Biomedical subjects

Jian Fei

Publications and source records attributed to Jian Fei.

At least 19 recordsLinked to original sources

PubMed2005

Macro-branched cell-penetrating peptide design for gene delivery.

Many kinds of macromolecular compounds that comprise a series of branches around an inner core have been made and showed some promises as DNA- and drug-delivery systems. In this study, a method for peptide polymer design having macro-branched peptide structure was adapted and it was applied for plasmid DNA delivery for the first time; acryloyl chloride was used by introducing the double bond into the N-terminal of HIV-1 tat (47-57, YGRKKRRQRRR). After being cleaved from resin, it was transformed to a polymer which possesses a poly-propionyl core matrix with tat branches and was named polytat by radical polymerization. After further separation and purification by Sephadex G150, polytat P1, polytat P2 and polytat P3 were acquired and the average molecular weight of the different macromolecules are 240, 178 and 82 kDa. We found that all these compounds complexed with plasmid DNA and showed significant transfection capabilities in a variety of mammalian cell lines, but acrylyl-tat or tat alone showed no significant transfection capability. Further study showed that polytat could be applied for plasmid DNA delivery for cell lines even with serum by endocytosis-mediated pathways.

Animals↗

PubMed2005

Expression and transport function of the glutamate transporter EAAC1 in Xenopus oocytes is regulated by syntaxin 1A.

The function of several membrane proteins is regulated by interaction with the SNARE protein syntaxin 1A; this includes regulation of GAT1, the transporter for the dominating inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Here we demonstrate that also EAAC1, the transporter for the dominating excitatory neurotransmitter, is down-regulated by interaction with syntaxin 1A. This is shown by coexpression of EAAC1 and syntaxin 1A in Xenopus oocytes. Total EAAC1 expression is not significantly affected by the coexpression of syntaxin 1A, but more proteins become targeted to the membrane as demonstrated by biotinylation. Colocalization by coimmunoprecipitation suggests direct interaction between the two proteins. In contrast to the number of transporters, the glutamate transport activity becomes reduced, and even stronger inhibition is observed for the EAAC1-mediated conductance uncoupled from glutamate translocation. We conclude that the interaction of syntaxin 1A with EAAC1 particularly disrupts the structure of the conductance pathway of EAAC1.

Amino Acid Transport System X-AG↗

PubMed2004

Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1.

The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.

Amino Acid Sequence↗

PubMed2004

Cognitive impairment in mice over-expressing gamma-aminobutyric acid transporter 1 (GAT1).

There is increasing evidence that GABAergic system plays an important role in the neural control of learning and memory processes. GAT1 over-expressing mice (NA) were generated, in which GAT1 is under the control of a neuron-specific enolase (NSE) promoter, to investigate effects of GABA transporter on cognitive function. Our results revealed that NA mice displayed cognitive deterioration in associative learning ability and new object recognition retention, compared with the wild-type littermates (WT2). However, the impaired cognitive function of transgenic mice could be rescued after chronic administration of GAT1 selective inhibitor for 6 days. In addition, there was no change of the expression of NMDA receptors in NA mice. Taken together, we show a potentially important role for GAT1 in the neural control of cognitive processes, and indicate great potential for GAT1 as a clinical target of cognitive disorders.

Animals↗

PubMed2004

Impaired reproduction in transgenic mice overexpressing Gamma-aminobutyric acid transporter I (GAT1).

It is well documented that g-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis, and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.

Animals↗

PubMed2004

Identification of glutamate transporters and receptors in mouse testis.

AIM: To investigate the presence of glutamate transporters and receptors in mouse testis. METHODS: Glutamate uptake analysis was performed to study the function of glutamate transporters in mouse testis. Comparative RT-PCR technique and sequencing analysis were used to study the expression of glutamate receptors and transporters in mouse testis. RESULTS: Mouse testis possessed glutamate uptake capacity with sodium-dependence. Vmax value of glutamate uptake was (1.60 +/- 0.21) pmol/min per mg protein and Km value of glutamate uptake was (11.0+/-1.6) micromol/L in mouse testis according to saturation analysis. Furthermore, the uptake activity could be inhibited by DHK (GLT1 selective inhibitor) and THA (glutamate uptake inhibitor). In addition, RT-PCR results revealed that glutamate transporters (GLT1 and EAAC1) and ionotropic glutamate receptors (NR1, NR2B, GluR6 and KA2) were expressed in mouse testis. CONCLUSION: Glutamate transporters and receptors do exist in mouse testis.

