Preparation of Nanofibrous Silver/Poly(vinylidene fluoride) Composite Membrane with Enhanced Infrared Extinction and Controllable Wetting Property.

Nanofibrous silver (Ag)/poly(vinylidene fluoride) (PVDF) composite membranes were obtained from a two-step preparation method. In the first step, the electrospun silver nitrate (AgNO3)/PVDF membranes were prepared and the influence of the AgNO3 content on the electrospinning process was studied. According to scanning electron microscopy (SEM) results, when the electrospinning solution contained AgNO3 in the range between 3 to 7 wt.%, the nanofiber morphologies can be obtained. In the second step, the electrospun AgNO3/PVDF membranes were reduced by sodium borohydride to form the nanofibrous Ag/PVDF composite membranes. The resultant composite membranes were characterized by SEM, X-ray diffraction (XRD), energy-dispersive spectroscopy (EDS), differential scanning calorimetry, X-ray photoelectron spectroscopy (XPS), and Fourier-transform infrared. The XRD, XPS, and EDS characterizations proved the existence of Ag in the nanofibrous Ag/PVDF composite membranes. The crystallinity degree of PVDF for composite membranes declined with the increase in Ag content. More importantly, the nanofibrous Ag/PVDF composite membranes had obviously higher Rosseland extinction coefficients and lower thermal radiative conductivities in comparison with electrospun PVDF membrane, which demonstrates that such composite membranes with high porosity, low density, and good water vapor permeability are promising thermal insulating materials to block the heat transfer resulting from thermal radiation. In addition, three different methods for surface modification have been used to successfully improve the hydrophobicity of nanofibrous Ag/PVDF composite membranes.

Dissipation of excess excitation energy of the needle leaves in Pinus trees during cold winters.

Photooxidative damage to the needle leaves of evergreen trees results from the absorption of excess excitation energy. Efficient dissipation of this energy is essential to prevent photodamage. In this study, we determined the fluorescence transients, absorption spectra, chlorophyll contents, chlorophyll a/b ratios, and relative membrane permeabilities of needle leaves of Pinus koraiensis, Pinus tabulaeformis, and Pinus armandi in both cold winter and summer. We observed a dramatic decrease in the maximum fluorescence (F m) and substantial absorption of light energy in winter leaves of all three species. The F m decline was not correlated with a decrease in light absorption or with changes in chlorophyll content and chlorophyll a/b ratio. The results suggested that the winter leaves dissipated a large amount of excess energy as heat. Because the cold winter leaves had lost normal physiological function, the heat dissipation depended solely on changes in the photosystem II supercomplex rather than the xanthophyll cycle. These findings imply that more attention should be paid to heat dissipation via changes in the photosystem complex structure during the growing season.

A novel single WAP domain-containing protein isoform with antibacterial relevance in Litopenaeus vannamei.

Single WAP domain (SWD)-containing protein is a small protein containing a whey acidic protein (WAP) domain at the C-terminal region. SWD-containing protein exhibits structural similarity to the family of serine proteinase inhibitors. As of this writing, some SWD domain-containing proteins have been identified in crustaceans, and their functions included antibacterial and anti-proteinase activities. We identified a SWD protein isoform gene in Litopenaeus vanname (Lv-SWDi). Very high sequence similarity was found between Lv-SWDi and Lv-SWD. Results of time-course analysis for the gene expression profile showed that Lv-SWDi could produce a rapid feedback and an obvious upregulation at 12 h after Vibrio injection. Endogenous Lv-SWDi protein was obviously upregulated, and the highest expression level was reached at 24 h after Vibrio injection. The purified rLv-SWDi could directly bind to Gram-positive and Gram-negative bacteria. Results of the proteinase inhibitory assay also showed that rLv-SWDi could inhibit secretory protease activity from Bacillus subtilis. Lv-SWDi is a part of an important immunity-relevant gene and may serve important functions in defense against bacteria.

