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Objective: To construct chimerical DNA vaccine plasmid of human papillomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity., Methods: Molecular cloning techniques were used to construct recombinant plasmid pcDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection.IL-2 and gamma-INF secreted by immunized spleens lymphocyte and HPV 11 L1 or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay., Results: The chimerical DNA plasmid of pcDNA3 L1-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and gamma-INF were detected in vaccinated mice., Conclusion: Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

Objective: To investigate the cross-talk between nuclear factor kappaB (NF-kappaB) and P53 signal pathways in human epidermal keratinocytes (NHEKs) after ultraviolet B (UVB) irradiation., Methods: Normal NHEKs harboring wild p53 gene and immortal human keratinocytes of the line HaCaT harboring mutant p53 gene were cultured at 37 degrees C in an atmosphere containing 5% CO(2) and then underwent irradiation of UVB of the dose of 60 mJ/cm(2). Part of the NHEKs and HaCaT cells were pretreated with BAY11-7082, NF-kappaB inhibitor inhibiting IkB-a phosphorylation, of the final concentration of 5 micromol/L. Western blotting was used to detect the protein expression of NF-kappaB, P53, and P21. The transcriptional activity of NF-kappaB was analyzed by electrophoretic mobility shift assay (EMSA)., Results: UVB irradiation increased the protein expression of NF-kappaB, P53, and P21 and triggered the transcriptional activity of NF-kappaB. BAY11-7082 significantly inhibited the NF-kappaB protein expression induced by UVB irradiation in the NHEKs and HaCaT cells. The P53 protein expression of the NHEKs undergoing pretreatment of BAY11-7082 and UVB irradiation was 0.08 +/- 0.07, significantly lower than that of the NHEKs undergoing only UVB irradiation (0.78 +/- 0.32, P < 0.01). The P21 protein expression of the NHEKs undergoing pretreatment of BAY11-7082 and UVB irradiation was 0.65 +/- 0.22, significantly lower than that of the NHEKs undergoing only UVB irradiation (1.58 +/- 0.77, P < 0.05). However, the P53 and the P21 protein expression of the HaCaT cells undergoing pretreatment of BAY11-7082 and UVB irradiation was not significantly different from that of the HaCaT cells undergoing only UVB irradiation., Conclusion: There exists a UVB-induced cross-talk between NF-kappaB and P53 signal pathways in human epidermal keratinocytes. This cross-talk is correlated with P53 functional condition.

Objective: To explore the effects of antisense epidermal growth factor receptor (EGF-R) oligodeoxynucleotides on ultraviolet-induced c-jun activity of keratinocytes after EGF-R oligodeoxynucleotides transfect to HaCaT in vitro., Methods: c-jun DNA binding activity after ultraviolet-B (UVB) irradiation and EGF-R oligodeoxynucleotides transfection were determined with a highly sensitive and specific colorimetric method. After EGF-R oligodeoxynucleotides transfection, the mRNA level of EGF-R was detected by reverse transcription polymerase chain reaction method., Results: Compared with control groups, c-jun activity increased significantly in UVB (10, 20, 30 mJ/cm2) irradiation groups (P < 0.05). EGF-R mRNA and c-jun activities induced by UVB were inhibited after the keratinocytes were transfected with EGF-R antisense oligodeoxynucleotides at 2, 4 and 8 microg/ml concentrations (P < 0.01)., Conclusion: The ultraviolet-induced c-jun activity of keratinocytes can be mediated by EGF-R and inhibited by EGF-R antisense oligodeoxynucleotides, which is transfected to keratinocytes and mediated by lipofectamine.

Background: Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1alpha on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins) mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts., Methods: Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1alpha. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA)., Results: Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1alpha increased MAP kinase activity and c-Jun mRNA expression, IL-1alpha also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1alpha increased MMP-1 production in UVA-irradiated fibroblasts., Conclusions: UVB-irradiated keratinocytes and IL-1alpha indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

Background: It is known that ultraviolet irradiation can affect cellular function through a number of signaling pathways. (-)-epigallocatechin-3-gallate (EGCG) is the major effective component in green tea and can offer protection from ultraviolet-induced damage. In this study, we investigated the protective mechanism of EGCG on human dermal fibroblasts damaged by ultraviolet A (UVA) in vitro., Methods: Transcription factor Jun protein levels were measured by Western blot. Matrix metalloproteinase 1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA were studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in conjunction with computer-assisted image analysis. MMP-1 and TIMP-1 proteins were quantified by enzyme-linked immunosorbent assay (ELISA)., Results: EGCG decreased transcription activity of Jun protein after induction by UVA. Both the mRNA and protein levels of MMP-1 were increased by UVA irradiation, while no significant changes were observed in TIMP-1 levels. The ratio of MMP-1 to TIMP-1 showed statistically significant differences compared with the control. EGCG decreased the ratio of MMP-1 to TIMP-1 by inhibiting UVA-induced MMP-1 expression (P < 0.05)., Conclusion: EGCG can protect human fibroblasts against UVA damage by downregulating the transcription activity of Jun protein and the expression of MMP-1. The ratio of MMP-1 to TIMP-1, rather than the levels of MMP-1 or TIMP-1 alone, may play a significant role in human skin photodamage.

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.