Amino Acid Transport System X-AG↗

PubMed2004

Adipose tissue-derived stromal cells express neuronal phenotypes.

BACKGROUND: Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons. METHODS: Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE). RESULTS: Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies. CONCLUSIONS: The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

Adipose Tissue↗

PubMed2004

[Effective expression of ribonuclease-onconase in Escherichia coli and assaying its cytotoxic potential].

Onconase, a ribonuclease purified from Rana pipiens oocytes, has cytotoxic activity against several tumor cell lines in vitro. With the characteristic of containing four pairs of disulfide bonds internal and N terminal sequence attributing mostly to its biological functions, it is difficult to obtain the active Onconase from Escherichia coli expression system with normal strategy. Here, we fused the cDNA coding for Onconase in frame with the pelB signal sequence in pET22b(+) expression vector. Onconase can be effectively expressed in inclusion body without additional residues at N terminal under the proper inducing condition. After refolding and purifying process, we can get the recombined Onconase which has a similar ribonuclease activity as the one isolated from oocytes. The recombined Onconase has a pronounced effect on proliferation of Hut-78 cells (IC50=0.5 micromol/L).

Animals↗

PubMed2004

[Ethanol affects the function of gamma-aminobutyric acid transporter I in mice brain].

It has been suggested that GABAergic system was involved in ethanol tolerance and addiction. Our previous work indicated both acute and chronic ethanol treatment enhanced the activity of GABA transporters in mouse brain. However, the expression level of GAT1 in CNS does not change, neither does the affinity of GABA transporters. In the present study, we demonstrated that GAT1 distribution on presynaptic membrane was altered after acute and chronic ethanol administration. Furthermore, the phosphorylation levels of GAT1 on three sites including Ser, Tyr and Thr were decreased. These data suggested that ethanol enhanced the function of GATs by means of inducing GAT1 redistribution in the cell and dephosphorylation may play a role in this process.

Animals↗

PubMed2004

[Establishment of mouse melanoma model expressing beta-galactosidase and its application in the research of DNA vaccines against tumor].

We established a mouse melanoma model expressing beta-galactosidase for the study of tumor immunotherapy. The recombinant vector p3gal was constructed by inserting a beta-galactosidase gene into the MCS of plasmid pcDNA3. The vector then transfected the B16 cells. Through selection with 500 microg/ml G418 and in situ X-Gal staining, the melanoma cell line galB16, stably expressing beta-galactosidase was obtained. The melanoma model was successfully established after inoculation in mouse with galB16 cells. In situ X-Gal staining showed that the tumor cells expressed beta-galactosidase in vivo. With the model, we designed animal experiments for mouse tumor immunotherapy. Twenty mice were randomly assigned to four parallel groups. They received i.m. injection with saline, DNA vaccine p3gal (100 microg/mouse), adjuvant CpG 1826 (20 microg/mouse), or p3gal+CpG 1826 respectively. Our result suggested that the DNA vaccine containing beta-galactosidase gene could protect mice against the galB16 tumor challenge. In addition, when combining with the adjuvant CpG 1826, the effect was increased prominently.

Adjuvants, Immunologic↗

PubMed2003

Decrease of morphine-induced reward effects and withdrawal symptoms in mice overexpressing gamma-aminobutyric acid transporter I.

Morphine addiction has been shown to result from neural adaptations produced by repeated drug exposure, but the mechanism is still unclear. In the present study, we found that gamma-aminobutyric acid (GABA) uptake was increased in mouse brain 120 min after, but not 20 min after, morphine (10 mg/kg, s.c.) injection. We generated GABA transporter I (GAT1)-overexpressing mice to investigate whether the GABAergic system and GABA transporter are involved in morphine-induced reward effects and withdrawal symptoms. Our results revealed that the rewarding effects induced by morphine were significantly decreased in GAT1-overexpressing mice as measured by the conditioned place preference (CPP) paradigm. Moreover, both somatic and vegetative signs of naloxone-induced morphine withdrawal symptoms were substantially reduced in GAT1-overexpressing mice. In addition, the decreased morphine rewarding in transgenic mice could be recovered when mice were coinjected with NO-711 (a GAT1 selective inhibitor) in the CPP paradigm. These findings suggest that the GABAergic system plays an important role in morphine addiction and point to the possibility of developing drugs that target GAT1 and extend the clinical application of opiates.

Adaptation, Physiological↗

PubMed2003

Hyperalgesic effects of gamma-aminobutyric acid transporter I in mice.

