Biomedical subjects

Jian Fei

Publications and source records attributed to Jian Fei.

At least 19 recordsLinked to original sources

PubMed2007

Reduced aggression in mice lacking GABA transporter subtype 1.

Dysregulation of the brain GABAergic system has been implicated in the pathophysiology of violence and aggression. As a key regulator of central GABAergic activity, dysfunction of the GABA transporter subtype 1 (GAT1) represents a potential mechanism mediating pathologic aggression. We provide evidence that GAT1-/- mice and GAT1+/- mice exhibit lower aggressive behavior both in home cage resident-intruder test and neutral arena resident-intruder test, compared to wild-type mice (GAT1+/+). The pharmacologic effects of the GAT1 inhibitor, tiagabine and the GABA(A) receptor antagonist, bicuculline have been assessed in GAT1+/+ mice: tiagabine inhibits attacks but bicuculline induces attacks. Compared to GAT1+/- and +/+ mice, the GAT1-/- mice displayed a normal circadian pattern of home cage activity, but more activity overall. Meanwhile, reduced testosterone concentration was found in GAT1-/- mice compared to GAT1+/+ mice but not in GAT1+/+ mice treated with tiagabine, suggesting that testosterone is not directly involved in GAT1 mediated aggressive behavior regulation. These results showed that GAT1 is an important target involved in the regulation of aggressive behavior in mice, and long-term dysfunction of GAT1 may also result in the alteration of testosterone secretion.

Aggression↗

PubMed2006

Reduced anxiety and depression-like behaviors in mice lacking GABA transporter subtype 1.

Gamma-aminobutyric acid (GABA) transporter subtype 1 (GAT1), which transports extracellular GABA into presynaptic neurons, plays an important regulatory role in the function of GABAergic systems. However, the contributions of the GAT1 in regulating mental status are not fully understood. In this paper, we observed the behavioral alterations of GAT1 knockout (GAT1(-/-)) mice using several depression- and anxiety-related models (eg, the forced-swimming test and the tail-suspension test for testing depression-related behaviors; the open-field test, the dark-light exploration test, the emergence test, and the elevated plus maze (EPM) test for anxiety-related behaviors). Here we found that GAT1(-/-) mice showed a lower level of depression- and anxiety-like behaviors in comparison to wild-type mice. Furthermore, GAT1(-/-) mice exhibited measurable insensitivity to selected antidepressants and anxiolytics such as fluoxetine, amitriptyline, buspirone, diazepam, and tiagabine in the tail-suspension test and/or the EPM test. Moreover, the basal level of corticosterone was found to be significantly lower in GAT1(-/-) mice. These results showed that the absence of GAT1 affects mental status through enhancing the GABAergic system, as well as modifying the serotonergic system and the hypothalamic-pituitary-adrenal (HPA) activity in mice.

Animals↗

PubMed2006

Reserpine-induced model of stress suppresses mucosal immunity.

Stress contributes significantly to the development of many diseases. In clinical studies, a strong correlation between depression and immune dysfunction has been shown. Our previous studies indicated that sympathetic innervation can regulate intestinal mucosal immunity through sympathetic synapses, but the mechanism in stress/depression-induced intestinal immune deficiency was unclear. Using a mouse model in which behavioural stress/depression is chemically induced by reserpine, it is found that there is a substantial deficiency of intestinal local humoral and particularly specific antibody response to the antigen stimulation in reserpine-treated group. No significant difference of CD4+, CD8+ or Mac1+ cells between reserpine-treated and control groups was detected in the intestine. This deficiency is closely correlated with stress/depression. A possible correlation between stress, cytokine secretion and humoral immunity in vivo is postulated.

Animals↗

PubMed2006

[Effects of Nurr1 down-regulation on the expression of tyrosine hydroxylase and neurite extension in dopaminergic cells.].