Attenuated Salmonella typhimurium carrying shRNA-expressing vectors elicit RNA interference in murine bladder tumors.

AIM: To examine whether attenuated Salmonella typhimurium (S typhimurium) could be used as an anti-cancer agent or a tumor-targeting vehicle for delivering shRNA-expressing pDNA into cancer cells in a mouse tumor model. METHODS: Mouse bladder transitional cancer cell line (BTT-T739) expressing GFP was used, in which the GFP expression level served as an indicator of RNA interference (RNAi). BTT-T739-GFP tumor-bearing mice (4-6 weeks) were treated with S typhimurium carrying plasmids encoding shRNA against gfp or scrambled shRNA. The mRNA and protein expression levels of GFP were assessed 5 d after the bacteria administration, and the antitumor effects of S typhimurium were evaluated. RESULTS: In BTT-T739-GFP tumor-bearing mice, S typhimurium (1×10(9) cfu, po) preferentially accumulated within tumors for as long as 40 d, and formed a tumor-to-normal tissue ratio that exceeded 1000/1. S typhimurium carrying plasmids encoding shRNA against gfp inhibited the expression of GFP in tumor cells by 73.4%. Orally delivered S typhimurium significantly delayed tumor growth and prolonged the survival of tumor-bearing mice. CONCLUSION: This study demonstrates that attenuated S typhimurium can be used for both delivering shRNA-expressing vectors into tumor cells and eliciting RNAi, thus exerting anti-tumor activity, which may represent a new strategy for the treatment of solid tumors.

Recombinant attenuated Salmonella harboring 4-1BB ligand gene enhances cellular immunity.

OBJECTIVE: To transfect antigen presenting cells (APCs) with 4-1BB ligand DNA by attenuated Salmonella enterica serovar Typhimurium in vivo, and to observe the effects of ectogenous 4-1BBL on the immune functions of infected rats. METHODS: Attenuated Salmonella typhimurium (vaccine strain) carrying plasmids pIRES2-EGFP-4-1BBL was constructed and used to infect HepG2 hepatoma cells. The expression of reporter gene, green fluorescent protein (GFP) and rat 4-1BBL in the transfected cells was detected by double-immunofluorescence staining. Rats were fed with the recombinant bacteria intragastrically on three occasions in 2 weeks, and were then sacrificed. The transcription and expression of GFP and 4-1BBL genes in splenocytes were measured by RT-PCR and flow cytometry. The phenotypes of T cells in peripheral blood and splenocytes were determined by flow cytometry. The content of IFN-gamma in the cultural supernatant of splenocytes stimulated by PHA was measured by ELISA. RESULTS: The recombinant bacteria harboring 4-1BBL had the same invasive abilities as the original bacteria, and it was able to deliver exogenous genes into HepG2 cells, where the GFP and 4-1BBL were successfully expressed. There were significant upregulations of CD3(+)CD8(+) T cells (P=0.018) and CD3(+)CD25(+) T cells (P=0.019) in the peripheral blood cells as well as CD3(+)CD8(+) T cells (P=0.022), and CD3(+)CD25(+) T cells (P=0.008) in splenocytes of the infected rats. The rats had more 4-1BBL expression detected in the spleen. IFN-gamma released by PHA-stimulated splenocytes increased significantly by the recombinant bacteria as compared with controls (P=0.002). CONCLUSION: Salmonella serovar Typhimurium containing 4-1BBL can transfect target genes into antigen presenting cells in vivo, and the expression of exogenous 4-1BBL enhances cellular immunity markedly.

Evaluation of the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste from biological research laboratories.

