Influence of Shengmaiyin Combined with Xuefu Zhuyu Tang on Hypercoagulable State Caused by Non-small Cell Lung Cancer / 中国实验方剂学杂志

Objective:To observe influence of dialectical addition and subtraction of Shengmaiyin combined with Xuefu Zhuyu Tang on fibrinolytic activity and coagulation active factor of patients with non-small cell lung cancer at hypercoagulable state. Method:One hundred and eighty patients were randomly divided into control group (58 cases) and observation group (60 cases) by random number table. Patients in control group got low molecular weight heparins calcium injection by subcutaneous injection for 3 weeks, 1.0 mL (5 000 AXa unit)/time, 1 time/day, and oral aspirin enteric-coated tablets, 100 mg/time, 1 time/day. Patients in observation group got dialectical addition and subtraction of Shengmaiyin combined with Xuefu Zhuyu Tang, 1 dose/day. A course of treatment was 8 weeks. And activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), platelet (PLT), fibrinogen (FIB), D-dimer (D-D), plasma tissue plasminogen activator (t-PA), plasminogen activator inhibitor in plasma-1 (PAI-1), von willebrand factor (vWF), P-selectin, basic fibroblast growth factor (bFGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF) were detected. And before and after treatment, scores of Qi deficiency and blood stasis syndrome and hemorheological indices were detected. Result:After treatment, APTT, PT and TT in observation group were longer than those in control group. Levels of PLT, D-D and PAI-1 were lower than those in control group (PPPPPPPConclusion:Dialectical addition and subtraction of Shengmaiyin combined with Xuefu Zhuyu Tang can ameliorate hypercoagulable state of NSCLC, relieve clinical symptoms, and can regulate fibrinolytic activity and coagulation activity factors, so it can mitigate dangers caused by deep venous thrombosis of NSCLC.

MACF1 knockdown in glioblastoma multiforme cells increases temozolomide-induced cytotoxicity / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ).</p><p><b>METHODS</b>TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts.</p><p><b>RESULTS</b>TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement.</p><p><b>CONCLUSION</b>MACF1 may be a potential therapeutic target for glioblastoma.</p>

RNA interference of PC4 and SFRS1 interacting protein 1 inhibits invasion and migration of U87 glioma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of small interfering RNA (siRNA)-mediated silencing of PC4 and SFRS1 interacting protein 1 (PSIP1) on invasion and migration of human glioma U87 cells.</p><p><b>METHODS</b>Chemically synthesized siRNA targeting PSIP1 gene was transfected into U87 cells via lipofectamine, and the gene silencing effect was determined using real-time PCR. The changes in the invasion and migration abilities of the transfected cells were assessed with Transwell assay and wound healing assay, respectively. Western blotting was used to analyze the expression of N-cadherin, β-catenin and the transcription factor Slug.</p><p><b>RESULTS</b>The mRNA and protein level of PSIP1 was significantly reduced in U87 cells after transfection with PSIP1 siRNA (P<0.0001). PSIP1 knockdown in U87 cells resulted in significant suppression of cell invasion and migration abilities (P<0.01) and also reduced N-cadherin, β-catenin and Slug expressions.</p><p><b>CONCLUSION</b>s Silencing of PSIP1 impairs the invasion and migration abilities of glioma cells and lowers the expressions of N-cadherin, β-catenin and Slug, suggesting that PSIP1 may regulate Slug by classical Wnt/β-catenin signaling pathway to modulate epithelial-mesenchymal transition and promote the invasion and migration of glioma cells.</p>