In the experiment, we designed and synthesized two siRNAs based on the sequence of nuclear receptor-related factor 1 (Nurr1) mRNA. They were separately subcloned into the plasmid of pSilenCircle (pSC) containing U6 promoter. The pSC-Nurr1 vectors (pSC-N1 and pSC-N2) specific to Nurr1 gene and the negative control vector of short-hairpin RNA (shRNA) eukaryotic expression vector were constructed. We cultured the dopaminergic cell line MN9D and the verified vectors were transfected with LipofectamineTM 2000 in vitro. The positive cell clones transfected with pSC were obtained after being screened with 500 mug/ml G418. After that, the silencing effects of Nurr1 and TH mRNA or protein were detected by real time RT-PCR and Western blot. The neurite extension of MN9D cells was observed and photographed by inverted microscope. The results showed that Nurr1 mRNA expression in MN9D cells was specifically down-regulated by the vectors of pSC-N1 and pSC-N2, and the silencing effects were 62.3% and 45.6%, respectively. The dopaminergic phenotype of TH mRNA was also suppressed significantly and the silencing effects were 76.3% and 62.6%, respectively. Meanwhile, the expressions of Nurr1 and TH proteins were also significantly suppressed, and the silencing effects of Nurr1 and TH protein were 57.4%, 72.0% and 79.1%, 70.1% respectively. The negative control and liposome groups had no effect on the two genes. In conclusion, Nurr1 shRNA expressing vectors can inhibit the expressions of Nurr1 and TH mRNA or protein in MN9D cells, and Nurr1 might play a role in neurite extension of MN9D cells. Nurr1 shRNA expressing vector may provide a novel applicable strategy for the study on the function of the genes associated with Parkinson disease and the development of dopaminergic neuron.

Cell Line↗

PubMed2006

Mice with genetically altered GABA transporter subtype I (GAT1) expression show altered behavioral responses to ethanol.

It is widely accepted that the GABAergic system plays an important role in the action of ethanol in vivo. GABA transporter subtype 1 (GAT1) constructs high affinity reuptake sites in the CNS and regulates GABAergic transmissions. In this study, mice lacking the GAT1 were developed by homologous recombination. Both hetero- and homozygous GAT1 mutant mice were tested for ethanol, saccharin or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and ethanol-induced sedation/hypnosis. The GAT1(-/-) mice showed decreased ethanol aversion and ethanol reward, and insensitivity to both the sedative/hypnotic and the motor stimulant effects of ethanol, along with increased avoidance of quinine preference and consumption. GAT1(+/-) mice showed significantly increased consumption of ethanol and saccharin, however, enhanced the rewarding and preference effect of ethanol, increased avoidance of quinine, and higher sensitivity to the motor stimulant effect of ethanol. These results demonstrate that GAT1, perhaps in a bi-directional way, modulates some behavioral effects of ethanol. The GAT1 mutant mice provided us a very useful model to investigate the mechanisms of ethanol action in vivo.

Analgesics, Non-Narcotic↗

PubMed2006

Development of peptidic dopamine transporter inhibitors via aromatic modification-mediated conformational restriction.

The dopamine transporter plays an important role in the molecular mechanism of cocaine dependence. It is suggested that inhibitors of the dopamine transporter would have strong therapeutic potential. Here we report that aromatic modification can constrain a linear peptide into the beta-turn conformation, which is preferred by the dopamine transporter. On the basis of this finding, a novel selective and competitive peptidic inhibitor of the dopamine transporter was developed. The peptide binds to the dopamine- and cocaine-binding site of the dopamine transporter and has behavioral effects different from those of cocaine in mice.

Animals↗

PubMed2006

[Study on the position of genes responsible for gallstone disease in Chinese population].

