Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

D-β-Hydroxybutyrate Prevents MPP+-Induced Neurotoxicity in PC12 Cells

Numerous studies show that D-β-Hydroxybutyrate (DβHB) is neuroprotective. The present study was to explore the neuroprotective effects of DβHB against the cell death and apoptosis induced by 1-methyl-4-phenylpyridinium ion (MPP+) in PC12 cells. PC12 cells were pretreated with DβHB and followed by MPP+ exposure. The cell viability was determined by MTT assay. The morphological characteristics of apoptosis was observed by Acridine Orange (AO) staining and apoptotic rates were measured by flow cytometer. The product of lipid peroxidation, malondialdehyde (MDA), was measured using thiobarbituric acid method. The mitochondrial membrane potential (MMP), intracellular ROS and total glutathione were detected by microplate reader. In PC12 cells, pretreatment with DβHB significantly reduced MPP+-induced the decrease of cell viability. AO staining and flow cytometric analysis found DβHB inhibited MPP+-induced apoptosis. The measurement of MDA formation showed that DβHB alleviated lipid peroxidation induced by MPP+. The loss of MMP induced by MPP+ was preventive by DβHB. The changes of intracellular ROS and total glutathione induced by MPP+ were reversed by DβHB. DβHB protected PC12 cells against MPP+-induced death and apoptosis.     

Expression of αB-Crystallin in the Peripapillary Glial Cells of the Developing Chick Retina

Peripapillary glial cells (PPGCs) are a peculiar macroglia in avian species, located in the central retina adjacent to the optic nerve head. PPGCs have a similar shape and orientation to Müller cells, which traverse the entire layer of the retina; however, there are differences in protein expression between the two cell types. In the present study, we first demonstrated that PPGCs expressed αB-crystallin, which is not expressed in Müller cells, during retinal development. αB-crystallin was first faintly expressed in PPGCs of the E5 retina, adjacent to the optic nerve head. Further, αB-crystallin was exclusively expressed in PPGCs up to E14. The shape of these cells was bipolar with vitread and ventricular processes. The vitread processes of αB-crystallin+ PPGCs became finer at E18. Double labeling analysis clearly demonstrated that only vimentin+ or GFAP+ astrocytes were located in the optic nerve head and were demarcated from the retina by αB-crystallin+ PPGCs. Furthermore, we determined that αB-crystallin+ PPGCs, with a number of processes, completely wrapped the optic nerve head and were densely located in the junction of the optic nerve head and the retina in a whole mount preparation and in vertical-sectioned retinae. The results of present study, together with reports that retinal astrocytes migrate from the optic nerve head, suggest that PPGCs prevent astrocytes from migrating into the retina in avian species.     

Protective Effect of Bajijiasu Against β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-amyloid peptide (Aβ), a major protein component of senile plaques associated with Alzheimer’s disease (AD), is also directly neurotoxic. Mitigation of Aβ-induced neurotoxicity is thus a possible therapeutic approach to delay or prevent onset and progression of AD. This study evaluated the protective effect of Bajijiasu (β- d-fructofuranosyl (2–2) β- d-fructofuranosyl), a dimeric fructose isolated from the Chinese herb Radix Morinda officinalis, on Aβ-induced neurotoxicity in pheochromocytoma (PC12) cells. Bajijiasu alone had no endogenous neurotoxicity up to 200 μM. Brief pretreatment with 10–40 μM Bajijiasu (2 h) significantly reversed the reduction in cell viability induced by subsequent 24 h exposure to Aβ_25–35 (21 μM) as measured by MTT and LDH assays, and reduced Aβ_25–35-induced apoptosis as indicated by reduced annexin V-EGFP staining. Bajijiasu also decreased the accumulation of intracellular reactive oxygen species and the lipid peroxidation product malondialdehyde in PC12 cells, upregulated expression of glutathione reductase and superoxide dismutase, prevented depolarization of the mitochondrial membrane potential (Ψm), and blocked Aβ_25–35-induced increases in [Ca²⁺] _i . Furthermore, Bajijiasu reversed Aβ_25–35-induced changes in the expression levels of p21, CDK4, E2F1, Bax, NF-κB p65, and caspase-3. Bajijiasu is neuroprotective against Aβ_25–35-induced neurotoxicity in PC12 cells, likely by protecting against oxidative stress and ensuing apoptosis.     

