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Jun Yang

Publications and source records attributed to Jun Yang.

At least 361 records · Page 20Linked to original sources
PubMed2003

[Fermentation and purification of recombinant alpha-galactosidase from Pichia pastoris].

In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was eluted with 40% 1mol/L NaCl and showed a 41kD band on SDS-PAGE. After concentration, the final recovery was about 41%. The Michaelis constant of the recombinant alpha-galactosidase was determined as 0.275 mmol/L, which slightly lower than the nature enzyme and suggested a higher affinity with specific substrate. When human blood type B erythrocytes pretreated with 100u/mL recombinant alpha-galactosidase reacted with bood type B antiserum, no hemagglutination occurred. This suggested that the B antigens had been removed by the enzyme successfully. These results demonstrated that the recombinant alpha-galactosidase could be produced in largescale and made it possible to explore the application of alpha-galactosidase in more fields.

ABO Blood-Group System↗
PubMed2003

[Advances in analysis techniques of phosphoproteome].

In eukaryotes protein phosphorytion is a key event. By reversible protein phosphorylation eukaryotes control many cellular processes including signal transduction, gene expression, the cell cycle etc. Phosphoproteomics involves identification of phosphoproteins and phosphopeptides, localization of the exact residues that are phosphorylated and quantitation of phosphorylation. Because protein phosphorylation is a dynamic process, and it is present at low abundance within cells, and the phosphorylated sites on proteins might vary, and mass spectrometry (MS) signals from phosphopeptides are usually suppressed etc., so phosphoprotein analysis have more difficulties than nonphosphoprotein. In this article, we outline several analysis techniques for separation, identification and quantitation of phosphorylated proteins and peptides, and discuss the progress in these techniques. At present, MS is still an essential core identification technology for phosphoproteomic studies, To search better enrichment strategies are the main challenges in this rapidly evolving field. A major goal of quantitative proteomics is precise quantification and identification of proteins in complex mixtures. A common method for quantitative proteome analysis is the stable isotope labeling method. Today there is no single method that supersedes all others techniques for Phosphoproteomic studies. With continued development of sample preparation techniques and instrumentation, it should be possible to perform a global analysis of protein phosphorylation.

Animals↗
PubMed2003

[Expression of heat-labile enterotoxin and the strategy of purification and storage].

Heat-labile enterotoxin (LT) from Escherichia coli is a bacterial protein toxin with an AB5 hexamer structure. LT is a powerful mucosal adjuvant when co-administered with soluble antigens. However, its use in mucosal immunity is inconvenient because of its low yield and depolymerization during long-term storage under normal condition. In this study, we report an efficient expression system and optimized purification and storage strategy of LT. A gene encoding LT was cloned into the vector pET11c and transformed in E. coli BL21(DE3). By growing this strain on modified M9-CAA medium, LT was expressed efficiently. About 46mg/L LT could be purified from the supernatant of bacteria lysate. Using D(+)-Immobilized galactose column, LT could be purified at a wide pH range with various elution buffers. The optimized elution buffers are TEAN (pH 7.3) containing 0.3mol/L galactose and carbonate buffer (pH 10.4) containing 0.3mol/L galactose. After dried by freeze and placed in 4 degrees C, LT dissolved in TEAN (pH 7.3) and carbonate buffer (pH 10.4) were assayed by HPLC. The results indicated that the integrity of AB5 hexamer was kept well. LT could undergo long-term storage under this condition. This was proved to be an optimized strategy of LT storage. The results of GM1 binding assay and toxicity assay showed that the purified recombinant LT has normal biological character.

Animals↗
PubMed2002

Design of a fiber-optic quasi-distributed strain sensors ring network based on a white-light interferometric multiplexing technique.

A fiber-optic quasi-distributed strain sensors ring network has been designed based on a Mach-Zehnder optical paths interrogator. The optical paths matching for each sensor are discussed, and the optical power budgetary analysis is performed. The relation between the number of sensors and the intensity of the signals of the ring network is given for evaluation of the multiplexing capacity. Experimentally, a seven-sensor array ring network was realized under the condition of light source power 35 microW at 1310 nm, and the distribution strain test was also demonstrated.

Journal Article↗
PubMed2002

The effects of nicotinamide on energy metabolism following transient focal cerebral ischemia in Wistar rats.

This study examined the effects of nicotinamide on adenosine triphosphate (ATP) and nicotinamide adenine (NAD) levels and poly(adenosine diphosphate-ribose) (poly(ADP-ribose)) polymerase activity following ischemia and reperfusion in ketamine pretreated rats. Nicotinamide was administered at the end of the ischemic period. Nicotinamide protected against the depletion of ATP and NAD at 6 and 24 h of reperfusion. Nicotinamide is known to inhibit poly(ADP-ribose) polymerase at early time points, but was found to increase poly(ADP-ribose) polymerase activity at 24 h of reperfusion. It appears that nicotinamide can help maintain cellular energetics during reperfusion, thereby protecting cells from necrotic and apoptotic mechanisms.

