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Zhuan Zhou

Publications and source records attributed to Zhuan Zhou.

At least 19 recordsLinked to original sources
PubMed2006

SynDB: a Synapse protein DataBase based on synapse ontology.

A synapse is the junction across which a nerve impulse passes from an axon terminal to a neuron, muscle cell or gland cell. The functions and building molecules of the synapse are essential to almost all neurobiological processes. To describe synaptic structures and functions, we have developed Synapse Ontology (SynO), a hierarchical representation that includes 177 terms with hundreds of synonyms and branches up to eight levels deep. associated 125 additional protein keywords and 109 InterPro domains with these SynO terms. Using a combination of automated keyword searches, domain searches and manual curation, we collected 14,000 non-redundant synapse-related proteins, including 3000 in human. We extensively annotated the proteins with information about sequence, structure, function, expression, pathways, interactions and disease associations and with hyperlinks to external databases. The data are stored and presented in the Synapse protein DataBase (SynDB, http://syndb.cbi.pku.edu.cn). SynDB can be interactively browsed by SynO, Gene Ontology (GO), domain families, species, chromosomal locations or Tribe-MCL clusters. It can also be searched by text (including Boolean operators) or by sequence similarity. SynDB is the most comprehensive database to date for synaptic proteins.

Animals↗
PubMed2006

Calcium influx through If channels in rat ventricular myocytes.

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I(f))/neuronal (I(h)) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I(h) channels in neurons. This raises the possibility of Ca(2+) permeation in I(f), the I(h) counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca(2+) signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I(f) in rat ventricular myocytes. We observed Ca(2+) influx when HCN/I(f) channels were activated. Ca(2+) influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I(f) channel blocker, inhibited I(f) and Ca(2+) influx at the same time. Quantitative analysis revealed that Ca(2+) flux contributed to approximately 0.5% of current produced by the HCN2 channel or I(f). The associated increase in Ca(2+) influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I(f) current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca(2+) chelator), preactivation of I(f) channels significantly reduced the action potential duration, and the effect was blocked by another selective I(f) channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I(f) channels had no effects on action potential duration. Our data extend our previous discovery of Ca(2+) influx in I(h) channels in neurons to I(f) channels in cardiac myocytes.

Animals↗
PubMed2006

Identification of derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization.

Endothelial cells play an important regulatory role in embryonic development, reproductive functions, tumor growth and progression. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify differentially expressed genes between non-stimulated endothelial cells and activated endothelial cells. Following mRNA isolation of non-stimulated and hepatocellular carcinoma homogenate-stimulated cells, cDNAs of both populations were prepared and subtracted by suppressive PCR. Sequencing of the enriched cDNAs identified a couple of genes differentially expressed, including derlin-1. Derlin-1 was significantly up-regulated by tumor homogenates, VEGF, and endothelial growth supplements in a dose-dependent manner. Knock-down of derlin-1 triggered endothelial cell apoptosis, inhibited endothelial cell proliferation, and blocked the formation of a network of tubular-like structures. Our data reveal that derlin-1 is a novel growth factor-responsive endothelial antigen that promotes endothelial cell survival and growth.

Antigens, Neoplasm↗
PubMed2006

The timing of endocytosis after activation of a G-protein-coupled receptor in a sensory neuron.

Endocytosis is a fundamental cellular event in membrane retrieval after exocytosis and in the regulation of receptor-mediated signal transduction. In contrast to the well-studied depolarization-induced membrane recycling, little is known about the kinetics of ligand-induced endocytosis of G-protein-coupled receptors in neurons. Here we investigated the kinetics of ligand-receptor binding-induced endocytosis in rat sensory neurons using a membrane capacitance assay. The time constant of ADP-induced endocytosis of P2Y-receptors was determined as 1.7 s. The ADP-induced endocytosis was blocked by antagonists against P2Y, phosphorylation, and clathrin. However, block of dynamin was without effect. The ADP-induced endocytosis was confirmed independently by a single vesicle image technique using a styryl FM2-10. Finally, the receptors were internalized in response to ADP, as determined by GFP-labeled P2Y. We conclude that ligand-receptor binding leads to rapid endocytosis in the cytoplasm of rat dorsal root ganglion neurons.

Action Potentials↗
PubMed2006

Ca2+ sparks and Ca2+ glows in superior cervical ganglion neurons.

