Overexpression of p27^(KIP1)induced cell cycle arrest in G_1 phase and subsequent apoptosis in HCC-9204 cell line

AIM We have previously reported that inducible over-expresaion of Bak may prolong cell cycle in G1 phase and lead to apoptosis in HCC-9204 cells. This study is to investigate whether p27KIP1 plays an important role in this process. MEHODS In order to elucidate the exact function of p27KIP1 in this process, a zinc inducible p27KIP1 stable transfectant and transient p27KIP1- GFP fusion transfectant were constructed. The effects of inducible p27KIP1 on cell growth, cell cycle arrest and apoptosis were examined in the mock, control pMD vector, and pMD-KIP1 transfected HCC-9204 cells. RESULTS This p27KIP1-GFP transfectant may transiently express the fusion gene. The cell growth was reduced by 35% at 48 h of p27KIP1 induction with zinc treatment as determined by trypan blue exclusion assay. These differences remained the same after 72 h of p27KIP1 expression, p27KIP1 caused cell cycle arrest after 24 h of induction, with 40% increase in G1 population. Prolonged p27KIP1 expression in this cell line induced apoptotic cell death reflected by TUNEL assay. Fourty-eight h and 72 h of p27KIP1 expression showed a characteristic DNA ladder on agarose gel electrophoresis.

Senescence-like changes induced by expression of p21^(Waf1/Cipl)in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.

Senescence—like changes induced by expression of p21^Waf1／Cip1 in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.

Screening of Epstein-Barr Virus Early Antigen Expression Inducers from Chinese Medicinal Herbs and Plants

Ether extrilcls of 1693 Chinesc medicinal herbs and plilnts from 268 families werestudied for the induction of Epstcin-Barr viral (EBV ) early antigcn (EA ) expression in theRaji cell line. Fifty-two from 18 families were found to have inducing activity. Twenty-fiveand seven of them were from Euphorbiaccae and Thymclaeaceae, respectively. Some ofthem, such as Croton tiglium, Euphorbia kansui, Daphnc genkwa, Wikstrocmia chamacdaphen, Wikstroemia indica, Prunus mandshurica Koehne and Achyranthes bidentata arecommonly used drugs. The significance of these herbs in the activation of EBV in vivo andtheir relation to the development of nasopharyngeal carcinoma were discussed.

Prenatal Exposure to Perfluorooctane Sulfonate impairs Placental Angiogenesis and Induces Aberrant Expression of LncRNA Xist

Perfluorooctane sulfonate （PFOS） is a class of stable organic compounds with wide industrial,commercial, and consumer applications, such as in textiles, paper, pesticides, and shampoos;. It is readily absorbed, but poorly eliminated, with the elimination half-life of approximately 5 years;.Hence, there have been concerns regarding its potential damage to human health. Some studies

Expression of Peroxiredoxins and Pulmonary Surfactant Protein A Induced by Silica in Rat Lung Tissue

Silicosis is one of the most serious occupational diseases in China and dates back to centuries ago. In this study, we successfully established a rat model of silicosis by intratracheal silica injection for 28 days and determined hydroxyproline levels to evaluate collagen metabolism in lung homogenates. Oxidative stress status was evaluated by detecting catalase and glutathione peroxidase activities.

Trichloroethylene Induces Biphasic Concentration-dependent Changes in Cell Proliferation and the Expression of SET-Associated Proteins in Human Hepatic L-02 Cells

Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TCE cytotoxicity have been made in cultured liver cells. However, the molecular mechanisms by which TCE induces hepatotoxicity are not well understood. SET （also known as protein phosphatase 2A inhibitor, 12PP2A, or template-activating factor-I, TAF-D is a nuclear protein that regulates histone modification, gene transcription, DNA replication, nucleosome assembly,

Induced in vitro Expression of Human Lactoferrin in Goat Mammary Gland Epithelial Cell

[Objective]The aim of this study was to explore the technical system of induced expression in vitro of goat mammary gland epithelial cell,and evaluate expression efficiency of mammary gland specific vector and foreign protein at the cell level.[Method]Goat mammary gland epithelial cell transfected by human lactoferrin gene was inducted by culturing in DMEM/F12 medium supplemented with 5 mg/L insulin,5 mg/L prolactin and 1 mg/L hydrocortisone.Supernatant was collected per 6 hours and concentrated.Expression situation of foreign protein were detected by SDS-PAGE and Western blotting.[Result]There was target protein expression in the induced culture medium,which molecular weight was about 42 kD.[Conclusion]The method used in this study can induce goat mammary gland epithelial cell to express foreign gene,it lays a foundation for researching heterologous expression of foreign gene and producing mammary gland bioreactor.

