MiR-130/SNAP-25 axis regulate presynaptic alteration in anterior cingulate cortex involved in lead induced attention deficits.

Brain volume decrease in the anterior cingulate cortex (ACC) after lead (Pb) exposure has been linked to persistent impairment of attention behavior. However, the precise structural change and molecular mechanism for the Pb-induced ACC alteration and its contribution to inattention have yet to be fully characterized. The present study determined the role of miRNA regulated synaptic structural and functional impairment in the ACC and its relationship to attention deficit disorder in Pb exposed mice. Results showed that Pb exposure induced presynaptic impairment and structural alterations in the ACC. Furthermore, we screened for critical miRNA targets responsible for the synaptic alteration. We found that miR-130, which regulates presynaptic vesicle releasing protein SNAP-25, was responsible for the presynaptic impairment in the ACC and attention deficits in mice. Blocking miR-130 function reversed the Pb-induced decrease in the expression of its presynaptic target SNAP-25, leading to the redistribution of presynaptic vesicles, as well as improved presynaptic function and attention in Pb exposed mice. We report, for the first time, that miR-130 regulating SNAP-25 mediates Pb-induced presynaptic structural and functional impairment in the ACC along with attention deficit disorder in mice.

Maternal Lead Exposure Impairs Offspring Learning and Memory via Decreased GLUT4 Membrane Translocation.

Lead (Pb) can cause a significant neurotoxicity in both adults and children, leading to the impairment to brain function. Pb exposure plays a key role in the impairment of learning and memory through synaptic neurotoxicity, resulting in the cognitive function. Researches have demonstrated that Pb exposure plays an important role in the etiology and pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. However, the underlying mechanisms remain unclear. In the current study, a gestational Pb exposure (GLE) rat model was established to investigate the underlying mechanisms of Pb-induced cognitive impairment. We demonstrated that low-level gestational Pb exposure impaired spatial learning and memory as well as hippocampal synaptic plasticity at postnatal day 30 (PND 30) when the blood concentration of Pb had already recovered to normal levels. Pb exposure induced a decrease in hippocampal glucose metabolism by reducing glucose transporter 4 (GLUT4) levels in the cell membrane through the phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt) pathway. In vivo and in vitro GLUT4 over-expression increased the membrane translocation of GLUT4 and glucose uptake, and reversed the Pb-induced impairment to synaptic plasticity and cognition. These findings indicate that Pb exposure impairs synaptic plasticity by reducing the level of GLUT4 in the cell membrane as well as glucose uptake via the PI3K-Akt signaling pathway, demonstrating a novel mechanism for Pb exposure-induced neurotoxicity.

Low-level Gestational Lead Exposure Alters Dendritic Spine Plasticity in the Hippocampus and Reduces Learning and Memory in Rats.

Lead (Pb) is known to impair children's cognitive function. It has been previously shown that developmental Pb exposure alters dendritic spine formation in hippocampal pyramidal neurons. However, the underlying mechanism has not yet been defined. In this study, a low-level gestational Pb exposure (GLE) rat model was employed to investigate the impact of Pb on the spine density of the hippocampal pyramidal neurons and its regulatory mechanism. Pb exposure resulted in impaired performance of the rats in the Morris water maze tasks, and in decreased EPSC amplitudes in hippocampal CA3-CA1 regions. With a 3D reconstruction by the Imaris software, the results from Golgi staining showed that the spine density in the CA1 region was reduced in the Pb-exposed rats in a dose-dependent manner. Decreased spine density was also observed in cultured hippocampal neurons following the Pb treatment. Furthermore, the expression level of NLGN1, a postsynaptic protein that mediates synaptogenesis, was significantly decreased following the Pb exposure both in vivo and in vitro. Up-regulation of NLGN1 in cultured primary neurons partially attenuated the impact of Pb on the spine density. Taken together, our resultssuggest that Pb exposure alters spine plasticity in the developing hippocampus by down-regulating NLGN1 protein levels.

Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

BACKGROUND: Mushrooms have been used in Asia as traditional foods and medicines for a long time. Ergosta-4,6,8(14),22-tetraen-3-one (ergone) is one of the well-known bioactive steroids, which exists widely in various medicinal fungi such as Polyporus umbellatus, Russula cyanoxantha, and Cordyceps sinensis. Ergone has been demonstrated to possess cytotoxic activity. However, the molecular mechanisms by which ergone exerts its cytotoxic activity are currently unknown. METHODS: In the present study, ergone possessed a remarkable anti-proliferative activity toward human hepatocellular carcinoma HepG2 cells. We assayed the cell cycle by flow cytometry using PI staining; investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V/PI staining; observed the nuclear fragmentation by Hoechst 33258 staining and studied the protein expression of Bax, Bcl-2, p-53, procaspase-3, -8, -9, PARP and cleaved PARP by Western blotting analysis. RESULTS: Cells treated with ergone showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. Furthermore, PARP-cleavage; activation of caspase-3, -8, -9; up-regulation of Bax and down-regulation of Bcl-2 were observed in HepG2 cells treated with ergone, which show that both the intrinsic and extrinsic apoptotic pathways are involved in ergone-induced apoptosis in HepG2 cells. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner. GENERAL SIGNIFICANCE: In this study, we reported for the first time that ergone-induced apoptosis through activating the caspase. These results would be useful for the further utilization of many medicinal fungi in cancer treatment.