The present study focused on the involvement of gamma-aminobutyric acid transporter I (GAT1) in pain. We found that GABA uptake was increased in mouse spinal cord at 20 min and 120 min after formalin injection and in mouse brain at 120 min, but not 20 min, after formalin injection. In addition, the antinociceptive effects of GAT1-selective inhibitors were examined using assays of thermal (tail-flick) and chemical (formalin and acetic acid) nociception in C57BL/6J mice. The GAT1-selective inhibitors, ethyl nipecotate and NO-711, exhibited significant antinociceptive effects in these nociceptive assays. To study further the effects of GAT1 on pain, we used two kinds of GAT1-overexpressing transgenic mice (under the control of a CMV promoter or a NSE promoter) to examine the nociceptive responses in these mice. In the thermal, formalin, and acetic acid assays, both kinds of transgenic mice displayed significant hyperalgesia after nociceptive stimuli. In addition, the micro opioid receptor antagonist naloxone had no influence on nociceptive responses in wild-type and transgenic mice. The results indicate that GAT1 is involved in the regulation of pain processes, and point to the possibility of developing analgesic drugs that target GAT1 other than opioid receptors.

Analgesics, Opioid↗

PubMed2003

Effects of green tea polyphenols on dopamine uptake and on MPP+ -induced dopamine neuron injury.

As antioxidants, polyphenols are considered to be potentially useful in preventing chronic diseases in man, including Parkinson's disease (PD), a disease involving dopamine (DA) neurons. Our studies have demonstrated that polyphenols extracted from green tea (GT) can inhibit the uptake of 3H-dopamine (3H-DA) and 1-methyl-4-phenylpyridinium (MPP(+)) by DA transporters (DAT) and partially protect embryonic rat mesencephalic dopaminergic (DAergic) neurons from MPP(+)-induced injury. The inhibitory effects of GT polyphenols on 3H-DA uptake were determined in DAT-pCDNA3-transfected Chinese Hamster Ovary (DAT-CHO) cells and in striatal synaptosomes of C57BL/6 mice in vitro and in vivo. The inhibitory effects on 3H-MPP(+) uptake were determined in primary cultures of embryonic rat mesencephalic DAergic cells. Inhibition of uptake for both 3H-DA and 3H-MPP(+) was dose-dependent in the presence of polyphenols. Incubation with 50 microM MPP(+) resulted in a significant loss of tyrosine-hydroxylase (TH)-positive cells in the primary embryonic mesencephalic cultures, while pretreatment with polyphenols (10 to 30 microg/ml) or mazindol (10 microM), a classical DAT inhibitor, significantly attenuated MPP(+)-induced loss of TH-positive cells. These results suggest that GT polyphenols have inhibitory effects on DAT, through which they block MPP(+) uptake and protect DAergic neurons against MPP(+)-induced injury.

1-Methyl-4-phenylpyridinium↗

PubMed2003

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand.

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT) mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times. After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITC-conjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.

Animals↗

PubMed2003

[Adipose tissue-derived stromal cells differentiate into neuron-like cells].

OBJECTIVE: To investigate the possibility of inducing adipose tissue-derived stromal cells to differentiate into neuron-like cells, and to explore a new cell source for central nervous system transplantation. METHODS: beta-mercaptoethanol was adopted to induce the cells to differentiate; undifferentiated cells and differentiated cells were identified with immunocytochemistry. RESULTS: A population of adipose tissue-derived stromal cells were isolated from adult rat adipose tissue; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. beta-mercaptoethanol induced the stem cells to express nestin, characteristic of neuronal precursor stem cells at early stage of differentiation, and at late stage they exhibited a neuronal phenotype, expressing neuron-specific enolase (NSE) and neurofilament(NF); with an optimal differentiation protocol, almost 60%-85% of the cells expressed NSE and NF. CONCLUSION: The data support the hypothesis that adult adipose tissue contains stem cells capable of differentiating into neurons.

Adipose Tissue↗

PubMed2003

Involvement of dopamine D3 and neuropeptide Y Y5 receptors in diabetic gastroparetic rats without response to erythromycin.