Maternal Lead Exposure Induces Down-regulation of Hippocampal Insulin-degrading Enzyme and Nerve Growth Factor Expression in Mouse Pups

Lead exposure is a known potential risk factor for neurodegenerative diseases such as Alzheimer’s disease （AD）. Exposure to lead during the critical phase of brain development has been linked with mental retardation and hypophrenia in later life.

Cytokines Expression in Lymphocytes From Rats With Allergic Asthma Induced by Pleurotus sapidus besidiospores

Allergic asthma caused by mushroom spores (Pleurotus sapidus besidiospores) is a common health problem among mushroom-cultivating workers in China. An animal model of allergic asthma through the challenge of Pleurotus sapidus besidiospores in primed rats was developed for investigating the role of cytokines in the pathogenesis of the disease. In the study a series of related cytokines and their receptors, including their activity and mRNA levels of spleen lymphocytes isolated from asthmatic rats, were measured. Determined by 3H-TdR incorporation assay and NAG microcolorimetric assay, Con A-induced spleen lymphocyte proliferation and IL-2 activity in culture supernatants of spleen lymphocytes from 7-day challengedrats with allergic asthma increased significantly by 261% and 208%, respectively, as compared with those in the control. Cytokines and their receptor expression at mRNA levels were determined by RNA/cDNA hybridization, using (α-32P-dCTP radiolabeled cDNA probes for different cytokines and their receptors in vitro. The results showed that mRNA expression of IL-4, GM-CSF, IL-6, IL-2 and IL-2R, except IL-6R, in lymphocytes of 7-day-challenged asthma-suffering rats, increased significantly by 54%, 45%, 170%, 83% and 76%, respectively. In 2-day challenged-rats, mRNA levels of IL-4, IL-6 and IL-2 increased by 37%,58% and 125%, respectively, whereas mRNA leve1s of GM-CSF, IL-2R and IL-6R remained unchanged. Thus, the experimental results suggested a significant increase in TH2type cytokines in the pathogenesis of Pleurotus sapidus besidiospore-induced allergic asthma and IL-4 may play an essential role.

Suppressive effect of dexamethasone on the neutrophil expression of CD18 in rats with radiation induced brain edema

BACKGROUND： Stereo-tactic radiation therapy （SRT） is widely used to treat intracranial diseases, but some patients suffered from radiation induced brain edema after SRT. Once radiation induced brain edema occurs, the treatment is quite difficult, and it always leads to a poor outcome. Dexamethasone has certain therapeutic effect on traumatic brain edema, but the biological mechanism is still unclear. OBJECTIVE ： To observe the effect of dexamethasone on the neutrophil expression of CD18.DESIGN ： A randomized control observation.SETTING： Changhai Hospital of the Second Military Medical University of Chinese PLA. MATERIALS ： The experiment was carried out in Changhai Hospital of the Second Military Medical University of Chinese PLA from January 1999 to December 1999. Twenty SD rats （male and female each in half） weighing （250±50） g were used. METHODS： Twenty SD rats were divided into four groups at random. ① Blank control group （n=5）： The rats were not treated without dexamethasone or irradiation;② Irradiation group （n=5）： The rats were given irradiation but no dexamethasone treatment; ③ Irradiation＋1 mg/kg dexamethasone group （n=5）; The rats were treated with irradiation and dexamethasone of 1 mg/kg; ④Irradiation＋5 mg/kg dexamethasone group （n=5）： The rats were treated with irradiation and dexamethasone of 5 mg/kg. The heads of the rats were irradiated with 10 MeV X-ray （30 Gy）, and brain tissue was removed after 2 weeks to observe the pathological changes. Blood samples were taken from the carotid artery, gradient centrifugation was used, and neutrophile layer was obtained, the level of neutrophile expression of CD18 mRNA and quantity of membrane proteins in blood were detected with Northern blot and flow cytometry respectively. MAIN OUTCOME MEASURES： ① Blood cell count; ② Pathological results; ③ level of neutrophile expression of CD18 mRNA and quantity of membrane proteins. RESULTS ： All the 20 SD rats were involved in the analysis of results without deletion. At 2 weeks after irradiation, obvious cell injury could be observed under light microscope. The level of neutrophile expression of CD18 mRNA and quantity of membrane proteins in blood were obviously increased, but the severity of cell injury was relieved in the irradiation＋1 and 5 mg/kg dexamethasone groups, and the CD18 expression was markedly suppressed （P 〈 0.05）, and the suppression was more obvious in the irradiation＋5 mg/kg dexamethasone group than in the irradiation＋1 mg/kg dexamethasone group （P 〈 0.01 ）. CONCLUSION： Dexamethasone can reduce the radiation induced brain edema by inhibiting the expression of CD18.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.

