[Application of NIR spectroscopy to estimate of MFA and fiber length of Neosinocalamus a f finis].

Near infrared spectroscopy was applied to rapidly predict microfibril angle (MFA) and fiber length of Neosinocalamus a f finis Keng by using a fiber-optic probe in diffuse reflectance mode. The MFA and fiber length were measured by X-ray diffractometry and optical microscope, respectively. Partial least squares (PLS) was used to build models based on raw and pretreated spectra, including noise spectra and noise combined with orthogonal signal correction (OSC) spectra. The results showed that the PLS models of MFA and fiber length, based on noise combined with OSC spectra, gave the strongest correlations, with correlation coefficient (R) of 0.8936 and 0.9883 and root mean standard error of prediction (RMSEP) of 0.2920 and 0.1460 in prediction set. The correlations between NIR predicted and MFA/fiber length actual values are very good. Therefore, it is concluded that MFA and fiber length of N. a f finis can be estimated by NIR spectroscopy with sufficient accuracy.

Promoter cloning and characterization of the rabbit BK channel beta1 subunit gene.

The beta1 subunit of the voltage-dependent and Ca(2+)-activated large-conductance K(+) channel (BK) in mammalian smooth muscle cells (SMCs) plays an important role in regulating smooth muscle tone and is closely linked with a series of smooth muscle tone associated diseases. However, knowledge of the transcriptional regulation of the BK beta1 is still largely unclear. For the first time, we cloned and characterized the full-length genomic sequence of the rabbit BK beta1 containing a 5'-flanking region of 2021 bp. The full-reading frame of the BK beta1 spans ~7.7 kb and is organized into 4 exons and 3 introns. All of the exon/intron junction sequences contain the GT/AG consensus junction sequence. The transcription initiation site (+1G) is located at 447 bp upstream of the translation initiation codon. Bioinformatics analysis indicated that, without any canonical TATA-box, the 5'-flanking region possesses a high GC content and contains a number of putative transcription factor binding sites. 5'-deletion analysis demonstrated that the region of -93/+30 potentially functions as a core promoter region. A gel mobility shift assay and chromatin immunoprecipitation assay revealed that Sp1 specifically interacts with a putative Sp1-binding site (-91/-85) in vitro and in vivo. Mutation of this site significantly diminished the promoter activities. Over-expression of Sp1 in smooth muscle cells of rabbit sphincter of Oddi enhanced the promoter activities of the BK beta1 in a dose-dependent manner. Thus, we suggest that the Sp1-binding site (-91/-85) is essential to the basal transcription of the rabbit BK beta1. Our studies provide a basic knowledge of the transcription regulation of the rabbit BK beta1.

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

OBJECTIVE: To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). METHODS: HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. RESULTS: Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. CONCLUSION: The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

[Preparation of anti-Red antisera and its subcellular localization].

AIM: To prepare rabbit anti-Red antisera. METHODS: The bet, exo and gam genes of lambda phage were amplified by PCR from genomic DNA and cloned into the expression vector pDH2, respectively. Red proteins were induced to express at 42 degrees C. The expressed proteins were analyzed by PAGE and thin-layer scanning. The antisera were prepared by immunizing rabbits with the three Red proteins, respectively. The titers and specificities of the antisera were detected by Western blot. RESULTS: Beta, Exo and Gam proteins accounted for about 40.3%, 49.2% and 73.4% of total bacterial protein, respectively. The titers of the antisera were about 1:2,000. Western blot analysis indicated that the three antisera all had good specificities to the corresponding proteins. CONCLUSION: Specific anti-Red antisera are prepared successfully.

[Effect of specific siRNA targeting against bcr-abl chimeric gene on chronic myelogenous leukemia cells].

