Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b6／f Complex

An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco suspension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei, evidenced by DNA electrophoresis and fluorescence microscopy observations, Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still exhibited the apoptosis-inducing activity, but the supernatant had no effect, which indicates that the apoptosisinducing effector in the chloroplast preparation is water-insoluble. The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO, the specific inhibitor of Caspase-3, implying involvement of a Caspase-3-1ike protease during the process. Furthermore, extensive apoptosis in nuclei was induced by cytochrome b6/f on the thylakoid membrane, indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro， PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3.1His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group B

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro, PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3. I/His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Glucose-induced Ca2+ signals in rat pancreatic β cells

Using microfluorometry to assay intracellular Ca2+ , the influences of varied factors on glucose induced Ca22+ signals, such as glucose-induced initial decline phase (GIDP), Ca2+ oscillation, and Ca2+ release from internal stores, were investigated in single rat pancreatic β cells. Glucose was able to evoke GIDP even at non-stimulus concentration (5 mol/L), which is insufficient to induce Ca2+ spikes. GIDP was dependent on neither membrane depo larization nor extraeellular Ca2+ . However, GIDP was inhibited by thapsigargin, indicating a dependence on Ca2+ up take by Ca22+ stores. The glucose-induced calcium oscillation was inhibited when external Ca2+ was removed. However, thapsigargin could not block the Ca2+ oscillation. These results suggest that maintenance of Ca22+ oscillation requires ex tracellular Ca2+ but not Ca2+ stores. Glucose was able to evoke Ca2+ signals even in the absence of external Ca2+ . The glucose-induced Ca2+ release from intracellular Ca2+ stores was blocked by TTX. However, TTX had no effect on high K--induced Ca2+ store release, suggesting that membrane depolarization can directly release Ca2+ from some internal Ca2+ stores in β cells.     

N-Terminal Truncation of TACO Inhibits PMA-Induced U937 Cell Adhesion

The effect of TA001-299, the N-terminal truncation of TACO, on phorbol 12-myristate 13-acetate （PMA）-induced U937 cell adhesion was investigated. Full-length TACO and several truncations were overexpressed in U937 cells. The effects of the expressed proteins on U937 cell adhesion mediated by PMA-induced differentiation were observed by fluorescence microscopy. The results show that the overexpression of TACO1-299 inhibits cell adhesion while overexpressions of the other proteins do not have this effect. The actin-binding capability of TACO1-299 was investigated and the results show that TACO1-299 lacks the ability of TACO to bind F-actin. The inhibitive effect of TACO1-299, the functional domain of TACO, suggests that TACO may play a role in cell differentiation mediating adhesion of monoblastic leukemia cells.     

Inactivation of Giardia by Ozone and Its Disinfect Mechanisms

This study was to investigate the effect of ozone(O3)inactivation on Giardia in water by the fluorescence staining method.In order to elucidate the dominant mechanisms of inactivation,cell surface or inner cell components damage were comparatively examined by scanning electron microscopy(SEM).Results suggested that O3had a stronger effect on inactivating capability.Firstly,when the concentration of O3was above 2.0 mg/L and the contact time was up to 5 min,it showed a significant inactivating effect.Secondly,the effect of turbidity on inactivation was also found to be significant in synthetic water;when turbidity increased,the inactivating effect decreased.Thirdly,the inactivating rates were improved with a temperature increase from 5 to 25℃,but decreased when the temperature were out of this range.The inactivating capability of O3was stronger under acidic conditions than alkalic conditions.Lastly,when the concentration of organic matter in the reactive system was increased,probably due to the competition between Giardia and organics on O3,the inactivating rate was decreased;in addition,the cellular morphology of Giardia varied with different contact times.At contact time of 30 s,cells were rotundity and sphericity;at 60 s they became folded,underwent emboly,and burst;and at 240 s,the cell membrane of Giardia shrinked and collapsed completely.     