Adenosine Triphosphate↗
PubMed2002

Artificial neural network classification based on high-performance liquid chromatography of urinary and serum nucleosides for the clinical diagnosis of cancer.

Nucleosides in human urine and serum have frequently been studied as a possible biomedical marker for cancer, acquired immune deficiency syndrome (AIDS) and the whole-body turnover of RNAs. Fifteen normal and modified nucleosides were determined in 69 urine and 42 serum samples using high-performance liquid chromatography (HPLC). Artificial neural networks have been used as a powerful pattern recognition tool to distinguish cancer patients from healthy persons. The recognition rate for the training set reached 100%. In the validating set, 95.8 and 92.9% of people were correctly classified into cancer patients and healthy persons when urine and serum were used as the sample for measuring the nucleosides. The results show that the artificial neural network technique is better than principal component analysis for the classification of healthy persons and cancer patients based on nucleoside data.

Biomarkers, Tumor↗
PubMed2002

Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet.

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Despite being the largest cytoskeleton, its structural composition has remained unknown. Here, we report a novel 220-kD protein, designated rootletin, found in the rootlets of ciliated cells. Recombinant rootletin forms detergent-insoluble filaments radiating from the centrioles and resembling rootlets found in vivo. An mAb widely used as a marker for vertebrate rootlets recognizes an epitope in rootletin. Rootletin has a globular head domain and a tail domain consisting of extended coiled-coil structures. Rootletin forms parallel in register homodimers and elongated higher order polymers mediated by the tail domain alone. The head domain may be required for targeting to the basal body and binding to a kinesin light chain. In retinal photoreceptors where rootlets appear particularly robust, rootlets extend from the basal bodies to the synaptic terminals and anchor ER membranes along their length. Our data indicate that rootlets are composed of homopolymeric rootletin protofilaments bundled into variably shaped thick filaments. Thus, rootletin is the long-sought structural component of the ciliary rootlet.

Animals↗
PubMed2002

Differential display identifies neuroendocrine-specific protein-A (NSP-A) and interferon-inducible protein 10 (IP-10) as ethanol-responsive genes in the fetal rat brain.

Fetal alcohol exposure is the most common nonhereditary cause of mental retardation in the western world. Rats prenatally treated with ethanol liquid diet exhibit extensive defects in the brain that accurately model those observed in humans. To analyze the ethanol effects on gene expression during brain development, we performed mRNA differential display and two-dimensional electrophoresis on gestational day (G) 13 and G 16 brain from rats treated with ethanol liquid diet. Using mRNA differential display followed by a variety of quantitative analyses, three genes were confirmed to be ethanol-responsive. Among them was Neuroendocrine-Specific Protein-A (NSP-A), which is known to be affected by thyroid hormone in the cortex at this developmental time. However, two additional genes known to be thyroid hormone-responsive were unaffected by ethanol, indicating that interference with thyroid hormone action may not be a predominant pathway by which alcohol induces damage in the fetal brain. The observation that interferon-inducible protein-10 (IP-10) is up-regulated in ethanol-treated fetal brain may indicate the presence of a disease process recruiting CD8+ T-cells capable of interfering with myelination. The result of two-dimensional (2D) electrophoresis and Western analyses demonstrated that few changes in the abundance of individual proteins or the phosphorylation of proteins at threonine and tyrosine were induced by prenatal ethanol exposure. A critical analysis of the approaches used in the present study may be important for future studies in this field.

Animals↗
PubMed2002

Reexamination of neomycin B degradation: efficient preparation of its CD and D rings as protected glycosyl donors.

The degradation of neomycin B was reexamined, and a novel protocol was established to prepare the properly masked neomycin CD ring as a glycol donor in excellent yield. Glycosylation of the CD ring with glycol acceptors provided a facile access to versatile intermediates that could be utilized to synthesize a variety of novel neomycin B mimetics for RNA recognition. [reaction: see text]

Anti-Bacterial Agents↗
PubMed2002

Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM.

RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse-chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device.

We have developed a microfluidic device for on-chip monitoring of cellular reactions. The device consists of two primary analytical functions: control of cell transport and immobilization, and dilution of an analyte solution to generate a concentration gradient. In this device, a dam structure in parallel to the fluid flow was constructed for docking and alignment of biological cells, which allows the fragile cells to move in the microfluidic channels and to be immobilized with controllable numbers in desired locations. The cells docked on the parallel dam structure are exposed to minimal stress caused by fluidic pressure. Additionally, a network of microfluidic channels was designed to generate a concentration gradient by controlled fluid distribution and diffusive mixing. An analyte solution could be diluted to different gradients as a function of distance along the dam. We used the ATP-dependent calcium uptake reaction of HL-60 cells as a model for on-chip measurement of the threshold ATP concentration that induces significant intracellular calcium signal. The results have demonstrated the feasibility of using the microchip for real-time monitoring of cellular processes upon treatment of a concentration gradient of a test solution. The integration of cell manipulation and solution manipulation on a microchip allows the measurement of concentration-dependent biological responses within a confined microscale feature.

