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Biomedical subjects

J Yan

Publications and source records attributed to J Yan.

At least 109 records · Page 6Linked to original sources
PubMed2001

[Molecular cloning and expression in E. coli of the surface-exposed lipoprotein LipL41 gene of Leptospira lai].

OBJECTIVE: To construct a recombinant expressive plasmid using LipL41 gene of leptospira lai for further research of subunit vaccine of leptospira. METHODS: A pair of oligonucleotide primers were designed based on the sequence of LipL41 of leptospira kirschneri RM 52 in Genbank. With genomic DNA of leptospira lai 017 as template, a fragment was amplified by PCR and DNA sequencing analysis showed this fragment to be the gene that encodes LipL41. A recombinant vector was constructed using plasmid pGEX1 lambda T and the expression of LipL41 gene was tested. RESULTS: The production of PCR was LipL41 gene. The recombinant plasmid was constructed and testified by nuclease digestion and PCR, and LipL41 gene could express in E. coli. CONCLUSION: The recombinant plasmid has been constructed successfully and LipL41 gene can express stably at high level.

Bacterial Outer Membrane Proteins↗
PubMed2001

[Calcium dependent synaptic plasticity].

Recent work shows that the intracellular free Ca2+ concentrations ([Ca2+]i) of the presynaptic and postsynaptic neurons play crucial signaling roles in short- and long-term synaptic plasticity. Residual [Ca2+]i followed conditioning stimulation may cause short-term synaptic enhancement. Presynaptic [Ca2+]i could influence the replacing of presynaptic depressed vesicles, as well as encode the precise relative timing of presynaptic input and postsynaptic activity and generate long-term synaptic modification of opposite polarity(LTP or LTD).

Animals↗
PubMed2001

A case of Erdheim-Chester disease with bilateral orbital involvement.

PURPOSE: To describe a case of Erdheim-Chester disease with bilateral orbital involvement. METHODS: A 43-year-old female with bilateral proptosis was presented. Its clinical features, image findings, pathological character and therapeutic effect were evaluated. RESULTS: CT demonstrated bilateral, diffuse orbital mass. Histopathologic assessment revealed a diffuse xanthogranulomatous process with clusters of lipidladen histocytes. Numerous Touton giant cells were scattered throughout the lesion. Renal and heart failure happened during a 6-year follow-up period. Long bones roentgenogram demonstrated diffuse symmetrical sclerosis with extensive, lytic lesions. Systemic administration of corticosteroids, chemotherapy, immunoglobulin and traditional Chinese medicine showed good therapeutic result. CONCLUSIONS: An administration of systemic corticosteroids, chemotherapy, immunoglobin and traditional Chinese medicine can control Erdheim-Chester disease. Further exploration of its pathogenesis and collection of useful clinical data are required.

Adult↗
PubMed2001

[Simultaneous determination of three components in antongding injection by Kalman filtering method].

Kalman filtering method was applied to determine three components in antongding injection without separation. The average recovery and relative standard deviation of amidopyrine, phenazone, barbital obtained by Kalman filtering method were 99.8%, 0.93%, 99.7%, 0.82%, 99.7%, 1.5% respectively. It was showed by t-test that there was no significant difference between the results of Kalman filtering method and standard method.

Aminopyrine↗
PubMed2000

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation.

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The effect of CNTF on glutamate-induced increases in intracellular free Ca2+ in hippocampal neurons.

Ciliary neurotrophic factor (CNTF) acts through the JAK/STAT signal transduction pathway. However, the rapid action of CNTF cannot readily be explained by reference to this pathway. Using the fluorophore, Fura 2-AM, and fluorescence imaging, the effect of CNTF on glutamate-induced increases in hippocampal intraneuronal free Ca2+ ([Ca2+]i) was investigated. Glutamate induces a rapid increase in [Ca2+]i. Incubation of hippocampal neurons with CNTF for 5 min inhibited the glutamate-induced increase in [Ca2+]i. In the absence of glutamate, CNTF had no effect on [Ca2+]i. Pertussis toxin (PTX), a G-protein antagonist, partially blocked the effect of CNTF. This suggests that CNTF may act via an alternative signal transduction pathway besides the generic JAK/STAT pathway.