OBJECTIVE: To search the susceptibility genes of gallstone disease in Chinese population. METHODS: A genome wide scan was performed in twelve families with gallstone disease using fluorescence-labeled microsatellite markers. Genehunter and Batchlink of Linkage package were used for non- parameter and parameter linkage analysis to search the linkage loci on chromosomes. RESULTS: Four loci of D3S1266, D4S406, D9S1682 and D11S902 showed suggestive evidence for linkage. nonparametric linkage analysis (NPL)-score of D4S406 and D9S1682 was 1.77 (P = 0.05) and 1.92 (P = 0.04) respectively. The corresponding logarithm of the odds ratio (LOD)-score of D3S1266, D9S1682 were 1.35 and 2.07, and showed a rise of LOD-score from 1.35 to 2.71, 2.07 to 2.40 respectively when families with later-found patients or with higher triglyceride level were analyzed alone. Transmitted disequilibrium test of D11S902 showed a P-value of 0.0027. CONCLUSIONS: Chromosome 3, 4, 9 and 11 may contain genes involved in gallstone disease in Chinese population, and chromosome 3, 9 may hide genes that are liked to gallstone disease in families with later-found patients or with higher triglyceride concentration.

Age Factors↗

PubMed2006

Protopine inhibits serotonin transporter and noradrenaline transporter and has the antidepressant-like effect in mice models.

The protopine isolated from a Chinese herb Dactylicapnos scandens Hutch was identified as an inhibitor of both serotonin transporter and noradrenaline transporter in vitro assays. 5-hydroxy-DL-tryptophan(5-HTP)-induced head twitch response (HTR) and tail suspension test were adopted to study whether protopine has anti-depression effect in mice using reference antidepressant fluoxetine and desipramine as positive controls. In HTR test, protopine at doses of 5, 10, 20 mg/kg dose dependently increase the number of 5-HTP-induced HTR. Protopine at doses of 3.75 mg/kg, 7.5 mg/kg and 30 mg/kg also produces a dose-dependent reduction in immobility in the tail suspension test. The present results open up new possibilities for the use of protopine in the treatment of mood disorders, such as mild and moderate states of depression.

Analysis of Variance↗

PubMed2006

Wnt pathway is involved in pleomorphic adenomas induced by overexpression of PLAG1 in transgenic mice.

Pleomorphic adenoma gene 1 (PLAG1) was found frequently rearranged and activated in human salivary gland pleomorphic adenomas. It encodes a developmentally regulated transcription factor. Ectopic overexpression of PLAG1 has been proposed to play a crucial role in tumorigenesis of salivary gland pleomorphic adenomas. It was reported that PLAG1 can activate the transcription of insulin-like growth factor 2 (IGF2), functioning as a protooncogene. In this report, we show that the salivary gland tumors developed in PLAG1 transgenic mice share major histopathologic features with human pleomorphic adenomas. It was found that beta-catenin, the key component of Wnt signaling pathway, was upregulated at transcriptional level in tumors developed in 3 independent transgenic mouse lines. Immunohistochemical staining revealed that expression of beta-catenin as well as c-myc, downstream of beta-catenin in Wnt signaling pathway, was highly upregulated with overexpression of PLAG1 transgene in tumor and normal transgenic salivary gland tissues. Moreover, we found that PLAG1 can activate the transcription of mouse but not human beta-catenin in the 3T3 cells cotransfected with reporter constructs. Sequence analysis shows there are 4 PLAG1 consensus binding sites in mouse beta-catenin promoter region but not in human. Our findings provide the first in vivo evidence for the oncogenic activity of PLAG1 in pleomorphic adenoma tumorigenesis, reveal a valued animal model for human salivary gland tumors and suggest that Wnt signaling pathway may also contribute to the development of pleomorphic adenomas in transgenic mice.

3T3 Cells↗

PubMed2006

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.

Amino Acid Sequence↗

PubMed2006

[Generation of transgenic mouse of dentin sialoprotein and transgene expression analysis].