Epidemiological features on 3 important viral diarrhea among children in Zhuhai during winter and spring / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the epidemiological characteristics of viral diarrhea of norovirus (NV), sapovirus (SV) and astrovirus (AstV) among children in Zhuhai during winter and spring.</p><p><b>METHODS</b>Stool specimens were collected from children with viral diarrhea in Maternal and Child Health Hospital of Zhuhai from November 21, 2009 to April 3, 2010. Nucleic acid of NV, SV and AstV from negative specimens of rotavirus and adenovirus were detected by using Reverse transcription-polymerase chain reaction (RT-PCR), and the types of positive samples of NV were also classified at the same time.</p><p><b>RESULTS</b>The total detection rate of the three viruses is 21.49 percent, the highest detection rate is 29.05% in December 2009, the lowest detection rate is 12.20% in February 2010, 87.96% of positive specimens were from children patients aged from 0 to 30 months. The season detection rate of NV, SV and AstV are 14.70%, 2.75% and 4.04% respectively. There were significant differences of NV and SV detection rates in every month of the season, whereas the AstV detection rate was comparatively stable. The highest detection rate of NV is 34.09% in children patients aged from 12 to 18 months, the highest SV detection rate is 12.5% in children patients aged from 60 to 120 months, and the highest AstV detection rate is 16.67% in children patients aged from 24 to 30 months. All the NV were belong to G II genogroup.</p><p><b>CONCLUSIONS</b>NV is one of the main pathogens causing viral diarrhea among children in Zhuhai during winter and spring, SV and AstV are also important pathogens. So we should strengthen the monitoring of viral diarrhea caused by NV, SV and AstV in infants and young children.</p>

Dingxin recipe ( ) prevents ischemia/reperfusion-induced arrhythmias via up-regulating prohibitin and suppressing inflammatory responses / 中国结合医学杂志

<p><b>OBJECTIVE</b>To identify the underlying mechanisms of the protective effects of Dingxin Recipe (: , DXR), a Chinese compound prescription that has been used clinically in China for more than 20 years, on ischemia/reperfusion (I/R)-induced arrhythmias in rat model.</p><p><b>METHODS</b>A total of 30 rats were randomly divided into three groups: sham group, I/R group, and DXR-pretreated I/R (DXR-I/R) group. Rats in the DXR-DXRI/R group were intragastrically administrated with DXR (12.5 g/kg per day) for consecutive 7 days, while rats I/in the sham and I/R groups were administrated with normal saline. Arrhythmias were introduced by I/R and electrocardiograms (ECG) were recorded. Two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used to identify assisted differentially expressed proteins. Immunohistochemistry, real-time quantitative polymerase chain reaction (RQ-RQPCR), Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to analyze proteins PCR), obtained in the above experiments.</p><p><b>RESULTS</b>DXR significantly reduced the incidence and mean duration of ventricular tachycardia and ventricular fibrillation and dramatically decreased the mortality, as well as arrhythmia score, compared with those of the I/R group. Among successfully identified proteins, prohibitin (PHB) and heart fatty acid binding protein (hFABP) were up-regulated in DXR-pretreated I/R rats compared with those of the I/R rats. In addition, compared with the I/R group, the level of glutathione (GSH) was elevated accompanied by reduced expressions of interleukin-6 (IL-6) and neutrophil infiltration in I/R rats with DXR pretreatment.</p><p><b>CONCLUSIONS</b>DXR could alleviate I/R-induced arrhythmias, which might be related to increased expression of PHB. The enhanced expression of PHB prevented against the depletion of GSH and consequently inhibited apoptosis of cardiomyocytes. Furthermore, up-regulation of PHB might ameliorate I/R-induced cell death and leakage of hFABP by suppressing neutrophil infiltration and IL-6 expressions.</p>

Prognostic value of ultra-sensitive pregnancy associated plasma protein-A in patients with acute coronary syndrome / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the prognostic value of ultra-sensitive pregnancy associated plasma protein-A (PAPP-A) level in the early phase of acute coronary syndrome (ACS) attack.</p><p><b>METHODS</b>Patients diagnosed as ACS were enrolled and the level of circulatory PAPP-A was measured within 12 hours after ACS attack. The patients were followed at the time of 1st, 6th, and 12th months post-ACS attack in order to observe the incidence of the cardiovascular adverse events. According to the highest quintile, the patients were divided into 2 groups: high level (≥26.08 μg/L) group and low level (<26.08 μg/L) group, to evaluate the association between the level of PAPP-A and the incidence of the cardiovascular events.</p><p><b>RESULTS</b>Compared with the low level group, the incidence of the composite outcome is significantly increased in the high level group, and the values of OR are 4.76, 4.38, 3.75 for 1st, 6th, 12th months respectively (P=0.000). For myocardial infarction (MI) + cardiac death (CD) the values of OR were 9.81, 6.08, 4.12 (P<0.01). Multivariate logistic regression analysis demonstrates that PAPP-A was an independent risk factor for the cardiovascular adverse events in the early, median, and late phase of ACS (P<0.05).</p><p><b>CONCLUSION</b>In the early phase of ACS attack, the elevation of PAPP-A is an independent risk factor for the occurrence of cardiovascular adverse events.</p>