Animals↗
PubMed2000

Escape from apoptosis after prolonged serum deprivation is associated with the regulation of the mitochondrial death pathway by Bcl-x(l).

Bcl-x(l) and Bax play important roles in the regulation of apoptosis. This study investigated the involvement of the mitochondrial death pathway and the role of Bcl-x(l) and Bax in the escape from apoptosis after prolonged serum deprivation in Madin-Darby canine kidney (MDCK) cells. Low level apoptosis and basal activity of the mitochondrial death pathway were detectable in normal cell growth. In serum deprivation, mitosis was partially suppressed, and the mitochondrial activity was stimulated. The level of apoptosis continuously rose over 48 h. This rise was concomitant with the increasing presence of cytochrome c in cytosol. However, both apoptosis and cytosolic cytochrome c fell dramatically at 72 h. Elevation of whole cell Bcl-x(l) and redistribution of Bcl-x(l) protein from cytosol to the membrane at 48 h and 72 h was observed. Redistribution of Bax protein from the membrane to cytosol occurred at 24 h, and remained steady to 72 h. Bax/Bcl-x(l) coimmunoprecipitation by anti-Bax antibody showed reduced Bax/Bcl-x(l) interaction at the membrane at 72 h, but not at 24 or 48 h. These results suggest that apoptosis upon serum withdrawal results from the leakage of cytochrome c to cytosol. Amelioration of the leakage of cytochrome c and apoptosis requires not only the increase of Bcl-x(l)/Bax ratio, but also the release of Bcl-x(l) from Bax at the membrane.

Animals↗
PubMed2000

Integrin-linked kinase (ILK): a "hot" therapeutic target.

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.

Animals↗
PubMed2000

Multiresidue analysis of seventeen pesticides in wine by gas chromatography with mass-selective detection.

We have developed a multiresidue method permitting the simultaneous quantitation of 17 pesticides in wine: dicloran, dimethoate, diazinon, chlorpyrifos-methyl, vinclozolin, carbaryl, methiocarb, dichlofluanid, parathion-ethyl, triadimefon, procymidone, myclobutanil, iprodione, imidan, dicofol, phosalone and azinphos-methyl. Solid-phase extraction of 0.5 ml of wine sample is followed by direct injection of 1 microl of the eluent onto a DB-5 MS gas chromatographic column followed by mass-selective detection using one target and two qualifier ions for each pesticide. The extraction and injection steps are carried out with automatic instrumentation. Good resolution of all compounds was achieved with a run-time approximating 23 min. Detection and quantitation limits were around 2 microg/l and 10 microg/l, respectively, with linear calibration curves up to 3 mg/l for most constituents. Recovery in half the compounds was >90%, and >80% in most of the remainder. Imprecision (relative standard deviation) was <10% for most pesticides and <18% in all. Further analytes can be added to the repertoire without difficulty. The method merits consideration together with four other multiresidue methods now available that offer similar analytical characteristics, slower run-times, and a different selection of analytes.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

Phytoreovirus T = 1 core plays critical roles in organizing the outer capsid of T = 13 quasi-equivalence.

The structures of the double-shelled rice dwarf virus and of its single-shell core have been determined by cryoelectron microscopy and image reconstruction. The core carries a prominent density located at each of the icosahedral faces of its T = 1 lattice. These protrusions are formed by outer shell trimers, tightly inserted at the threefold positions of the core. Such configuration of the core may guide the assembly of the outer shell, aided by lateral interactions between its subunits, into a T = 13 lattice. The organization of the phytoreovirus capsid elucidates for the first time a general model for assembling two unique T numbers of quasi-equivalence.

Animals↗
PubMed2000

[Isolation and purification of Lycium barbarum polysaccharides and its antifatigue effect].

A purified component of lycium barbarum polysaccharide (LBP-X) was isolated from lycium barbarum L. by DEAE ion-exchange cellulose and sephacryl gel chromatography. LBP-X was tested on five different doses (5, 10, 20, 50 and 100 mg.kg-1.d-1) in mice. The results showed that LBP-X induced a remarkable adaptability to exercise load, enhanced resistance and accelerated elimination of fatigue. LBP-X could enhance the storage of muscle and liver glycogen, increase the activity of LDH before and after swimming, decrease the increase of blood urea nitrogen (BUN) after strenuous exercise, and accelerate the clearance of BUN after exercise. The dosage of LBP-X 10 mg.kg-1.d-1 was the best amount among the five tested doses.