The discharge of recombinant DNA waste from biological laboratories into the eco-system may be one of the pathways resulting in horizontal gene transfer or "gene pollution". Heating at 100 degrees C for 5-10 min is a common method for treating recombinant DNA waste in biological research laboratories in China. In this study, we evaluated the effectiveness and the safety of the thermo-treatment method in the disposal of recombinant DNA waste. Quantitative PCR, plasmid transformation and electrophoresis technology were used to evaluate the decay/denaturation efficiency during the thermo-treatment process of recombinant plasmid, pET-28b. Results showed that prolonging thermo-treatment time could improve decay efficiency of the plasmid, and its decay half-life was 2.7-4.0 min during the thermo-treatment at 100 degrees C. However, after 30 min of thermo-treatment some transforming activity remained. Higher ionic strength could protect recombinant plasmid from decay during the treatment process. These results indicate that thermo-treatment at 100 degrees C cannot decay and inactivate pET-28b completely. In addition, preliminary results showed that thermo-treated recombinant plasmids were not degraded completely in a short period when they were discharged into an aquatic environment. This implies that when thermo-treated recombinant DNAs are discharged into the eco-system, they may have enough time to re-nature and transform, thus resulting in gene diffusion.

[Mechanisms and enzymatic characters of hexavalent chromium reduction by Bacillus cereus S5.4].

A strain of bacteria identified as Bacillus cereus S5.4, which was isolated from the electroplating sludge of Baosteel Corporation, Shanghai, was able to completely reduce 2 mmol/L Cr6+ in LB liquid after 72 hours. Experiments on concentrations of hexavalent and total chromium inside and outside bacterial cells after hexavalent chromium reduction, and hexavalent chromium reduction abilities of different cellular components, and SEM analysis of cellular morphology before and after hexavalent chromium reduction, showed that cellular walls and membranes, which were able to prevent hexavalent chromium out from living cells, were the main sites where hexavalent chromium reduction took place, and changes of their permeability would take effect on the function of hexavalent chromium reductase. The reductase was nonsecretive and took effect on the inner side of living cells. Determination of the activity and stability of hexavalent chromium reductase showed that the optimal temperature range and pH of the reductase was 25 - 37 degrees C and pH 7, respectively. Cu2+ had some effect on the promotion of reductase activity. At the temperature of 37 degrees C, Km and Vmax of the reductase was 125.61 micromol/L and 7.68 nmol/(min mg), respectively.

[The analysis method and progress in the study of codon bias].

Codon bias refers to the nonrandom usage of synonymous codons for encoding amino acids in organisms. As it is related to the carrier molecular of genetic information (DNA) and functional molecular (protein) of life, this phenomenon implicates important biological sense. In this review, we summarize the basic theories and analysis methods about codon bias; and present the softwares and websites which are usually used for codon usage analysis. The related fields about codon bias and the research progress are also introduced.

[Phylogenetic analysis of bacterial diversity in Pacific Arctic sediments].

Using PCR-DGGE (denaturing gradient gel electrophoresis) methods, bacterial phylogenetic diversity in three Pacific Arctic sediment samples were investigated, taken from different depths in the range of 47 m to 3850 m. DGGE profiles of different layers in the same sediment sample are not completely same. 16S rDNA sequences corresponding to 50 excised bands from three sediment samples were analyzed and fell into seven lineages of the domain Bacteria: alpha- beta-, gamma-, delta-, epsilon- Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides (CFB) group and Actinobacteria. However, the composition of bacterial phylotypes in three sediments is different. Fourteen sequences obtained from sediment B78 collected from the Canadian Basin belong to beta-, gamma- Proteobacteria, Comamonadaceae and Acidobacteria. Bacterial phylotypes in submarine plateau sediment P24 are alpha-, gamma-, delta-Proteobacteria; While seventeen sequences from sediment S11 colleted from continental slop in the Chukchi Sea are grouped into alpha-, gamma-, delta-, epsilon-Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides (CFB) group and Actinobacteria. It is suggested the different characteristics of three sediments may cause the difference in the composition of bacterial phylotypes. 16S rDNA sequences from members of gamma-Proteobacteria dominated three sediments samples. The majority of the sequences were most closely related to uncultured marine environmental sequences, especially marine sediment environmental sequences (88% - 100%).