PURPOSE: To generate the transgenic mouse model of DSP and perform transgene expression analysis by RT-PCR. METHODS: Plasmid pcDNA3.1-CX was constructed by substituting promoter cbeta-actin for CMV promoter of pcDNA3.1, and the ultimate transgenic vector, pcDNA3.1-CX-dsp, was constructed by cloning DSP coding sequence into pcDNA3.1-CX. The pcDNA3.1-CX-dsp plasmid was linearized and microinjected into the male pronucleus of the zygotes. The tail DNA of pups was tested by PCR and Southern blot. A member of F1 generation of one positive mouse was used to perform transgene expression analysis by RT-PCR. RESULTS: 717 embryos were implanted to 29 recipient pseudopregnant mice, 4 of the 67 pups carrying the transgene. Expression of DSP was detected in a member of F1 generation of one positive mouse by RT-PCR. CONCLUSION: Founders of the DSP transgenic mouse were obtained successfully, and the expression of DSP was primarily confirmed.

Animals↗

PubMed2006

[The establishment of conditional MT transgenic mice model].

OBJECTIVE: To construct the doxycycline-inducible MT transgenic mice model, and provide a basis for the study of hemangioma as well as MT molecular function in vivo. METHODS: Tetracycline-controlled expression systems were employed to this study. A conditional transgenic vector combining the two transcriptional units on a single plasmid was constructed, and the MT gene was subcloned into this vector. To minimize any potential interference, the two elements were spaced with a 1.2 kb cHS4 insulator. To shield the transgene from the affection of chromosomal position effect and improve its expression efficiency, another cHS4 insulator was inserted into the upstream of transgene cassette. After transient transfection of cells in vitro, and analyzing the relative quantification of MT transcripts (target) in mRNA samples by semi-quantitative RT-PCR method, the pronuclear microinjection technique was used to introduce the purified transgene into the chromosomes of fertilized mice eggs, in order to obtain transgenic positive animals. The MT expression in positive mouse was induced through adding deoxycycline in drinking water. Phenotype analysis was done by pathology, and MT expression was confirmed by RT-PCR. RESULTS: The conditional transgenic vector was constructed successfully, and the expression of MT in vitro was regulated by doxycycline. Five transgenic positive mice were obtained through pronuclear microinjection. After MT induction, one transgenic mice developed hemangiomas, and the expression of MT was confirmed by RT-PCR method. The others were active and in breeding. CONCLUSION: Conditional MT transgenic animal model was constructed successfully, and may provide platform for the experimental research of hemangioma as well as the MT molecular function in vivo.

Animals↗

PubMed2006

[New option for gene-targeting vector construction Red/ET recombination].

The study of human new gene's function has become an increasingly active academic field basically relying on the gene knockout (KO) mouse. The construction of targeting vector economically and efficiently has turned into the key step to acquire a KO mouse because of the low efficiency of recombination with traditional constructed targeting vector. For study of the function of new gene-Resp18, we brought in a new DNA engineering platform-Red/ET recombination to construct Resp18 targeting vector. Red/ET recombineering differs from the conventional ways of vector construction (e.g., PCR, restriction enzyme digestion and ligation) and achieves genetic modification by acquisition, insertion, fusion or replacement of the target gene through small fragments mediated homologous recombination. Now Resp18 targeting vectors of three strategies were yielded successfully through two homologous recombination processes of retrieve and neo-targeting. Red/ET recombination has the advantage of getting longer homology regions without mutation, which makes it a new and reliable alternative to the construction of a targeting vector today.

Bacteriophage lambda↗

PubMed2005

A novel strategy to identify the regulatory DNA-organized cooperations among transcription factors.

To identify the functional contributions of cooperations among transcription factors on regulatory DNA is critical for understanding transcription activation. But so far there is a great lack of effective identifying methods. Here we describe a novel strategy, based on comprehensively perturbed experiments and a computational model, to identify the cooperations among NF-kappaB (p65), CREB, and AP-1 in transcription activation of human cytomegalovirus major IE1 promoter/enhancer (MIEP). In this strategy, functional profiles of protein-MIEP association and RNA synthesis are achieved through comprehensively perturbing the association of p65, CREB or AP-1 with MIEP and then subjected to the computational model. Consequently, the 'real' cooperations contributing to MIEP activation are found to comprise five but not seven types of potential cooperations. Thus, our research provides a facile systematic approach to identifying the DNA-organized cooperations among transcription factors and understanding transcription activation.