AIM: Ca2+ release from the endoplasmic reticulum (ER) is an integral component of neuronal Ca2+ signaling. The present study is to investigate properties of local Ca2+ release events in superior cervical ganglion (SCG) neurons. METHODS: Primary cultured SCG neurons were prepared from neonatal rats (P3-P7). Low concentration of caffeine was used to induce Ca2+ release from the ER Ca2+ store, and intracellular Ca2+ was recorded by high-resolution line scan confocal imaging and the Ca2+ indicator Fluo-4. RESULTS: Two populations of local Ca2+ release events with distinct temporal characteristics were evoked by 1.5 mmol/L caffeine near the surface membrane in the soma and the neurites of SCG neurons. Brief events similar to classic Ca2+ sparks lasted a few hundreds of milliseconds, whereas long-lasting events displayed duration up to tens of seconds. Typical somatic and neurite sparks were of 0.3- and 0.52-fold increase in local Fluo-4 fluorescence, respectively. Typical Ca2+ glows were brighter (deltaF/F0 approximately 0.6), but were highly confined in space. The half maximum of full duration of neurite sparks was much longer than those in the soma (685 vs 381 ms). CONCLUSION: Co-existence of Ca2+ sparks and Ca2+ glows in SCG neurons indicates distinctive local regulation of Ca2+ release kinetics. The local Ca2+ signals of variable, site-specific temporal length may bear important implications in encoding a 'memory' of the trigger signal.

Animals↗
PubMed2006

"Kiss-and-run" exocytosis in astrocytes.

Neurons and glia are the principal cellular components of the nervous system. Although the glia are 10 times more numerous than neurons, until recently they were thought to be passive cells that monitor and support the active neurons by taking up used neurotransmitters from the synapses. In the past few years, this concept has been challenged by the findings that Ca(2+) waves spread from one astrocyte to another via Ca(2+)-and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent gliotransmitter release in pure cultures of astrocytes, raising the possibility that glia are not so passive as previously thought. This hypothesis was further advanced by two recent reports, which demonstrated that astrocytes release glutamate via vesicular exocytosis in response to stimuli. The kinetics of single vesicle exocytosis is distinct from its neural equivalent, because in response to physiological stimulation, gliotransmitter release is exclusively in the mode of "kiss and run." These advances were made possible by newly available techniques for single vesicle recordings, which will also be briefly reviewed here.

Animals↗
PubMed2005

"Kiss-and-run" glutamate secretion in cultured and freshly isolated rat hippocampal astrocytes.

Under physiological conditions, astrocytes not only passively support and nourish adjacent neurons, but also actively modulate neuronal transmission by releasing "glial transmitters," such as glutamate, ATP, and D-serine. Unlike the case for neurons, the mechanisms by which glia release transmitters are essentially unknown. Here, by using electrochemical amperometry and frequency-modulated single-vesicle imaging, we discovered that hippocampal astrocytes exhibit two modes of exocytosis of glutamate in response to various stimuli. After physiological stimulation, a glial vesicle releases a quantal content that is only 10% of that induced by nonphysiological, mechanical stimulation. The small release event arises from a brief (approximately 2 ms) opening of the fusion pore. We conclude that, after physiological stimulation, astrocytes release glutamate via a vesicular "kiss-and-run" mechanism.

Animals↗
PubMed2005

Activation of GPCRs modulates quantal size in chromaffin cells through G(betagamma) and PKC.

Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.

Action Potentials↗
PubMed2005

Ca2+ sparks and secretion in dorsal root ganglion neurons.

Ca(2+) sparks as the elementary intracellular Ca(2+) release events are instrumental to local control of Ca(2+) signaling in many types of cells. Here, we visualized neural Ca(2+) sparks in dorsal root ganglion (DRG) sensory neurons and investigated possible role of DRG sparks in the regulation of secretion from the somata of the cell. DRG sparks arose mainly from type 3 ryanodine receptor Ca(2+) release channels on subsurface cisternae of the endoplasmic reticulum, rendering a striking subsurface localization. Caffeine- or 3,7-dimethyl-1-(2-propynyl)xanthine-induced store Ca(2+) release, in the form of Ca(2+) sparks, triggered exocytosis, independently of membrane depolarization and external Ca(2+). The spark-secretion coupling probability was estimated to be between 1 vesicle per 6.6 sparks and 1 vesicle per 11.4 sparks. During excitation, subsurface sparks were evoked by physiological Ca(2+) entry via the Ca(2+)-induced Ca(2+) release mechanism, and their synergistic interaction with Ca(2+) influx accounted for approximately 60% of the Ca(2+)-dependent exocytosis. Furthermore, inhibition of Ca(2+)-induced Ca(2+) release abolished endotoxin-induced secretion of pain-related neuropeptides. These findings underscore an important role for Ca(2+) sparks in the amplification of surface Ca(2+) influx and regulation of neural secretion.