Apoptosis Induced by High Concentrations of Nicotinamide in Tobacco Suspension Cells

As an inhibitor of poly(ADP-ribose) polymerase (PARP), nicotinamide has a restraining effect on apoptosis at certain low concentrations. In our present study, apoptosis induced by high concentrations of nicotinamide was observed in tobacco suspension cells. When cells were preincubated with 250 mmol/L nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180-200 bp，the condensation and peripheral distribution of nuclear chromatin, and a positive reaction to the TUNEL assay. At the same time, the degradation of PARP and the reduction in the potential of the inner membrane of mitochondria appeared in apoptotic cells induced by high concentrations of nicotinamide. This result indicates that apoptosis induced by high concentrations of nicotinamide is associated with caspase-3-1ike activity and with the opening of mitochondrial permeability pores. These results partially support the hypothesis that high concentrations of PARP inhibitor could force cells to enter an apoptotic pathway by delay of DNA repair in replicating cells.     

Increase in electron transfer activity in photosystem Ⅱ of spinach thylakoids caused by conversion of phosphatidyl- glycerol to phosphatidic acid molecules

The techniques of oxygen electrode polarography, sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) were employed to investigate the effect of phospholipase D treatment on physiological function of spinach thylakoids. It was shown that the phospholipase D treatment on thylakoid resuited in the degradation of phosphatidylglycerol (PG) and occurrence of phosphatidic acid (PA). The changes of PG to PA molecules caused an increase in oxygen evolution in photosystem Ⅱ (PS Ⅱ), which was accompanied by an uncoupling effect on thylakoid membrane. It was revealed that the head-groups of PG molecules play an important role in themaintenance of the appropriate physiological activity of thylakoid membrane.     

Establishment of the model of vascular endothelial cell membrane chromatography and its preliminary application

A model of vascular endothelial cell membrane chromatography was established by using an ECV304 cell membrane stationary phase (ECV304 CMSP) prepared by immobilizing the ECV304 cell membrane onto the surface of silica carrier. The surface and chromatographic characteristics of ECV304 CMSP were studied. The active component from Caulophyllum robustum was screened by using the model of vascular endothelial cell membrane chromatography. The interaction between the active component and membrane receptor was determined by using a replace experiments. The effect of the active component was tested by using tube formation of ECV304 cell. The results indicated that the model of ECV304 cell membrane chromatograph (ECV304 CMC) can stimulate the interaction between drug and receptor in vitro and the retention characteristics of taspine as active component was similar to that of model molecule in the model of ECV304 CMC. And therefore, taspine acted on VEGFR2 and inhibited the tube formation of ECV304 cell induced by VEGF. This model can be used to screen definite active component as a screening model.     

Protection of phosphatidyl-choline to photosystem II membrane during heat treatment

Photosystem II membrane was reconstituted with phosphatidylcholine (PC) with different kinds of fatty acyl chains and the protection of PC to photosystem II (PS II) membrane during heat treatment was investigated using oxygen electrode, variable fluorescence and circular dichro-ism (CD) spectroscopy. Heat treatment decreased the oxygen evolution rate and the F'v/Fm' ratio of PS II membrane and influenced CD spectra of PS II membrane, but PC inhibited the effect of heat treatment on the oxygen evolution rate, the F'v/F'm ratio and CD spectra of PS II membrane. The results indicate that PC can protect PS II membrane against heat treatment and the alterations in the unsaturated fatty acid extent in PC can cause the changes of the protection ability.     

Amyloid precursor protein-mediated modulation of capacitive calcium entry

Alzheimer’s disease(AD) is a progressive neurodegenerative disorder.In the present study,the function of amyloid precursor protein(APP) in modulating capacitive calcium entry(CCE),a refilling mechanism for depleted intracellular calcium stores,was investigated.CCE in neural 2a(N2a) cells stably expressing wild-type human APP was lower than in wild-type N2a cells,while CCE in APP knockout mouse embryonic fibroblast(MEF) cells was higher than in their wild-type counterparts.We demonstrate that wild-type APP depresses CCE.Furthermore,using N2a cells transfected with C-terminal APP fragments,we show that these fragments anchored in the cell membrane play an important role in CCE depression.     