Effects of sophoridine on the growth and expressions of p53 and vascular endothelial growth factor of transplanted solid tumor SW480 in nude mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of sophoridine on the transplanted solid tumor SW480 and the expression of p53 and vascular endothelial growth factor (VEGF) in the tumor in nude mice.</p><p><b>METHODS</b>The nude mouse model bearing transplanted solid tumor SW480 was established, and the changes in the volume and weight of the tumor were determined after treatment of the mice with sophoridine. Western blotting and immunohistochemistry were employed to examine the expressions of p53 and VEGF proteins, respectively, and fluorescence quantitative PCR used to detect their mRNA expressions in the tumor tissue.</p><p><b>RESULTS</b>The volume and weight of the tumor xenograft in sophoridine group decreased in comparison with those in the control group. Sophoridine treatment resulted in lowered expressions of p53 and VEGF at both the protein and mRNA levels in the tumor explants as compared with the control group, with a tumor inhibition rate of 34.07% in nude mice.</p><p><b>CONCLUSION</b>Sophoridine can inhibit the growth of transplanted solid tumor of human SW480 cell line, the mechanism of which involves the inhibition of p53 and VEGF expression.</p>

Effect of Sanhuangyinchi decoction on liver damage and caspase-3 in rats with acute hepatic failure / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate effect of Sanhuangyinchi decoction (SHYCD) on liver damage and the pro-apoptotic factor caspase-3 in rats with acute hepatic failure.</p><p><b>METHODS</b>SD rats were randomly divided into blank control group, model group, Angongniuhuang group (AGNH) and SHYCD group. Acute hepatic failure was induced in the rats by intraperitoneal injections of D-GaLN and LPS, and the death rate, ALT, TBIL, PT and caspase-3 activity was observed or tested.</p><p><b>RESULTS</b>At 36 h after the injections, the death rate of the rats was 74.29% (26/35) in the model group, 31.43% (11/35) in AGNH group and 28.57%(10/35) in SHYCD group. The death rate, ALT, TBIL, PT and caspase-3 activity in AGNH and SHYCD groups were significantly lower than those in the model group (P<0.01). SHYCD showed stronger effect than AGNH in depressing TBIL and the activity of caspase-3 (P<0.05).</p><p><b>CONCLUSION</b>SHYCD can improve the liver function and inhibit the activity of caspase-3 in rats, which can be the possible mechanism for its therapeutic effect against acute hepatic failure.</p>

High mobility group box-1 stimulates proinflammatory cytokine production in endothelial cells via MAP kinases / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.</p><p><b>METHODS</b>HUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.</p><p><b>RESULTS</b>The levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng<ml) or HMGB1 (15 ng/ml) alone resulted in significantly increased levels of IL-8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.</p><p><b>CONCLUSION</b>HMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.</p>

Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fusion with Hela cell membrane / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.</p><p><b>METHODS</b>S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.</p><p><b>RESULTS</b>The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.</p><p><b>CONCLUSIONS</b>The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.</p>