Animals↗
PubMed2005

A simulation study on the Ca2+-independent but voltage-dependent exocytosis and endocytosis in dorsal root ganglion neurons.

In patch-clamped somata of dorsal root ganglion (DRG) neurons, two types of secretion have been proposed: Ca(2+)-dependent secretion and Ca(2+)-independent but voltage-dependent secretion (CIVDS). The Ca(2+)-induced and the depolarization-induced membrane capacitance (C(m)) increases contribute 80 and 20% to the total C(m) increase, respectively (Zhang and Zhou in Nat Neurosci 5:425, 2002). In order to explore the mechanism of the voltage-dependent C(m) change (DeltaC(m)), we constructed a model with sequential states. The simulation with this model closely approximates all the experimental data. The model predicts that the majority of fusion events (approximately 80%) are so-called "kiss-and-run" events, which account for the fast recovery or the rapid retrieval feature of the signals. The remaining 20% are attributed to full fusion events, which account for a slow retrieval feature. On the basis of the model, one mechanism of the activity-dependent endocytosis has revealed a differential distribution of vesicles between the kiss-and-run and full fusion states at different stimulation frequencies. The quantitative model presented in this study may help us to understand the mechanism of the CIVDS and the tightly coupled endocytosis found in mammalian DRG neurons.

Animals↗
PubMed2005

BmP09, a "long chain" scorpion peptide blocker of BK channels.

A novel "long chain" toxin BmP09 has been purified and characterized from the venom of the Chinese scorpion Buthus martensi Karsch. The toxin BmP09 is composed of 66 amino acid residues, including eight cysteines, with a mass of 7721.0 Da. Compared with the B. martensi Karsch AS-1 as a Na(+) channel blocker (7704.8 Da), the BmP09 has an exclusive difference in sequence by an oxidative modification at the C terminus. The sulfoxide Met-66 at the C terminus brought the peptide a dramatic switch from a Na(+) channel blocker toaK(+) channel blocker. Upon probing the targets of the toxin BmP09 on the isolated mouse adrenal medulla chromaffin cells, where a variety of ion channels coexists, we found that the toxin BmP09 specifically blocked large conductance Ca(2+)- and voltage-dependent K(+) channels (BK) but not Na(+) channels at a range of 100 nm concentration. This was further confirmed by blocking directly the BK channels encoded with mSlo1 alpha-subunits in Xenopus oocytes. The half-maximum concentration EC(50) of BmP09 was 27 nm, and the Hill coefficient was 1.8. In outside-out patches, the 100 nm BmP09 reduced approximately 70% currents of BK channels without affecting the single-channel conductance. In comparison with the "short chain" scorpion peptide toxins such as Charybdotoxin, the toxin BmP09 behaves much better in specificity and reversibility, and thus it will be a more efficient tool for studying BK channels. A three-dimensional simulation between a BmP09 toxin and an mSlo channel shows that the Lys-41 in BmP09 lies at the center of the interface and plugs into the entrance of the channel pore. The stable binding between the toxin BmP09 and the BK channel is favored by aromatic pi -pi interactions around the center.

Adrenal Medulla↗
PubMed2005

Optimization of a multi-channel puffer system for rapid delivery of solutions during patch-clamp experiments.

In biological experiments, especially in neuroscience research, it is important to manipulate the extracellular environment efficiently. We have developed a micro-puffing system for local drug delivery to single cells in electrophysiological experiments, and validated the kinetic properties of this instrument. Based on our results, the kinetics of the delivery of solutions and the territory controlled by this system are influenced by several factors: (1) inner diameter (I.D.) of the guide tubing; (2) I.D. of the puffing tip; (3) angle of the puffing tip; and (4) gravity or external pressure applied to the solution. The system can fully control a territory of 200 x 600 micrometer2. The minimum delay in response to drug delivery is 10-20 ms. Switching between different solutions takes less than 100 ms. The minimum volume of solution required by the system is 0.2 ml. Taken together, our results provide useful data for designing and using an efficient drug/solution delivery system in electrophysiological experiments.

