To examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods. A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutantsD1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-N1, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both D1/D2-Fas and D3/D4 Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class II + Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.
Antigen-Presenting Cells ; immunology ; metabolism ; CD4 Antigens ; chemistry ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line ; Dimerization ; Fas Ligand Protein ; metabolism ; Histocompatibility Antigens Class II ; genetics ; immunology ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Protein Binding ; genetics ; Protein Multimerization

In order to investigate the mechanism of stat3 nuclear import. positioned a characterized NLS of the SV40 large T antigen into Stat3-GFP, Dstat3-GFP respectively between the C-terminus of Stat3 and the N-terminus of GFP to create Stat3-NLS-GFP and Dstat3-NLS-GFP. With NLS-GFP as the positive control, Expression of the Stat3-NLS-GFP without IL-6 stimulation and Stat3-GFP with IL-6 stimulation resulted in a predominantly nuclear localization in 293T cell. Expression of Stat3-GFP and Dstat3-NLS-GFP without IL-6 stimulation resulted in predominantly cytoplasm localization in 293T cell. The results suggest that latent Stat3 is not anchored in the cytoplasm, and that nuclear localization in response to IL-6 is facilitated by gain of an NLS function.
Active Transport, Cell Nucleus ; Cell Nucleus ; metabolism ; Nuclear Localization Signals ; Plasmids ; metabolism ; STAT3 Transcription Factor ; metabolism

Objective To study the inhibitory effect of pterin acid (PTA) against ricin and recombinant ricin A chain protein. Methods Luciferase protein synthesis inhibition assay in a cell-free system and in vitro cytotoxicity experiments were performed to assess the biological activity of ricin and rRTA treated with PTA.Results The result showed that PTA could significantly inhibit the activity of ricin and rRTA in a dose-dependent manner.Conclusion PTA might be used as a small molecular probe to develop an evaluating system for ricin/RTA small molecular inhibitor in vitro. The cell-free system adopted in the current study could also serve as a necessary basis for screening some novel small molecular compounds against ricin and RTA in the future.

To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.
Animals ; Drug Evaluation, Preclinical ; Enhancer Elements, Genetic ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Immunosuppressive Agents ; pharmacology ; Interleukin-2 ; genetics ; Jurkat Cells ; Luciferases ; genetics ; metabolism ; Mice ; Models, Biological ; NFATC Transcription Factors ; genetics ; metabolism ; Promoter Regions, Genetic ; Signal Transduction ; Tacrolimus ; pharmacology

Recent researches indicate that ubiquitin-protea some pathway plays an important role in apoptosis regulation. Proteasome inhibitors induce apoptosis in many kinds of neoplastic cells, thus provide a great opportunity for exploring synergy of proteasome inhibitors and other apoptosis-inducing agents. In this study, the effect of the proteasome inhibitor Z-LLL-CHO combined with etoposide (VP16) on leukemic cell lines M-07e and TF-1 was investigated by MTT assay, trypan blue exclusion, flow cytometry and Western blot. The results showed that the combination of Z-LLL-CHO and VP16 was much more effective than either agents alone in promoting cytotoxicity in both cell lines evaluated. Accumulation of cells in S + G2/M phase of the cell cycle was observed in the cells treated with VP16 and Z-LLL-CHO alone, while apparent increase of sub-G0/G1 fraction was detected in cells treated with combination of the agents. The cleavage of Bcl-2 into a shortened 22 kD fragment was detected in M-07e cells exposed to either agents alone, and the fraction of 22 kD fragment was increased in the cells treated with combination of the agents. In conclusion, the combination of Z-LLL-CHO and VP16 enhanced their individual cytotoxic effect by inducing apoptosis, in which increase of S + G2/M fraction in cell cycle as well as the enhanced cleavage of Bcl-2 are the possible mechanism of the additive effect on leukemic cells by Z-LLL-CHO and VP16.
Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cysteine Endopeptidases ; Etoposide ; pharmacology ; Humans ; Leukemia ; pathology ; Multienzyme Complexes ; antagonists & inhibitors ; Oligopeptides ; pharmacology ; Proteasome Endopeptidase Complex ; Proto-Oncogene Proteins c-bcl-2 ; analysis

OBJECTIVETo observe antisepsis, anti-swelling, and therapeutic effects of Fuxiye (FXY), a Chinese medical lotion for external wash in treating vaginitis model rats.

METHODSThe cervicitis rat model was induced by agar plate diffusion, ear auricle swelling induced by dimethylbenzene, and chemical stimulus. The in vitro antibiotic actions of FXY were observed. Besides, its effects on the swelling and inflammation in model rats were also observed.

RESULTSFXY at 25 mg/mL could completely inhibit the growth of Pseudomonas aeruginosa, Escherichia coli, pyogenic Streptococcus, and Streptococcus agalactiae. FXY at 50 mg/mL could completely inhibit the growth of Staphylococcus aureus and Candida albicans. It obviously restrained dimethylbenzene induced ear auricle swelling. It significantly alleviated cervicitis induced by chemical stiumli.