Effects of free sterols on the fluidity of polar lipids in rice dry embryo

Free sterols from rice dry embryo reduce the onset phase transition temperature and widen the transition range of total polar lipids and phosphatidylglycerol, which increases the membrane fluidity below their phase transition temperature and decreases the fluidity of membranes above their phase transition temperature. And 50% of free sterols result in a complete disappearance of phase transition. The fluidity of other main components of polar lipids is decreased by free sterols above 0℃. The results confirm that free sterols can modulate membrane lipid fluidity.     

Three-dimensional recon- struction and analysis of structure characteristics on senile plaques of Alzheimer’s disease

Alzheimer‘s disease is a progressive neurodegenerative disorder characterized by the presence of senile plaques primarily composed of amyloid β in brain. Abnor-mal secretion and aggregation of amyloid β are the key events in pathogenesis of Alzheimer‘s disease. Reduction of amyloid β production and inhibition of amyloid β aggregation to form senile plaques are hopeful strategies for the treatment and prevention of Alzheimer‘s disease. In the present study, the silver and immunohistochemical staining methods were applied to discover senile plaques in the hippocampus of Alzheimer‘s disease patients, and then images were processed and three-dimensionally reconstructed by Matlab and AVS software. The structure characteristics of senile plaques were measured through correlation function calculation and fractal dimension by a computer-aided method. Diffuse plaque had no amyloid center, but classic plaque presented compact central core structure; two types of plaques were both of porous structure, but the sizes of their pores were significantly different. Furthermore, there was difference in fractal dimension value between the diffuse plaque and classic plaque in the two staining methods. The comparison of structure characteristics between two types of plaques indicated that they developed independently. Establishment of the methods for reconstructing the three-dimensional structure of senile plaque and analyzing their structure characteristics is helpful for further study on the aggregation mechanism of senile plaque.     

Protection of phosphatidylcholine to photosystem Ⅱ membrane during heat treatment

Photosystem Ⅱ membrane was reconstituted with phosphatidylcholine (PC) with different kinds of fatty acyl chains and the protection of PC to photosystem Ⅱ (PS Ⅱ)membrane during heat treatment was investigated using oxygen electrode, variable fluorescence and circular dichroism (CD) spectroscopy. Heat treatment decreased the oxygen evolution rate and the F′v/Fm′ ratio of PS Ⅱ membrane and influenced CD spectra of PS Ⅱ membrane, but PC inhibited the effect of heat treatment on the oxygen evolution rate, the F′v/F′m ratio and CD spectra of PS Ⅱ membrane. The results indicate that PC can protect PS Ⅱ membrane against heat treatment and the alterations in the unsaturated fatty acid extent in PC can cause the changes of the protection ability.     

Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K_(in)~ channels in Arabidopsis guard cells

We explore nitric oxide (NO) effect on K in channels in Arabidopsis guard cells. We observed NO inhib- ited K in currents when Ca2 chelator EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) was not added in the pipette solution; K in currents were not sensitive to NO when cytosolic Ca2 was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO ele- vates cytosolic Ca2 by activating plasma membrane Ca2 channels firstly, then inactivates K in chan- nels, resulting in stomatal opening suppressed subsequently.     