Protective Effects of Pinostrobin on β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-Amyloid peptide (A??), a major protein component of brain senile plaques in Alzheimer??s disease (AD), has been considered as a critical cause in the pathogenesis of AD. Pinostrobin, a potent flavonoid inducer, is the major and most active ingredient of Folium cajani. The present study aimed to investigate whether pinostrobin could provide protective effect against A??_25-35-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The PC12 cells were pretreated with different concentrations of pinostrobin for 2?h, followed by the challenge with 20???M A??_25?C35 for 24?h. The results showed that pretreatment with pinostrobin significantly elevated cell viability, decreased the lactate dehydrogenase activity, the levels of intracellular reactive oxygen species and calcium, and mitochondrial membrane potential in A??_25?C35-treated PC12 cells. In addition, pinostrobin significantly suppressed the formation of DNA fragmentation and increased the ratio of Bcl-2/Bax. These results indicate that pinostrobin was able to exert a neuroprotective effect against A??_25?C35-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative damage and calcium overload, as well as suppressing the mitochondrial pathway of cellular apoptosis.     

Protective Effect of Isorhynchophylline Against β-Amyloid-Induced Neurotoxicity in PC12 Cells

Beta-amyloid peptide (Aβ), a major protein component of senile plaques, has been considered as a critical cause in the pathogenesis of Alzheimer’s disease (AD). Modulation of the Aβ-induced neurotoxicity has emerged as a possible therapeutic approach to ameliorate the onset and progression of AD. The present study aimed to evaluate the protective effect of isorhynchophylline, an oxindole alkaloid isolated from a Chinese herb Uncaria rhynchophylla, on Aβ-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells. The results showed that pretreatment with isorhynchophylline significantly elevated cell viability, decreased the levels of intracellular reactive oxygen species and malondialdehyde, increased the level of glutathione, and stabilized mitochondrial membrane potential in Aβ_25-35-treated PC12 cells. In addition, isorhynchophylline significantly suppressed the formation of DNA fragmentation and the activity of caspase-3 and moderated the ratio of Bcl-2/Bax. These results indicate that isorhynchophylline exerts a neuroprotective effect against Aβ_25-35-induced neurotoxicity in PC12 cells, at least in part, via inhibiting oxidative stress and suppressing the mitochondrial pathway of cellular apoptosis.     

17β-Estradiol Downregulated the Expression of TASK-1 Channels in Mouse Neuroblastoma N2A Cells

TASK channels, an acid-sensitive subgroup of two pore domain K⁺ (K2P) channels family, were widely expressed in a variety of neural tissues, and exhibited potent functions such as the regulation of membrane potential. The steroid hormone estrogen was able to interact with K⁺ channels, including voltage-gated K⁺ (Kv) and large conductance Ca²⁺-activated (BK) K⁺ channels, in different types of cells like cardiac myocytes and neurons. However, it is unclear about the effects of estrogen on TASK channels. In the present study, the expressions of two members of acid-sensitive TASK channels, TASK-1 and TASK-2, were detected in mouse neuroblastoma N2A cells by RT-PCR. Extracellular acidification (pH 6.4) weakly but statistically significantly inhibited the outward background current by 22.9 % at a holding potential of 0 mV, which inactive voltage-gated K⁺ currents, suggesting that there existed the functional TASK channels in the membrane of N2A cells. Although these currents were not altered by the acute application of 100 nM 17β-estradiol, incubation with 10 nM 17β-estradiol for 48 h reduced the mRNA level of TASK-1 channels by 40.4 % without any effect on TASK-2 channels. The proliferation rates of N2A cells were also increased by treatment with 10 nM 17β-estradiol for 48 h. These data implied that N2A cells expressed functional TASK channels and chronic exposure to 17β-estradiol downregulated the expression of TASK-1 channels and improved cell proliferation. The effect of 17β-estradiol on TASK-1 channels might be an alternative mechanism for the neuroprotective action of 17β-estradiol.     

Presence of DNase γ-Like Endonuclease in Nuclei of Neuronal Differentiated PC12 Cells