Construction and functional identification of red fluorescent protein reporter plasmid for human catalase gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a red fluorescent protein reporter gene driven by human catalase gene promoter.</p><p><b>METHODS</b>The red fluorescent protein reporter gene plasmid pDsRed-CATp containing human catalase gene promoter was constructed by gene recombination technique. The plasmid was transiently transfected into NIH/3T3 cells to observe their response to H(2)O(2) stimulation.</p><p><b>RESULTS</b>The plasmid was constructed correctly as verified by double enzyme digestion and sequence analysis. The plasmid was lowly expressed in resting NIH/3T3 cells, but the expression level increased obviously after stimulation by H(2)O(2). CONCLSIONS: A red fluorescent protein reporter gene plasmid driven by human catalase gene promoter has been constructed successfully with a sensitive response to H(2)O(2) stimulation. This system provides a convenient tool for the study of the regulatory mechanism of catalase gene expression.</p>

BRCA1 regulates progesterone receptors A and B protein expressions in breast cancer cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells.</p><p><b>METHODS</b>Breast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting.</p><p><b>RESULTS</b>The protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased.</p><p><b>CONCLUSION</b>In breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.</p>

Expression and localization of endogenous C-reactive protein in THP-1 monocytes and LO2 hepatocytes / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the expression and localization of endogenous C-reactive protein (CRP) in cells from different tissues under different conditions.</p><p><b>METHODS</b>Macrophages differentiated from THP-1 monocytes with phorbol ester (PMA) induction and human LO2 hepatocytes were stimulated with lipopolysaccharide (LPS). The culture supernatant of the LPS-stimulated THP-1 cells was collected and added into LO2 cell culture, and after incubation, the cells were lysed to extract the proteins for SDS-PAGE and Western blotting. The stimulated cells were also examined immunocytochemically for CRP expression.</p><p><b>RESULTS</b>Western blotting detected CRP in both of the unstimulated cell lysates, but in neither of the two cell supernatants. After LPS stimulation, CRP expression was significantly increased in the cell lysate of THP-1 cells with also a small amount present in the supernatant, but CRP expression and release in the LO2 cells showed no significant variation. Treatment of the LO2 cells with the culture supernatant of LPS-stimulated THP-1 cells resulted in positivity of CRP in the cell lysate and the culture supernatant. Immunocytochemistry identified CRP expression throughout the THP-1 cell body (most obvious in the nuclei), which increased after LPS stimulation. In LO2 hepatocytes, CRP expression was found only outside the nuclei and increased after stimulation with the culture supernatant of LPS-treated THP-1 cells, especially obvious around the membrane.</p><p><b>CONCLUSION</b>CRP can not be up-regulated directly by LPS treatment in LO2 cells, but can be induced by certain cytokines (IL-6) secreted from LPS-stimulated THP-1 cells. The localization of CRP represents the characteristics of secreted protein in LO2 cells, but in THP-1 cells, CRP is found mainly in the cell nuclei.</p>

Establishment and optimization of two-dimensional gel electrophoresis for separation of human kidney phosphoproteome / 第二军医大学学报

Objective: To separate human kidney phosphoproteome by two-dimensional gel electrophoresis. Methods: The phosphorylated proteins from human kidney tissues were enriched with phosphate metal affinity chromatography (PMAC) resin. After being concentrated and desalted, the samples were separated by isoelectric focusing on first dimension and SDS electrophoresis on second dimension. Results: The phosphorylated proteins were successfull y extracted from human kidney tissues and were separated by two-dimensional gel electrophoresis. Conclusion: Phosphoprotein enrichment technique combin ed with two-dimensional gel electrophoresis is an effective approach to study phosphoproteome, laying a foundation for further investigation of human kidney phosphoproteins.

Construction and identification of a vector expressing short hairpin RNA targeting annexin II gene / 第二军医大学学报