[A pedigree with myotonic dystrophy: non-CTG, non-CCTG repeat expansion].

OBJECTIVE: Two genetic loci are associated with the myotonic dystrophy (DM) phenotype: DM1 DMPK on chromosome 19, and DM2 ZNF9 on chromosome 3. The aim of this study was to investigate the molecular genetics of a pedigree with DM. METHODS: In twenty-six individuals from a family with DM, the CTG repeats in DMPK and CCTG repeats in ZNF9were evaluated genetically, using Long Expand trade mark Template polymerase chain reaction (PCR), Southern blotting and genomic scanning. RESULTS: The numbers of CTG and CCTG repeat were all in normal range. There was no significant difference between the CTG repeat size in DMPK gene and that 4 years later from the same individual. The Lod score values with short tandem repeats STR markers chosen in 19q and 3q were all smaller than 1, which suggested that no STR marker was linked with this DM family. CONCLUSION: There might be some other mutant in this DM pedigree. Further study should be done to find the genetic basis of this pedigree.

Attenuation of estrogen receptor alpha-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor.

Estrogen receptor alpha (ERalpha) signaling is paramount for normal mammary gland development and function and the repression of breast cancer. ERalpha function in gene regulation is mediated by a number of coactivators and corepressors, most of which are known to modify chromatin structure and/or influence the assembly of the regulatory complexes at the level of transcription initiation. Here we describe a novel mechanism of attenuating the ERalpha activity. We show that cofactor of BRCA1 (COBRA1), an integral subunit of the human negative elongation factor (NELF), directly binds to ERalpha and represses ERalpha-mediated transcription. Reduction of the endogenous NELF proteins in breast cancer cells using small interfering RNA results in elevated ERalpha-mediated transcription and enhanced cell proliferation. Chromatin immunoprecipitation reveals that recruitment of COBRA1 and the other NELF subunits to endogenous ERalpha-responsive promoters is greatly stimulated upon estrogen treatment. Interestingly, COBRA1 does not affect the estrogen-dependent assembly of transcription regulatory complexes at the ERalpha-regulated promoters. Rather, it causes RNA polymerase II (RNAPII) to pause at the promoter-proximal region, which is consistent with its in vitro biochemical activity. Therefore, our in vivo work defines the first corepressor of nuclear receptors that modulates ERalpha-dependent gene expression by stalling RNAPII. We suggest that this new level of regulation may be important to control the duration and magnitude of a rapid and reversible hormonal response.

[The alpha-synuclein gene microsatellite polymorphism and late-onset sporadic Parkinson's disease susceptibility].

OBJECTIVE: To explore the association of the microsatellite polymorphisms in the promoter region of alpha-synuclein gene with the late-onset sporadic Parkinson's disease (PD) susceptibility. METHODS: The microsatellite polymorphism of alpha-synuclein gene was analyzed with amplified fragment length polymorphism (Amp-FLP) and semiautomatic fluorescent labeled genotyping technique. Association analysis was performed in 135 unrelated late-onset sporadic PD patients and 170 age-matched healthy controls. RESULTS: The distribution of the alleles of the dinucleotide repeats variants of alpha-synuclein gene promoter region in PD cases was significantly different from that in the healthy controls. The most frequent allele in PD patients was allele 269 bp, but in controls it was the 271 bp allele. Alleles of

[Study on gamma-synuclein gene in patients with idiopathic Parkinson's disease].

OBJECTIVE: To evaluate the relationship between idiopathic Parkinson's disease (PD) and two polymorphisms (C243G and A377T) of the gamma-synuclein gene in a Chinese Han population of Shanghai area. METHODS: Polymorphic genotyping was performed with PCR-RPLP technique. Association analysis was carried out in 145 unrelated idiopathic PD patients and 184 age-matched healthy controls. RESULTS: The authors failed to detect any distributional difference of the C243G and A377T polymorphisms of the gamma-synuclein gene between PD cases and control subjects, nor did they find any association. CONCLUSION: These data do not support that gamma-synuclein gene C243G and A377T polymorphisms are involved in idiopathic PD onset in the Han population of Shanghai area.