CONCLUSIONFXY showed better effects on antisepsis, anti-inflammation, and treating cervicitis.

Animals ; Anti-Infective Agents ; administration & dosage ; pharmacology ; Anti-Inflammatory Agents ; administration & dosage ; pharmacology ; Dosage Forms ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Rats ; Rats, Sprague-Dawley ; Uterine Cervicitis ; drug therapy ; Vaginitis ; drug therapy

B-Cell Activating Factor ; antagonists & inhibitors ; metabolism ; B-Lymphocytes ; drug effects ; immunology ; metabolism ; Humans ; Lymphoma, B-Cell ; drug therapy ; immunology ; metabolism ; Models, Biological ; Multiple Myeloma ; drug therapy ; immunology ; metabolism ; Protein Binding ; Receptors, Tumor Necrosis Factor ; antagonists & inhibitors ; metabolism

Objective:To obtain a high specificity and high affinity anti-PcrV protein monoclonal antibody which can be used for the treatment of Pseudomonas aeruginosa infected.Methods: The PcrV gene was amplified by PCR using P.aeruginosa PAO1 genome DNA as the template.The expression vector(pET-28a-PcrV) was constructed and transformed into E.coli BL21(DE3).The re-combinant PcrV protein was expressed by IPTG induction and purified by Ni2+affinity chromatography.The specific binders of PcrV were screened by phage display.The genes encoding VH and VL were amplified respectively by PCR using the plasmid of positive clone as the template.Then the recombinant expression vectors were constructed and transfected into 293E cells.Monoclonal antibody were purified by the Protein A affinity resin from the culture supernatants.The affinity of antibody was detected by ELISA and the function of YG5 was verified in murine pneumonia model caused by P.aeruginosa.Results: Recombinant PcrV protein was expressed and purified.A full human monoclonal antibody(named as YG5) against PcrV was obtained by phage display.The results of ELISA showed that YG5 had a high affinity with EC50=61 ng/ml.Furthermore,it was found that YG5 could protect mice from infection caused by P.aeruginosa.Conclusion:Our findings present a novel human monoclonal antibody YG5 against PcrV,which inhibits the infection casued by P.aeruginosa and may be a potential drug for treatment of P.aeruginosa infection.

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. Recently, proteasome inhibitors have been shown to induce apoptosis in many kinds of human malignant cells. In this study, the mechanism of apoptosis induced by proteasome inhibitor in leukemic cells was examined. Evaluated by MTT assay, treatment of leukemic cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death in a dose-dependent manner. Appearance of the sub G(0)/G(1) fraction of cell cycle observed in flow cytometry assay suggested the induction of apoptosis, which was further proved by typical DNA ladder and morphological study. Western blot displayed the cleavage of bcl-2 into a shortened 22 kD fragment and the decrease in the levels of caspase-3 precursor. A highly sensitive colorimetric assay was employed and the elevation of caspase-3 activity was detected in both cell lines after treatment with Z-LLL-CHO. By comparison, these results showed that the leukemic cell line M-07e and KG-1a, which both express bcl-2 at a relative high level, had different susceptibility to undergo apoptosis induced by Z-LLL-CHO, which possibly due to their different levels of expression and activation of caspase-3 precursor, as well as their different degree of bcl-2 cleavage after treated by Z-LLL-CHO.

After treating with chemotherapy or immunosuppressant, malignant diseases of hematopoietic system such as leukemia, malignant lymphoma and aplastic anemia usually induced severe infection such as sepsis. Sepsis which is hard to be diagnosed causes high death rate. This study was purposed to establish an experimental sepsis mouse model so as to provide a basis for pathogenesis and intervention study. A classic caecal ligation and puncture (CLP) was used to establish experimental sepsis model. ELISA was used to detect levels of C5a, IL-6, TNFalpha, and IFN-gamma. Flow Cytometry was applied to measure apoptosis of lymphocytes in thymus and mesentery. The pathologic changes of thymus and spleen were confirmed by HE staining. The results showed that almost 70%-80% mice died at 72 hours after CLP. Only approximate 20% animal survived during finite time, mice in CLP group had significant weight lose. Meanwhile large release of different inflammatory mediators which are related with sepsis (C5a, IL-6, TNF-alpha, and IFN-gamma) was observed after CLP. Apoptosis of lymphocytes in thymus and mesentery lymphonodus was enhanced markedly after CLP. Significantly pathologic injury was also observed in thymus and spleen. It is concluded that a mouse model of experimental sepsis was successfully established by caecal ligation and puncture which can well mimic the clinical symptom of sepsis. The experimental sepsis mouse model provides an excellent tool for exploring the pathogenesis and intervention ways for sepsis accompanied with complicated malignant hematological diseases in vivo.
Animals ; Apoptosis ; Cecum ; injuries ; Complement C5a ; metabolism ; Disease Models, Animal ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sepsis ; metabolism ; pathology ; Spleen ; pathology ; Thymus Gland ; pathology ; Tumor Necrosis Factor-alpha ; metabolism