DNase , which cleaves chromosomal DNA into nucleosomal units (DNA ladder formation), has been suggested to be the critical component of apoptotic machinery. Using rat pheochromocytoma PC12 cells, which are differentiated to sympathetic neurons by nerve growth factor (NGF), we investigated whether DNase -like enzyme is present in neuronal cells and is involved in neuronal cell death. The nuclear auto-digestion assay for DNase catalyzing internucleosomal DNA cleavage revealed that nuclei from neuronal differentiated PC12 cells contain acidic and neutral endonucleases, while nuclei from undifferentiated PC12 cells have only acidic endonuclease. The DNA ladder formation observed in isolated nuclei from neuronal differentiated PC12 cells at neutral pH requires both Ca²⁺ and Mg²⁺, and is sensitive to Zn²⁺. The molecular mass of the neutral endonuclease present in neuronal differentiated PC12 cell nuclei is 32000 as determined by activity gel analysis (zymography). The properties of the neuronal endonuclease present in neuronal differentiated PC12 cell nuclei were similar to those of purified DNase from rat thymocytes and splenocytes. Interestingly, in neuronal differentiated PC12 cells, internucleosomal DNA fragmentation is observed following NGF deprivation, whereas undifferentiated PC12 cells fail to exhibit DNA ladder formation during cell death by serum starvation. These results suggest that the DNase -like endonuclease present in neuronal differentiated PC12 cell nuclei is involved in internucleosomal DNA fragmentation during apoptosis, induced by NGF deprivation.     

Carnosine Protects Against Aβ42-induced Neurotoxicity in Differentiated Rat PC12 Cells

(1) The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Aβ42-induced impairment in differentiated PC12 cells. (2) PC12 cells were exposed to Aβ42 (5 μM) for 24 h after carnosine (5 mM) applied for 18 h. Histamine receptor antagonists (diphenhydramine, zolantidine, thioperamide, clobenpropit) or histidine decarboxylase inhibitor (α-fluoromethylhistidine) were added 15 min before carnosine. Cell viability, glutamate release or cell surface expression of NMDA receptor was examined. (3) Aβ42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with carnosine ameliorated this impairment. This amelioration was reversed by the H₃ receptor antagonists thioperamide and clobenpropit, but not by either the H₁ receptor antagonist diphenhydramine or the H₂ receptor antagonist zolantidine. Further, α-fluoromethylhistidine, an irreversible inhibitor of histidine decarboxylase, also had no effect. In the presence of Aβ42, carnosine significantly decreased glutamate release and carnosine increased the surface expression of NMDA receptor. (4) These results indicate that the mechanism by which carnosine attenuates Aβ42-induced neurotoxicity is independent of the carnosine–histidine–histamine pathway, but may act through regulation of glutamate release and NMDA receptor trafficking.     

The Influence of Fluoride on the Expression of Inhibitors of Wnt/β-Catenin Signaling Pathway in Rat Skin Fibroblast Cells

The effective therapy of fluoride-induced bone diseases requires an understanding of the mechanism of the disorders. Changes in the inhibitors of the Wnt/β-catenin pathway, Dickkopf-1 (Dkk-1) and Sclerostin (SOST),were studied in supernatants harvested from rat skin fibroblasts cultured with varied doses of fluoride. The contents of SOST and Dkk-1 in fibroblast supernatants were assessed at four exposure time-points and investigated by using the method of ELISA. Compared to the relevant controls(0 mg F(?)/L), a significant decrease of the concentrations of SOST and Dkk-1 was observed as the fluoride concentration increased. Compared to the relevant time controls (24 h), a significant decrease of the concentrations of SOST and Dkk-1 was observed with the extension of time. Our results suggest that the Wnt/β-catenin pathway inhibitors Dkk-1 and SOST play an important role in skeletal fluorosis. They can be used as important indications for diagnosing bone metabolism changes caused by fluoride exposure and therapeutic targets in diseases resulting from fluoride exposure.     

Expression of Recombinant Carcinoembryonic-Antigen-Human-β-Defensin-2 Gene in Colon Cancer HCT116 Cells

The objective of this study was to construct a system driving high expression of human-β-denfensin-2 (HBD-2) system in eukaryotic cell. To construct recombinant retrovirus expression vector, CEA signal peptide-HBD-2 (mature peptide sequence) was cloned in the retrovirus expression vector PLNCX2. The retroviral vector was transfected into PT67 cells by DOTAP; after screening and amplifying single clone cell by G418, the virus particles were collected and infected to eukaryotic, colon cancer HCT116 cells. Furthermore, the HCT116 cells were also screened by using G418 and the resistance clones were obtained. Finally, the expression of HBD-2 was detected by Western blotting, which suggested a high level of expression of HBD-2 in HCT116 cells. In conclusion, the results indicate that high level of HBD-2 expression was obtained in HCT116 cells which will help in effective commercial production and purification of HBD-2 protein.     

Expression of the Cloned Hemagglutinin–Neuraminidase Gene of the Newcastle Disease Virus in Mouse Myeloma Cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin–neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoic fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.