Adenosine Triphosphate↗
PubMed2002

Apelin-immunoreactivity in the rat hypothalamus and pituitary.

With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.

Animals↗
PubMed2002

HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication.

Certain HIV-encoded proteins modify host-cell gene expression in a manner that facilitates viral replication. These activities may contribute to low-level viral replication in nonproliferating cells. Through the use of oligonucleotide microarrays and high-throughput Western blotting we demonstrate that one of these proteins, gp120, induces the expression of cytokines, chemokines, kinases, and transcription factors associated with antigen-specific T cell activation in the absence of cellular proliferation. Examination of transcriptional changes induced by gp120 in freshly isolated peripheral blood mononuclear cells and monocyte-derived-macrophages reveals a broad and complex transcriptional program conducive to productive infection with HIV. Observations include the induction of nuclear factor of activated T cells, components of the RNA polymerase II complex including TFII D, proteins localized to the plasma membrane, including several syntaxins, and members of the Rho protein family, including Cdc 42. These observations provide evidence that envelope-mediated signaling contributes to the productive infection of HIV in suboptimally activated T cells.

Animals↗
PubMed2002

An epithelial precursor is regulated by the ureteric bud and by the renal stroma.

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.

Animals↗
PubMed2002

Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca(2+) release channels.

The inositol trisphosphate (InsP(3)) receptor (InsP(3)R) is a ubiquitously expressed intracellular Ca(2+) channel that mediates complex cytoplasmic Ca(2+) signals, regulating diverse cellular processes, including synaptic plasticity. Activation of the InsP(3)R channel is normally thought to require binding of InsP(3) derived from receptor-mediated activation of phosphatidylinositol lipid hydrolysis. Here we identify a family of neuronal Ca(2+)-binding proteins as high-affinity protein agonists of the InsP(3)R, which bind to the channel and activate gating in the absence of InsP(3). CaBP/caldendrin, a subfamily of the EF-hand-containing neuronal calcium sensor family of calmodulin-related proteins, bind specifically to the InsP(3)-binding region of all three InsP(3)R channel isoforms with high affinity (K(a) approximately 25 nM) in a Ca(2+)-dependent manner (K(a) approximately 1 microM). Binding activates single-channel gating as efficaciously as InsP(3), dependent on functional EF-hands in CaBP. In contrast, calmodulin neither bound with high affinity nor activated channel gating. CaBP1 and the type 1 InsP(3)R associate in rat whole brain and cerebellum lysates, and colocalize extensively in subcellular regions in cerebellar Purkinje neurons. Thus, InsP(3)R-mediated Ca(2+) signaling in cells is possible even in the absence of InsP(3) generation, a process that may be particularly important in responding to and shaping changes in intracellular Ca(2+) concentration by InsP(3)-independent pathways and for localizing InsP(3)-mediated Ca(2+) signals to individual synapses.

Animals↗
PubMed2002

Generation of concentration gradient by controlled flow distribution and diffusive mixing in a microfluidic chip.

We have developed a simple method to generate a concentration gradient in a microfluidic device. This method is based on the combination of controlled fluid distribution at each intersection of a microfluidic network by liquid pressure and subsequent diffusion between laminas in the downstream microchannel. A fluid dynamic model taking into account the diffusion coefficient was established to simulate the on-chip flow distribution and diffusion. Concentration gradients along a distance of a few hundred micrometers were generated in a series of microchannels. The gradients could be varied by carefully regulating the liquid pressure applied to the sample injection vials. The observed concentration gradients of fluorescent dyes generated on the microfluidic channel are consistent with the theoretically predicted results. The microfluidic design described in this study may provide a new tool for applications based on concentration gradients, including many biological and chemical analyses such as cellular reaction monitoring and drug screening.

Journal Article↗
PubMed2002

Nicotinamide and ketamine reduce infarct volume and DNA fragmentation in rats after brain ischemia and reperfusion.

The possible ability of nicotinamide and ketamine to decrease infarction volume and DNA fragmentation was investigated in a middle cerebral artery occlusion rat model. DNA fragmentation was measured with an enzyme linked immunoassay. Control infarct volume was 223.8 +/- 10.6 mm(3). Ketamine alone did not alter infarct volume, 233.2 +/- 61.8 mm(3). Nicotinamide alone did not alter infarct volume, 235.2 +/- 62.8 mm(3). The combination of ketamine and nicotinamide decreased infarct volume to 83.8 +/- 35.2 mm(3). Ketamine produced hypothermia. Nicotinamide and ketamine decreased brain swelling and DNA fragmentation in the cerebral cortex, striatum and hippocampus in rats perfused for 6 or 24 h. Ketamine may synergize the actions of nicotinamide and partially prevent brain damage from ischemia and reperfusion.

Animals↗