Animals↗
PubMed2000

Biophysical characterization of interactions between the core binding factor alpha and beta subunits and DNA.

Core binding factors (CBFs) play key roles in several developmental pathways and in human disease. CBFs consist of a DNA binding CBFalpha subunit and a non-DNA binding CBFbeta subunit that increases the affinity of CBFalpha for DNA. We performed sedimentation equilibrium analyses to unequivocally establish the stoichiometry of the CBFalpha:beta:DNA complex. Dissociation constants for all four equilibria involving the CBFalpha Runt domain, CBFbeta, and DNA were defined. Conformational changes associated with interactions between CBFalpha, CBFbeta, and DNA were monitored by nuclear magnetic resonance and circular dichroism spectroscopy. The data suggest that CBFbeta 'locks in' a high affinity DNA binding conformation of the CBFalpha Runt domain.

Animals↗
PubMed2000

Active efflux kinetics of etoposide from rabbit small intestine and colon.

The aim of the present study was to investigate the directional transport kinetics of etoposide in rabbit intestinal tissues using side-by-side diffusion chambers. Etoposide is a routinely used mixed-mechanism 'efflux' inhibitor; however, its absorptive and secretory transport kinetics in rabbit intestinal tissues, a commonly used animal model, have not yet been reported. Kinetic studies revealed that the apical (AP) to basolateral (BL) (i.e. absorptive) transport of etoposide was not apparently mediated by specialized transporters, whereas secretion (i.e. BL to AP transport) by intestinal tissues was concentration dependent and saturable. Half-saturation constant values (K(m), mean+/-standard deviation (S.D.)) ranged from 53.6+/-35.8 microM to 168.7+/-127.3 microM, consistent with previous results from our group in intestinal tissues from other species and Caco-2 cell monolayers. Secretory permeability was greatest in the ileum, whereas values in the upper small intestine and colon were approximately equal, and represented only 50% of the value in the ileum. The ileal secretory transport of etoposide was temperature dependent, with the activation energy (E(a)) >4 kCal/mole at 5 microM, suggesting the involvement of the active, energy dependent mechanism. Etoposide inhibition by verapamil and saquinavir, known inhibitors of intestinal secretion, was characterized as competitive with K(i)'s equal to 193.0+/-164.4 microM and 72.6+/-53.5 microM, respectively. The current results demonstrate that the absorptive transport of etoposide in rabbit tissue was not mediated by specialized carriers, and that secretory transport was regionally dependent, mediated by a transporter or transporters, the K(m)'s were in the micromolar range, and involved the energy dependent mechanism(s). The relatively low k(m) of etoposide compared with its aqueous solubility (0.25-0.34 mM, pH 5-6.5, 25 degrees C) makes it the excellent mixed-mechanism competitive inhibitor for determining the secretory transport properties of putative drug substrates. Understanding the in vitro secretory transport kinetics of etoposide provides a mechanistic basis for ongoing studies exploring the functional role of 'efflux' in vivo.

ATP-Binding Cassette Transporters↗
PubMed2000

One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase.

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.

Animals↗
PubMed2000

Construction and identification of recombinant adenovirus vector containing the hVEGF165 gene.

To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF165), the cDNA for hVEGF165 was subcloned into pACCMV.pLpA. Subsequently, this recombinant pACCMV.hVEGF was co-transfected into 293 cells together with pJM17 to obtain the replication-deficient recombinant adenovirus containing hVEGF gene--AdCMV.hVEGF. The VEGF gene expression was detected by using RT-PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV.hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV.hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV.hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro. Successfully prepared AdCMV.hVEGF165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.

Adenoviridae↗
PubMed2000

Co-culture of early embryo with human decidual stromal cells in vitro by improvement of early embryo development.

An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM + 0.4% bovine serum albumin (BSA) and 163 embryos cultured in MEM + 15% FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73% developed to the morula stage and 67.21% cavitated to blastocysts with 59.74% hatching, as compared with 61.34% to morula stage, 48.47% to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

Adult↗