Cyclic AMP Response Element-Binding Protein↗

PubMed2005

[Risk factors of familial gallstone disease: study of 135 pedigrees].

OBJECTIVE: To explore the role of both inheritable and epidemiological factors in the pathogenesis of familial gallstone disease in pedigrees. METHODS: 135 pedigrees, with 695 members aged > or = 18 (18 - 83, with a mean of 50 +/- 14), 282 males and 413 females, including 370 patients, at least one patient existing in every generation, were investigated. Inquiry was carried out to collect the personal and medical history. Physical examination and ultrasonography were conducted. Blood samples were collected to detect the total cholesterol (TCh), triglyceride (TG), high-density lipoprotein cholesterol (HDL-Ch), and apolipoprotein (Apo) A1 and ApoB. The concentration of low-density lipoprotein cholesterol (LDL-Ch) was calculated by the formula: Tch-TG/5 + HDL-Ch. The inheritable characteristics and heritability were analyzed. RESULTS: The prevalence of gallstone disease was 53.24%, 56.66% among the females, significantly higher than that among the males (48.23%, P = 0.03). The heritability in the first-degree relatives was 138% +/- 7%, and the concordance rate of monozygotic twins was 100%. The significant risk factors were female gender (P = 0.03), body mass index (P < 0.01) and diet (P < 0.008). The history of hypertension, hyperlipidemia, and diabetes were correlated to gallstone disease (P = 0.008, < 0.008, and 0.03 respectively). Serum HDL-Ch and ApoA1 concentrations were lower in the stone group than in the non-stone group (P = 0.003 and 0.005 respectively). There were no significant differences in TCh, TG, LDL-Ch, and ApoB between the 2 groups. CONCLUSION: Genetic factors play a major role in the pathogenesis of familial gallstone disease, characterized by autosomal dominant inheritance. Female gender, obesity, diet, hypertension, hyperlipidemia, diabetes may be the risk factors of gallstone disease. Dyslipidemia is a characteristic of gallstone disease.

Adolescent↗

PubMed2005

Expression profiling suggests a regulatory role of gallbladder in lipid homeostasis.

AIM: To examine expression profile of gallbladder using microarray and to investigate the role of gallbladder in lipid homeostasis. METHODS: 33P-labelled cDNA derived from total RNA of gallbladder tissue was hybridized to a cDNA array representing 17,000 cDNA clusters. Genes with intensities > or =2 and variation <0.33 between two samples were considered as positive signals with subtraction of background chosen from an area where no cDNA was spotted. The average gray level of two gallbladders was adopted to analyze its bioinformatics. Identified target genes were confirmed by touch-down polymerase chain reaction and sequencing. RESULTS: A total of 11 047 genes expressed in normal gallbladder, which was more than that predicted by another author, and the first 10 genes highly expressed (high gray level in hybridization image), e.g. ARPC5 (2 225.88+/-90.46), LOC55972 (2 220.32+/-446.51) and SLC20A2 (1 865.21+/-98.02), were related to the function of smooth muscle contraction and material transport. Meanwhile, 149 lipid-related genes were expressed in the gallbladder, 89 of which were first identified (with gray level in hybridization image), e.g. FASN (11.42+/-2.62), APOD (92.61+/-8.90) and CYP21A2 (246.11+/-42.36), and they were involved in each step of lipid metabolism pathway. In addition, 19 of those 149 genes were gallstone candidate susceptibility genes (with gray level in hybridization image), e.g. HMGCR (10.98+/-0.31), NPC1 (34.88+/-12.12) and NR1H4 (16.8+/-0.65), which were previously thought to be expressed in the liver and/or intestine tissue only. CONCLUSION: Gallbladder expresses 11 047 genes and takes part in lipid homeostasis.

Adult↗

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↗