The Expression of RcLEA Gene Improves Tolerance of E. coli Cells to Abiotic Stress

[Objective] This study was to reveal the heat induced expression model of RcLEA gene and its tolerance to various abiotic stresses.[Method] Heat resistant and heat sensitive varieties of Rosa hybrida L.were subjected to heat shock treatment at 38 ℃ for 3 h;then RcLEA gene from both varieties treated was cloned and transformed into Escherichia coli strain BL21;finally recombinant colonies were separately cultured at 4 ℃ and 50 ℃ under the stresses of LiCl,NaCl,Na2CO3,CdCl2 and H2O2 to study the responses of recombinant E.coli strains to high temperature,low temperature and some other abiotic stresses.[Result] After heat shock treatment at 38 ℃ for 3 h,RcLEA gene expressed highly in ＇Schloss mannieim＇（SM）and ＇Las vegas＇（LV）variety,but weakly or even not expressed in ＇Kordes＇ Perfecta＇（KP）,indicating that this gene is closely related with heat resistance of R.hybrida.Compared with WT strains,recombinant clones showed higher tolerance to abiotic stresses including high temperature,low temperature,heavy metal,high salt,high pH value and oxidation,suggesting that RcLEA is concerned with the response of R.hybrida to abiotic stresses mentioned above.[Conclusion] These results provide thoughts for increasing heat resistance by introducing RcLEA into heat sensitive R.hybrida varieties and studying the heat-resistant mechanism of R.hybrida,and also provide theoretical support for selecting heat resistant variety of landscape and ornamental plants like R.hybrida.

Identification of an intestine-specific promoter and inducible expression of bacterial α-galactosidase in mammalian cells by a lac operon system

Background： o-galactosidase has been widely used in animal husbandry to reduce anti-nutritional factors （such as o-galactoside） in feed. Intestine-specific and substrate inducible expression of a-galactosidase would be highly beneficial for transgenic animal production. Methods： To achieve the intestine-specific and substrate inducible expression of o-galactosidase, we first identified intestine-specific promoters by comparing the transcriptional activity and tissue specificity of four intestine-specific promoters from human intestinal fatty acid binding protein, rat intestinal fatty acid binding protein, human mucin-2 and human lysozyme. We made two chimeric constructs combining the promoter and enhancer of human mucin-2, rat intestinal trefoil factor and human sucrase-isomaltase. Then a modified lac operon system was constructed to investigate the induction of o-galactosidase expression and enzyme activity by isopropyl p-D-]-thiogalactopyranoside （IPTG） and an a-galactosidase substrate, a-lactose. We declared that the research carried out on human （Zhai Yafeng） was in compliance with the Helsinki Declaration and experimental research on animals also followed internationally recognized guidelines. Results： The activity of the human mucin-2 promoter was about 2 to 3 times higher than that of other intestine-specific promoters. In the/ac operon system, the repressor significantly decreased （P 〈 0.05） luciferase activity by approximately 6.5-fold and reduced the percentage of cells expressing green fluorescent protein （GFP） by approximately 2-fold. In addition, the expression level of o-galactosidase mRNA was decreased by 6-fold and a-galactosidase activity was reduced by 8-fold. in line with our expectations, IPTG and a-lactose supplementation reversed （P 〈 O.O5） the inhibition and produced a 5-fold increase of luciferase activity, an 11-fold enhancement in the percentage of cells with GFP expression and an increase in o-galactosidase mfiNA abundance （by about 5-fold） and o-galactosidase activity （by about 7-fold）. Conclusions： We have successfully constructed a high specificity inducible lac operon system in an intestine-derived cell line, which could be of great value for gene therapy applications and transgenic animal production.

Construction of Expression Vector for Porcine Gastrin-releasing Peptide Fusion Protein

[Objectives]This study was conducted to obtain porcine gastrin-releasing peptide(GRP)fusion protein.[Methods]The constructed pET32a(+)-GRP plasmid was transformed into Escherichia coli BL21(DE3)competent cells to obtain the pET32a(+)-GRP-BL21(DE3)fusion protein expression strain,which was induced with 0.5 mM IPTG at 25℃and 150 r/min for 12 h,and the His-tagged GRP fusion protein was detected by SDS-Page gel electrophoresis and Western Blot.[Results]After optimizing the IPTG-induced expression conditions,it was confirmed that the porcine GRP fusion protein was obtained,and the porcine GRP fusion protein was soluble,stable and highly active.[Conclusions]This study lays a foundation for the subsequent preparation of anti-pig GRP antibodies.

Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND： Hypoxia inducible factor-1 alpha （HIF-1 （x） and erythropoietin（EPO）, possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance （IT）. OBJECTIVE：To observe the neuroprotective effect of cerebral ischemic preconditioning（IPC） of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN ： A randomized and controlled observation SETTING： Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University MATERIALS： Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd （Wuhan）; Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company （USA）. METHODS： This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot： IPC group （n=40）, sham-operation group （n=40） and control group （n=4）. In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1^st, 3^rd, 7^th, 14^th and 21^st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion （MCAO）. That was to say, after 10-minute preischemia, suture was exited to the extemal carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation（only common carotid artery and crotch were exposed, and MCAO by suture was omitted）, and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume （The total cerebral infarction area of each layerxinterspace ） was calculated. After brain tissue was stained by haematoxylin-esoin （HE）, the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α（and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES： ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS：Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group： Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively [（161.2±6.9） mm^3 vs （219.9±11.2） mm^3, （134.9±9.0） mm^3 vs （218.6±13.0） mm^3, （142.9±13.7） mm^3 vs （221.3±14.2） mm^3, t=-8.924, 10.587,7.947, P〈 0.01]. ② Observation of nerve cell form of brain tissue： HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus ： The expression of HIF-1αof IPC group was significantly higher than that of sham-operation group on the 1^st, 3^rd and 7^th days after reperfusion respectively （125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763,9.899,P 〈 0.05-0.01）. The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3^rd and 7^th days after perfusion respectively （141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18,t=5.499,3.970, P〈 0.05）. CONCLUSION ： ①IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.

Inheritance and Expression of Potato Proteinase Inhibitor Gene Ⅱ （pinⅡ） in Transgenic Rice

The inheritance and expression of bar gene and pinII gene were studied in three transgenic rice lines and their F2 hybrid populations, which were created through hybridization with a PGMS line, ZAU11S. By Basta painting, PCR analysis and determining of the inhibitory trypsin activity, the results show that bar gene and pinII gene in rice F2 population fit the simple Mendel's low of inheritance and close linkage, but a few plants in F2 have not sufficiently expressed. The wound inducible pin II gene has an expression regulated spatially and temporally, and the signal transduction pathway is not only upward, but also downward. The inducible expression of pin II in different rice transgenic lines is not completely coincident.

Establishment of the Eukaryotic Cell Lines for Inducible Control of SARS-CoV Nucleocapsid Gene Expression

In order to establish the eukaryotic cell lines for inducible control of SARS-CoV nucleocapsid gene expression.The recombinant plasmid of pTRE-Tight-SARS-N was constructed by using the plasmid p8S as the PCR template which contains a cDNA clone covering the nucleocapsid gene of SARS-CoV HKU-39449. Restriction enzymes digestion and sequence analysis indicated the recombinant plasmid of pTRE-Tight-SARS-N contained the nucleocapsid gene with the optimized nucleotide sequence which will improve the translation efficiency. Positive cell clones were selected by cotransfecting pTRE-Tight-SARS-N with the linear marker pPUR to BHK-21 Tet-on cells in the presence of puromycin. A set of double-stable eukaryotic cell lines (BHK-Tet-SARS-N) with inducible control of the SARS-CoV neucleocapsid gene expression was identified by using SDS-PAGE and Western-blot analysis. The expression of SARS-CoV nucleocapsid protein was tightly regulated by the varying concentration of doxcycline in the constructed double-stable cell line. The constructed BHK-Tet-SARS-N cell strains will facilitate the rescue of SARS-CoV in vitro and the further reverse genetic research of SARS-CoV.

Investigation on the Purification and Expression of Cell Ⅰ－Hep Ⅱ Recombinant FN Polypeptide in E．Coli

An anti-metastatic polypeptide, bifunctional-domain (Cell Ⅰ -Hep Ⅱrecombinant polypeptide of human fibronectin. was expressed in E. coli and purified. The expression level was found to be about 20% 30 % of the total cell proteins. In BL21 (DE3)/T7, an E. coli expressing system, lactose can be used as an inducer to substitute IPTG thereby reducing the cost by several hundredfold and it is suitable for the large-scale preparation of recombinant FN polypeptide. Cell Ⅰ -Hep Ⅱ fragment is an alkaline polypeptide. In BL21 (DE3)/T7 expressing system' better isolation was achieved if DEAE-52, instead of CM-52,was used for ion-exchange chromatography. The purified product was obtained after heparin-agarose affinity chromatography following ion-exchange chromatography.