 null mice but not affected in E2f2 null mice. This study identifies novel genes potentially important for muscle regeneration and reveals highly coordinated myogenic cell proliferation and differentiation programs in adult skeletal muscle regeneration in vivo.

Animals↗

PubMed2002

Dopamine D4 receptors modulate GABAergic signaling in pyramidal neurons of prefrontal cortex.

Dopaminergic neurotransmission in the prefrontal cortex (PFC) plays an important role in regulating cognitive processes and emotional status. The dopamine D4 receptor, which is highly enriched in the PFC, is one of the principal targets of antipsychotic drugs. To understand the cellular mechanisms and functional implications of D4 receptors, we examined the impact of D4 receptors in PFC pyramidal neurons on GABAergic inhibition, a key element in the regulation of "working memory." Application of the D4 agonist N-(methyl)-4-(2-cyanophenyl)piperazinyl-3-methylbenzamide maleate caused a reversible decrease in postsynaptic GABA(A) receptor currents; this effect was blocked by the D4 antagonist 3-[(4-[4-chlorophenyl]piperazine-1-yl)methyl]-[1H]-pyrrolo[2,3-b]pyridine but not by the D2 antagonist sulpiride, suggesting mediation by D4 receptors. Application of PD168077 also reduced the GABA(A) receptor-mediated miniature IPSC amplitude in PFC pyramidal neurons recorded from slices. The D4 modulation of GABA(A) receptor currents was blocked by protein kinase A (PKA) activation and occluded by PKA inhibition. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of PD168077 on GABA(A) currents. Furthermore, disrupting the association of the PKA/PP1 complex with its scaffold protein Yotiao significantly attenuated the D4 modulation of GABA(A) currents, suggesting that Yotiao-mediated targeting of PKA/PP1 to the vicinity of GABA(A) receptors is required for the dopaminergic signaling. Together, our results show that activation of D4 receptors in PFC pyramidal neurons inhibits GABA(A) channel functions by regulating the PKA/PP1 signaling complex, which could underlie the D4 modulation of PFC neuronal activity and the actions of antipsychotic drugs.

Animals↗

PubMed2002

Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+/calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons.

We have studied the regulation of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC). Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT(1A) receptor agonists and blocked by 5-HT(1A) antagonists, indicating the mediation by 5-HT(1A) receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT(1A) agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. We concluded that serotonin, by activating 5-HT(1A) receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2002

Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons.

Roscovitine is widely used for inhibition of cdk5, a cyclin-dependent kinase expressed predominantly in the brain. A novel function of roscovitine, i.e. an effect on Ca(2+) channels and transmitter release in central neurons, was studied by whole-cell voltage-clamp recordings and time-lapse fluorescence imaging techniques. Extracellular application of roscovitine markedly enhanced the tail calcium current following repolarization from depolarized voltages. This effect was rapid, reversible and dose dependent. Roscovitine dramatically slowed the deactivation kinetics of calcium channels. The deactivation time constant was increased 3- to 6-fold, suggesting that roscovitine could prolong the channel open state and increase the calcium influx. The potentiation of tail calcium currents caused by roscovitine and by the L-channel activator Bay K 8644 was not occluded but additive. Roscovitine-induced potentiation of tail calcium currents was significantly blocked by the P/Q-channel blocker CgTx-MVIIC, indicating that the major target of roscovitine is the P/Q-type calcium channel. In mutant mice with targeted deletion of p35, a neuronal specific activator of cdk5, roscovitine regulated calcium currents in a manner similar to that observed in wild-type mice. Moreover, intracellular perfusion of roscovitine failed to modulate calcium currents. These results suggest that roscovitine acts on extracellular site(s) of calcium channels via a cdk5-independent mechanism. Roscovitine potentiated glutamate release at presynaptic terminals of cultured hippocampal neurons detected with the vesicle trafficking dye FM1-43, consistent with the positive effect of roscovitine on the P/Q-type calcium channel, the major mediator of action potential-evoked transmitter release in the mammalian CNS.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2002

Activity-dependent bidirectional regulation of GABA(A) receptor channels by the 5-HT(4) receptor-mediated signalling in rat prefrontal cortical pyramidal neurons.

Emerging evidence has implicated a potential role for 5-HT(4) receptors in cognition and anxiolysis. One of the main target structures of 5-HT(4) receptors on 'cognitive and emotional' pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT(4) receptors on GABA(A) receptor channels in PFC pyramidal neurons. Application of 5-HT(4) receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABA(A) receptor beta3 or beta1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that beta3 and beta1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT(4) receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT(4) receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT(4) receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network.

4-Aminobenzoic Acid↗

PubMed2002

Expression of heat shock protein 70 in the cochlea in experimental autoimmune inner ear disease.

To explore whether the immune response of the inner ear could induce heat shock protein 70 (hsp70) in the guinea pig cochlea, we established a model of autoimmune inner ear disease (AIED) by systemically immunizing guinea pigs with homologous crude inner ear antigen (CIEAg). The immunized cochleas and normal control cochleas were examined for the expression of hsp70 by immunohistochemical analysis and in situ hybridization. It was found that 10 of 28 ears showed significant increases in hearing thresholds after the animals had been sensitized with CIEAg, and 67.9% of the ears from the immunized animals had modiolar vasculitis. The hsp70-like proteins were basically expressed in the normal cochleas, but dramatically increased expressions of hsp70 (perhaps also including hsp70-like molecules) and its messenger RNA were observed in the cochleas of the AIED models. The results suggest that the immune response of the inner ear induced the expression of hsp70 in the guinea pig cochleas.

Animals↗

PubMed2002

[The effect of multi-unit ribozymes on the growth inhibition and apoptosis induction of CML K562 cells].

OBJECTIVE: To investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype. METHODS: As bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis. RESULTS: Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells. CONCLUSION: Multi-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.

Apoptosis↗

PubMedDate not supplied

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications.

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT2 receptors in PFC pyramidal neurons inhibits GABAA-receptor currents via phosphorylation of GABAA receptor gamma2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT4 receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABAA-receptor beta subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT2, 5-HT1, and 5-HT3 receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.

Animals↗