Animals↗
PubMed2004

A novel scorpion toxin blocking small conductance Ca2+ activated K+ channel.

Small conductance calcium activated potassium channels (SK) are crucial in the regulation of cell firing frequency in the nervous system and other tissues. In the present work, a novel SK channel blocker, designated BmSKTx1, was purified from the scorpion Buthus martensi Karsh venom. The sequence of the N-terminal 22 amino acid residues was determined by Edman degradation. Using this sequence information, the full-length cDNA and genomic gene of BmSKTx1 were cloned and sequenced. By these analyses, BmSKTx1 was found to be a peptide composed of 31 amino acid residues with three disulfide bonds. It shared little sequence homology with other known scorpion alpha-KTxs but showed close relationship with SK channel blockers in the phylogenetic tree. According to the previous nomenclature, BmSKTx1 was classified as alpha-KTx14.1. We examined the effects of BmSKTx1 on different ion channels of rat adrenal chromaffin cells (RACC) and locust dorsal unpaired median (DUM) neurons. BmSKTx1 selectively inhibited apamin-sensitive SK currents in RACC with Kd of 0.72 microM and Hill coefficient of 2.2. And it had no effect on Na+, Ca2+, Kv, and BK currents in DUM neuron, indicating that BmSKTx1 was a selective SK toxin.

Amino Acid Sequence↗
PubMed2004

Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes.

AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor. METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence, cell cycle analysis and MTT assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio-informatics. RESULTS: The primary cDNA library consisted of 2 x 10(6) recombinants. Thirty-six positive clones were obtained from 6 x 10(5) independent clones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70, GRP58, GAPDH, SSB, S100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, S100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries. CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis were identified. The modified SEREX, xenogeneic functional serum screening, was demonstrated to be effective for isolation and identification of antigen genes of tumor endothelium, and also for other tumor cell antigen genes. These antigen genes obtained in this study could be a valuable resource for basic and clinical studies of tumor angiogenesis, thus facilitating the development of anti- angiogenesis targeting therapy of tumors.

Animals↗
PubMed2004

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap.

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.

Chromosomes, Plant↗
PubMed2004

Calcium- and dynamin-independent endocytosis in dorsal root ganglion neurons.

Synaptic vesicle endocytosis is believed to require calcium and the GTPase dynamin. We now report a form of rapid endocytosis (RE) in dorsal root ganglion (DRG) neurons that, unlike previously described forms of endocytosis, is independent of calcium and dynamin. The RE is tightly coupled to calcium-independent but voltage-dependent secretion (CIVDS). Using FM dye and capacitance measurements, we show that membrane depolarization induces RE in the absence of calcium. Inhibition of dynamin function does not affect RE. The magnitude of RE is proportional to that of preceding CIVDS and stimulation frequency. Inhibitors of protein kinase A (PKA) suppress RE induced by high-frequency depolarization, while PKA activators enhance RE induced by low-frequency depolarization. Biochemical experiments demonstrate that depolarization directly upregulates PKA activity in calcium-free medium. These results reveal a calcium- and dynamin-independent form of endocytosis, which is controlled by neuronal activity and PKA-dependent phosphorylation, in DRG neurons.

Action Potentials↗
PubMed2004

Calcium influx through hyperpolarization-activated cation channels (I(h) channels) contributes to activity-evoked neuronal secretion.

The hyperpolarization-activated cation channels (I(h)) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca(2+) can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and I(h) channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca(2+) imaging, we found that there is a Ca(2+) influx in proportion to I(h) induced by hyperpolarization in HEK293 cells. The I(h) channel blockers Cs(+) and ZD7288 inhibit both HCN current and Ca(2+) influx. Measurements of the fractional Ca(2+) current showed that it constitutes 0.60 +/- 0.02% of the net inward current through HCN4 at -120 mV. This fractional current is similar to that of the low Ca(2+)-permeable AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of I(h) for 30 s also resulted in a Ca(2+) influx and an elevated action potential-induced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for I(h) channels in modulating neuronal secretion by permitting Ca(2+) influx at negative membrane potentials.

Animals↗