Prokaryotic expression, localization and function of the infectious laryngotracheitis virus glycoprotein G

The infectious laryngotracheitis virus （ILTV） glycoprotein G （gG） gene of E3 and Zhonghai strains was cloned, sequenced and compared with the gG gene of other Type Ⅰ animal herpesviruses. To find the localization and the function of the gG in the infected cells, the 35 kD fusion protein （His-GG） was expressed by inserting the coding region of gG except for the signal peptide into pET30a （＋）. After purification of the His-GG fusion protein, the rats＇ antibody to the His-GG was prepared and purified by using the protein G Sepbarose. Results of laser scanning confocal microscopy （LSCM） detection showed that the ILTV gG was in the perinuclear region and membrane of chicken embryo liver （CEL） and kidney （CEK） cells, and that the gG accumulated more in the coalescent part than in the other parts of the adjacent CEL or CEK cells. The plaque size and the one-step growth curve tests suggested that the ILTV gG was required for viral growth by cell-to-cell direct infection in tissue-cultured CEL cells.     

Preparation and cytocompatibility of polylactic acid/hydroxyapatite/graphene oxide nanocomposite fibrous membrane

A series of polylactic acid (PLA) based nanocomposite fibrous membranes,including neat PLA,PLA/hydroxyapatite (HA) and PLA/HA/graphene oxide (GO),were fabricated via electrospinning method.The morphology and composition were investigated by scanning electron microscopy (SEM),X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) respectively.The thermal stability was determined by thermogravimetric analysis (TGA).To estimate the cytocompatibility of asprepared PLA/HA/GO fibrous membrane,MC3T3-E1 cells were cultured,and the corresponding cell adhesion and differentiation capability were investigated by fluorescence microscopy,SEM and MTT test.The electrospun ternary PLA/HA/GO membrane exhibited three-dimensional fibrous structure with relatively rough surface morphology,which made itself ideal for cell attachment and proliferation in bone tissue regeneration.The fluorescence microscopy,SEM and MTT test confirmed that the PLA/HA/GO nanocomposite fibrous membrane created a proper environment for the seeding and proliferation of MC3T3-E1 cells.     

Involvement of NADPH oxidase NtrbohD in the rapid production of H2O2 induced by ABA in cultured tobacco cell line BY-2

The mechanisms for the production of hydrogen peroxide （H2O2） induced by abscisic acid （ABA） were investigated in suspension culture cells of tobacco BY-2 cells. The results showed that the immediate generation of H2O2, which was mainly derived from super-oxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA resulted in a time-dependent quick increase in plasma membrane （PM） NADPH oxidase activity, which coin- cided on time and magnitude with the elevation in ABA-induced accumulation of H2O2. Moreover, these enhanced effects were pro- nouncedly inhibited by two NADPH oxidase inhibitors, diphenylene iodonium and imidazole, suggesting that PM NADPH oxidase is involved in the rapid accumulation of H2O2 in cultured tobacco cells. In addition, analysis of the expression level of NtrbohD, a PM NADPH oxidase gene in tobacco, by RT-PCR and protein gel blot revealed that the gene at both mRNA and protein levels was upregulated by ABA, indicating that NtrbohD participates in the ABA-stimulated rapid production of H2O2 in tobacco culture cells. Taken together, these findings suggest that ABA induces the rapid accumulation of reactive oxygen species via NADPH oxidase in sus-pension culture cells of tobacco, and that NADPH oxidase and H2O2 appear to be important components in ABA signal transduction pathway in plants.     

Preparation of A-type zeolite membranes on nonporous metal supports by using electrophoretic technique

A-type zeolite membranes were prepared on the nonporous metal supports by using electrophoretic technique. The as-synthesized membranes were characterized by XRD and SEM. The effect of the applied potential on the formation of the A-type zeolite membrane was investigated,and the formation mechanism of zeolite membrane in the electric field was discussed. The results showed that the negative charged zeolite particles could migrate to the anode metal surface homogenously and rapidly under the action of the applied electric field, consequently formed uniform and dense membranes in short time. The applied potential bad great effect on the membrane formation, and more uniform and denser zeolite membranes were prepared on the nonporous metal supports with 1 V potential.     

Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate

Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon （MB） specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.