[Dopamine beta hydroxylase gene polymorphism and Parkinson's disease].

OBJECTIVE: To investigate the correlation between the polymorphism of dopamine beta hydroxylase(DBH) gene and the susceptibility of Shanghai Chinese Han population to Parkinson's disease(PD). METHODS: Association study was performed in 144 PD patients and 188 healthy control subjects matched for age, sex and origin. Polymorphism of DBH gene was analyzed with polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The allelic frequency of A2 allele of DBH gene was significantly higher in PD patients than in controls(P<0.01).The risk of suffering from PD increased (OR=1.82) in the individual with A2 allele. And the genotypic frequency of A2/A2 was significantly higher in PD patients(OR=2.11, P<0.01),too. On the other hand, the allelic frequency of A1 allele and the genotypic frequency of A1/A2 genotype of DBH gene in PD patients were significantly lower(A1 alleles: OR=0.54, P<0.01; A1/A2 genotypes: OR=0.45, P<0.01). CONCLUSION: The polymorphism in DBH gene might play an important role in the susceptibility of Shanghai Chinese Han population to PD.

[Expression variation and mutation analysis of exon 9 and 10 in amyloid precursor protein gene in Alzheimer's disease].

To explore the expression differences of exon 9 and 10 in Amyloid Precursor Protein gene(APP9 approximately 10) in Alzheimer's disease,and detect the probable point mutation appeared in cDNA fragment of APP9 approximately 10 in the Shanghai Han people.semi-quantitative competitive RT-PCR technique was performed to detect the expression of APP9 approximately 10 in peripheral lymphocyte, and the Apolipoprotein E gene(ApoE) and Presenilin 1(PS1)gene were genotyped with PCR-RFLP method. We also analyzed the point mutation in APP9 approximately 10 cDNA through the denatured gel electrophoresis. The results are as follows:1. While compared with healthy controls,expression of APP9 approximately 10 mRNA was significantly enhanced in Alzheimer disease; 2.APOE*epsilon4 allele, the most common genetic risk factor for AD, did not affect the Expression of APP9 approximately 10 mRNA, whereas the APP9 approximately 10 mRNA expression might be increased by the allele 1 of PS1 gene, another probable susceptibility gene of AD.3. No point mutation in APP9 approximately 10 cDNA was detected. In our samples, the expression of APP9 approximately 10 mRNA in AD was significantly different from that of controls, suggesting that the change of peripheral APP9 approximately 10 mRNA expression might be another bio-marker used in clinical diagnosis for AD.

[Expression and purification of telomerase resever transcriptase motifs in E.coli].

Our project is designed to clone a 1.3kb gene fragment of telomerase catalytic subunit gene which contains seven reverse transcriptase motifs and specific region with conserved sequence termed "T motif". The gene fragment was amplified by PCR and was inserted into expression vector pET28-b. The recombinant plasmid was induced by IPTG for 4h and a 52KD recombinant protein was produced. Amount of hTRT recombinant protein expression was 20% of total bacterial protein in the form of inclusion. Inclusion was dissolved in 8 mol/L urea and 10 mmol/L DTT and carried out affinity purification under denaturing condition. The purified hTRT recombinant protein was conformed by Western-blot successfully.

Treatment of Tumor in Mice by Oral Administration of Cytosine Deaminase Gene Carried in Live Attenuated Salmonella.