Objective: To construct a vector encoding short hairpin RNA(shRNA) targeting annexin H and to observe its influence on annexin II expression. Methods: Annexin II-targeted hairpin RNA1 and RNA2 were devised according to the Gen-Bank annexin II sequence. Meanwhile, a nonspecific sequence was taken as negative control. The oligonucleotide strands of DNA fragments encoding the above shRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into human pGenesil1 plasmid followed by amplification in E. coli and sequencing. The 3 pGenesil1 constructs were then transfected into HEK293 cells and the expression of annexin II was examined by real-time fluorescence quantitative RT-PCR and Western bolt. The non-transfected cells were taken as blank control. Results: Restrictive enzyme digestion and sequencing verified that the 3 recombinants (pGenesil1-annexin II shRNA1, pGenesil1-annexin II shRNA2, and pGenesil1-negative shRNA) were correct. The annexin II expression was significantly lower in HEK293 cells transfected with pGenesil1-annexin II shRNA2 compared with those transfected with pGenesil1-annexin II shRNA1, pGenesil1-negative shRNA, and blank control (P<0.05); and there was no significant difference between the latter 3. Conclusion: We have successfully generated an annexin II-targeted shRNA construct, which can significantly inhibit the expression of annexin II.

Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology.</p><p><b>METHODS</b>The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method.</p><p><b>RESULTS</b>The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2.</p><p><b>CONCLUSION</b>This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.</p>

Application of two-color infrared fluorescence for detection of protein phosphorylation / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the value of two-color infrared fluorescence imaging system in detecting protein phosphorylation in comparison with chemiluminescent detection.</p><p><b>METHOD</b>The lung tissue homogenate of mice treated with lipopolysaccharide (LPS) for different time lengths were prepared to separate the proteins by SDS-polyacrylamide gel electrophoresis followed by transfer of the proteins onto PVDF membranes. The membranes were incubated with the antibodies against total p42/44 MAPK/phospho-p42/44 MAPK, and then with goat anti-mouse or anti-rabbit secondary antibodies conjugated to Alexa Fluor 680, IRDye 800 or horseradish peroxidase. The blotted proteins were detected and quantified using Odyssey infrared imaging system or chemiluminescent detection.</p><p><b>RESULTS</b>LPS treatment rapidly induced p42/44 MAPK phosphorylation, which reached the peak level 1 h after the treatment and resumed the baseline level at 12 h. Consistent results were obtained by the two detection methods, but two-color infrared fluorescence imaging system showed better sensitivity in detecting the target protein, and was easy to manipulate with good efficiency and capable of analyzing two proteins simultaneously.</p><p><b>CONCLUSION</b>Two-color infrared fluorescence system is a powerful system for detecting phosphorylation of proteins.</p>

Construction and functional study of a cell penetrating peptide-based expression vector for targeted delivery of proteins into the cell nuclei / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct an cell penetrating peptide-based expression vector capable of targeted delivery of proteins into the cell nuclei and study its function of protein transduction.</p><p><b>METHODS</b>The fusion protein expression vector pET14b-HC(L)NE (pET14-b-His-CPP-Linker-NLS-EGFP) incorporating cell penetrating peptide (CPP), nuclear localization signal(NLS), linker and enhanced green fluorescent protein (EGFP) was constructed based on His-tagged pET14b-HE (pET14b-His-EGFP) by site-directed mutagenesis PCR method. After identification by enzyme digestion and DNA sequencing, the recombinant plasmid was transformed into BL21(DE(3)) strain. The HC(L)NE fusion protein was expressed following IPTG induction and purified with Ni(2+)-NTA affinity chromatography. After dialysis and filtration, the HC(L)NE fusion protein was added into cultured eukaryotic cells. The protein transduction in the living cells was observed under fluorescence microscope and analyzed by Western blotting.</p><p><b>RESULTS</b>Enzyme digestion and DNA sequencing confirmed successful construction of the pET14b-HC(L)NE vector, and the fusion protein efficiently expressed in E. coli. Protein transduction experiments in eukaryotic cells revealed that the fusion protein could rapidly penetrate the cell membrane and reach the cell nucleus, and this internalization was time- and concentration-dependent.</p><p><b>CONCLUSION</b>The cell penetrating peptide-based expression vector for targeted protein delivery to the cell nucleus has been successfully constructed, and a transport system that can delivery exogenous proteins or polypeptides into the cytoplasm and cell nucleus is established, which provides an economical and efficient means for functional study of the proteins and polypeptide in cells and targeted drug delivery.</p>