[Analysis of full-length molecule of human lipopolysaccharide responsed gene and its expression in four cell lines].

AIM: To clone full-length human lipopolysaccharide responsive gene(hlrp) and predict its function by bioinformatics analysis; to observe full-length hlrp protein and quantify its relative gene expression in four cell lines. METHODS: Total RNA was extracted from LPS-stimulated human embryonic kidney cells HEK293 and the full-length hlrp was obtained by RT-PCR. Function of hlrp was predicted by bioinformatics analysis with Internet and GenBank database. Expression full-length hlrp protein in HEK293, HepG2, HeLa and PDC was observed by laser scanning confocal fluorescence microscope and compared. RESULTS: Full-length hlrp of 2 045 bp was amplified and sequenced. Leucine zipper was found in the hlrp series that may have an important function. hlrp gene have been mapped to a particular chromosome location in Xp22.2. Laser scanning confocal fluorescence microscope showed hlrp protein was expressed in the cell lines (HEK293, HepG2, HeLa and PDC). CONCLUSION: hlrp has been successfully cloned and its function has been predicted. Expression of hlrp has been detected in 4 cell lines. Present result would provide data for the further study of hlrp.

[Expression of mouse lipopolysaccharide response protein LRP in E. coli and preparation of rabbit anti-mLRP serum].

AIM: To express mouse lipopolysaccharide response protein (mLRP) and prepare rabbit anti-mLRP serum. METHODS: The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs. Then the primers were designed. mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide (LPS) was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp. The His-TAT-mLRP fusion protein was expressed in E. coli BL21(DE3) and was used to immunize the rabbits to get rabbit anti-mLRP serum. The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot. RESULTS: The predicted length of mlrp cDNA was 1905 bp. The encoding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT. The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein. CONCLUSION: The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the foundation for further study of the function of mLRP.

[Study on the presence of hepatitis B virus in first-trimester villi in pregnant women with hepatitis B surface antigen positive].

OBJECTIVE: To observe the presence of hepatitis B virus (HBV) in first-trimester villi cells from pregnant women carrying HBsAg. METHODS: Immunohistochemical streptavidin-biotin peroxidase complex (SABC) staining with monoclonal HBsAg, hepatitis B core antigen (HBcAg) and PCR, in situ hybridization were used for detection of HBV infection markers in villi. Positive villi ultramicrostructures were observed with transmission electron microscope. RESULTS: HBV was detected in 8 of 25 villi of HBsAg positive pregnant women, the positive rate was 32%. HBsAg was located in the decidual cell, trophoblastic cell and villous mesenchymal cell. HBV analog was detected in rough endoplasmic reticulum of trophoblastic cell. CONCLUSIONS: HBV may infect villous cells in first-trimester pregnancy. It would be impossible for HBV to transmit the desmosomes.

[Cloning, expression and antitumor effect of mouse costimulatory molecule 4-1BBL].

AIM: To clone mouse 4-1BBL gene, construct its eukaryotic expression vector, and evaluate antitumor activity of the expression product. METHODS: RT-PCR was used to amplify mouse 4-1BBL gene from total RNA of C57BL/6 splenocytes stimulated by PHA. Then m4-1BBL cDNA was subcloned into eukaryotic expression vector pcDNA3.1(+) and transfected into mouse hepatocellular carcinoma cell line Hepa1-6. The expression of m4-1BBL in transfected cells was detected by RT-PCR, indirect immunofluorescence staining, and flow cytometry. Non-adherent splenocytes from non-immunized C57BL/6 mice were incubated with mitomycin-treated non-transfected Hepa1-6(Hepa1-6-wt) or transfected Hepa1-6 cells (Hepal-6-m4-1BBL), respectively. Then the lymphocytes were tested for cytotoxic activity to Hepa1-6-wt cells. RESULTS: The Hepa1-6 cells transfected by pcDNA3.1(+)-m4-1BBL could efficiently express m4-1BBL. As compared with Hepa1-6-wt cells,Hepa1-6-m4-1BBL cells could induce more efficiently cytotoxic activity of lymphocytes to Hepa1-6-wt cells (P<0.01). CONCLUSION: The expression of m4-1BBL by tumor cells is effective in inducing antitumor immune response.