To study the possibility of oral gene therapy using live attenuated Salmonella, eukaryotic expression vectors EGFPN1, pLCDSN were introduced into a live attenuated AraA(-) auxotrophic mutant of Salmonella typhimurium (SL3261) and were administered orally to BALB/c and C57BL/6 mice. After six weeks, these mice were challenged with 4T(1) and Lewis cancer cells. Until the tumors reached to about 10 mm in diameter, 5-fluorocytosine was given through intraperitoneal injection. Flow cytometry, confocal microscopy and PCR methods were used to detect the integration and expression of the genes. The inhibition of the tumor and the survival time of the mice were also investigated. Results showed that cytosine deaminase gene integration could be detected in almost all kinds of mice tissue. And the GFP expression was much stronger in spleen and tumor than in other tissues. Cytosine deaminase/5-fluorocytosine system had significant antitumoractivities in vivo. The anti-tumor activities of cytosine deaminase/5-fluorocytosine at 500 mg/kg on 4T(1) and Lewis carcinoma in BALB/c and C57BL/6 mice were more potent than the efficiency of 5-fluorouracil 10 mg/kg(P 0.05). Therefor, this experiment demonstrates the potential value of live attenuated Salmonella as carrier for oral gene therapy.

SCAIF80, a Novel Inhibitor of Angiogenesis, and Its Effect on Tumor Growth.

A novel inhibitor of angiogenesis named SCAIF80 (shark cartilage-derived angiogenesis inhibitory factor) from shark cartilage has been isolated and characterized. SDS-PAGE analysis followed by silver staining revealed a single band with molecular weight (M(r)) of 80 kD. To determine whether this protein was capable of inhibiting angiogensis, it was assayed in endothelial cell (EC) proliferation and migration assay. The results showed that SCAIF80 significantly suppressed EC proliferation and migration in a dose-dependent manner. In the chick chorioallantoic membrane (CAM) assay, SCAIF80 also showed a potent inhibitory activity on angiogenesis in vivo. In animal tests, the growth of tumor was potently suppressed by SCAIF80 therapy. Lewis lung carcinoma was inhibited by 93.83 % at a dose of 5 mg/(kg.d). These findings suggest that shark cartilage may produce a novel protein with anti-angiogenic and anti-tumor activity.

Cloning and Analysis of Intron 3 of Human Telomerase Reverse Transcriptase (hTERT) Gene.

Total RNA was extracted from telomerase-positive cell line SPC-A, and reverse transcribed to cDNA. The long template PCR was performed using this cDNA as template and the hTERT cDNA specific oligonucleotides as primers. A long fragment of about 2.2 kb was produced as well as a short fragment of about 150 bp. The long fragment was purified from the gel, cloned into T-easy vector and sequenced from two directions. The sequencing result and homologous comparison indicated that the fragment contained the intron 3 of hTERT gene. Further assay showed that the precursor of this fragment is premature mRNA (pre-mRNA) of hTERT gene and the copy number varied among different cell lines, as verified in this study. These results suggest the feasibility of cloning the intron of eucaryotic gene by the combination of reverse transcription and long template PCR system. And the different splicing efficiency of the intron 3 from the hTERT pre-mRNA was also implied from this assay.

Purification and Functional Characterization of a Shark Cartilage Factor Inhibitory to Angiogenesis.

SCAIF-I, an inhibitor of angiogenesis from shark cartilage was purified to homogeneity. The 4 mol/L guanidinium chloride extract of shark cartilage was fractionally precipitated with 35%-65% acetone, then purified by Resource Q ion exchange chromatography, Sephacryl S-300 gel filtration, and reverse-phase high performance liquid chromatography. The pure inhibitor was homogeneous as a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. SCAIF-I had an molecular weight of 18 kD. It specifically inhibited proliferation of endothelial cells, and strongly blocked endothelial cell movement and angiogenesis in the chorioallantoic membrane of chick embryos. Systemic administration of SCAIF-I at the dose of 5 mg/kg.d suppressed 87.93% of the growth of Lewis lung carcinoma implanted in C57BL/6 mice.