Erythromycin may accelerate gastric emptying in animals and human probably as an motilin agonist, but its prokinetic effects show obvious individual disparity. This study was to find the mechanism of this phenomenon. Microarray analysis was used to screen genes that might be involved in the response of diabetic gastroparesis rats to erythromycin. It was found that erythromycin accelerated gastric emptying of diabetic rats with great individual disparity. Through microarray analysis we screened differential expression genes that might be involved in the effect of erythromycin. Among 10 genes screened out, dopamine D3 receptor (DRD3) and neuropeptide Y Y5 receptor (NPYY5) genes were submitted to RT-PCR quantification and showed consistent results with microarray. It can be concluded that erythromycin promote gastric emptying of gastroparetic rats; DRD3 and NPYY5 may be involved in prokinetic action of erythromycin; and targets other than motilin receptor of erythromycin might exist as prokinetics.

Animals↗

PubMed2003

Increase in drug-induced seizure susceptibility of transgenic mice overexpressing GABA transporter-1.

AIM: The changes of seizure susceptibility of transgenic mice overexpressing GABA transporter-1 (GAT-1) were studied to clarify the possible role of GABAergic transmission in epileptogenesis. METHODS: Seizures were induced by intraperitoneal administration of pentylenetetrazol (PTZ), picrotoxin (PIC), or kainic acid (KA) respectively. The anticonvulsant effect of ethyl nipecotate was tested by its intraperitoneal injection 15 min before the administration of the epileptogenic agents. RESULTS: The percentages of occurrence of clonic seizures induced by PTZ 45 mg/kg, PIC 2.5 mg/kg, or KA 20 mg/kg in GAT-1 transgenic mice were 88.9 %, 100 %, and 83.3 % respectively, whereas those in control C57BL/6J mice were 42.9 %, 57.1 %, and 33.3 %. The percentages of occurrence of tonic seizures induced by PTZ 45 mg/kg, PIC 2.5 mg/kg, or KA 20 mg/kg in transgenic mice were 88.9 %, 100 %, and 83.3 % respectively, and whereas those in control mice were 28.6 %, 42.9 %, and 16.7 %. The latencies of both clonic and tonic seizures onset in transgenic mice were markedly shortened compared with those in control animals. The results indicated that GAT-1 transgenic mice showed increased susceptibility to seizures induced by the anti-GABAergic convulsive drugs (PTZ, PIC), as well as glutamic receptor agonist (KA). Ethyl nipecotate, inhibitor of GAT-1, inhibited PTZ-induced seizures in both GAT-1 transgenic and C57BL/6J mice. The incidence of seizures was decreased after the application of ethyl nipecotate, and the latencies to the onset of clonic or tonic seizures were also prolonged. CONCLUSION: The increase in seizure susceptibility of transgenic mice over-expressing GAT-1 is an evidence for involvement of GABAergic transmission in epileptogenesis, and this transgenic mouse might be a useful animal model for study on the role of GABAergic transmission in epileptogenesis.

Animals↗

PubMed2003

[Development of salivary gland tumors in pleomorphic adenoma gene 1 transgenic mice].

OBJECTIVE: Activation and overexpression of pleomorphic adenoma (PLAG1) gene due to t(3;8)(p21;q12) translocation are associated with the development of human pleomorphic adenomas of the salivary glands. This study was conducted to generate ubiquitously-expressed or tissue-specific expressed PLAG1 transgenic mice and to elucidate the role of PLAG1 gene in tumorigenesis in vivo. METHODS: Human PLAG1 cDNA was cloned from salivary gland tumor or placenta tissues by RT-PCR. Ubiquitous expression vector pCMV-EGFP/PLAG1 driven by CMV promoter and tissue-specific expression vector pMMTV-PLAG1 driven by MMTV LTR were constructed. NIH3T3 cells transiently transfected with pCMV-EGFP/PLAG1 showed high expression of PLAG1 in nucleus. Transgenes were microinjected into pronucleus of zygotes to generate transgenic mice. RESULTS: It was found that the human PLAG1 cDNA cloned from several salivary gland tumor and normal placenta tissues consistently showed a variation of a single nucleotide at the same position when compared with the human PLAG1 cDNA sequence in Genbank (Accession No. U65002), which led to T458P at protein level. It might be a single nucleotide polymorphism (SNP)locus. Fused EGFP/PLAG1 protein was found to be localized in the nucleus of NIH3T3 cells transiently transfected with pCMV-EGFP/ PLAG1. Several pCMV-EGFP/PLAG1 and pMMTV-PLAG1 transgenic mouse lines were obtained respectively. As might be expected, pMMTV-PLAG1 transgenic mice spontaneously developed salivary gland tumors in three independent lines, among which, line 42 showed tumorigenic phenotype in 100% of transgenic mice within three months after birth. CONCLUSION: Overexpression of PLAG1 gene plays a crucial role in tumorigenesis of salivary gland tumors.

Animals↗