OBJECTIVE: To investigate the effect of specific small interfering RNA (siRNA) targeting against bcr-abl chimeric gene on the biological traits of chronic myelogenous leukemia (CML) cells. METHODS: CML cells of the line K561 transcribing a type of b3: a2 mRNA of bcr-abl chimeric gene were cultured. A 21nt siRNA targeting against the chimeric location of the b3: a2 mRNA of bcr-abl chimeric gene was designed, synthesized, and transfected into the K562 cells as RNA interference group. Another K562 cells were transfected with fluorescein enzyme gene specific siRNA as indifferent controls, or with lipid alone as blank vector controls. Some K562 cells without treatment were used as normal controls. 48 hours after the transfection Western blotting was used to detect the expression of P210bcr-abl fusion protein. 3H-TdR incorporation was used to detect the proliferation activity of K562. Annexin V-fluorescencein isothiocyanate (FITC)/phosphatidylinositol (PI) staining was used to detect the apoptosis of K2562 cells. Flow cytometry was used to observe the cell cycle of K562 cells. Benzidine staining was used to detect the differentiation of K562 cells towards erythrocytic series. Western blotting was used further to detect the expression of apoptosis-related protein Bcl-xL/Bax. RESULTS: (1) In contrast with the control groups, the expression level of bcr-abl chimeric gene was much lower in the RNAi group. (2) (3)H-TdR incorporation test showed time-dependent inhibition of proliferation of K562 cells, reflected in decrease of counts per minute (CPM) value in RNAi group 24 h, 48 h, 72 h, and 96 h after siRNA transfection by 33.06%, 52.25%, 57.64%, and 70.87% respectively (F=17.7, P < 0.01). (3) About 43.2% of K562 cells in the RNAi group were apoptotic 48 h after siRNA transfection (F=13.6, P < 0.01). (4) In contrast with the control groups, the expression of apoptosis-associated protein Bcl-xL was greatly down-regulated; however, the expression of Bax protein showed little change. (5) The percentage of benzidine-positive cells in the RNAi group was 23.5% +/- 3.2%, significantly higher than those in the indifferent control group, blank vector group, and normal control group (2.4% +/- 0.3%, 4.5% +/- 0.5%, and 3.6% +/- 0.2% respectively, all P < 0.01), which meant that part of the K562 cells differentiated towards erythrocytic series. (6) The percentage of G1 phase of K561 cells in the RNA1 group was significantly higher than those of the other groups (F = 6.2, P < 0.05), showing a capture in G1-phase of cell cycle. CONCLUSION: The specific siRNA distinctly inhibits the expression of bcr-abl chimeric gene and influences essential biological traits of K562 cells, which will ultimately result in differentiation or apoptosis of K562 cells.

[Cloning, expression and antitumor effect of mouse costimulatory molecule 4-1BBL].

AIM: To clone mouse 4-1BBL gene, construct its eukaryotic expression vector, and evaluate antitumor activity of the expression product. METHODS: RT-PCR was used to amplify mouse 4-1BBL gene from total RNA of C57BL/6 splenocytes stimulated by PHA. Then m4-1BBL cDNA was subcloned into eukaryotic expression vector pcDNA3.1(+) and transfected into mouse hepatocellular carcinoma cell line Hepa1-6. The expression of m4-1BBL in transfected cells was detected by RT-PCR, indirect immunofluorescence staining, and flow cytometry. Non-adherent splenocytes from non-immunized C57BL/6 mice were incubated with mitomycin-treated non-transfected Hepa1-6(Hepa1-6-wt) or transfected Hepa1-6 cells (Hepal-6-m4-1BBL), respectively. Then the lymphocytes were tested for cytotoxic activity to Hepa1-6-wt cells. RESULTS: The Hepa1-6 cells transfected by pcDNA3.1(+)-m4-1BBL could efficiently express m4-1BBL. As compared with Hepa1-6-wt cells,Hepa1-6-m4-1BBL cells could induce more efficiently cytotoxic activity of lymphocytes to Hepa1-6-wt cells (P<0.01). CONCLUSION: The expression of m4-1BBL by tumor cells is effective in inducing antitumor immune response.

The severe acute respiratory syndrome (SARS) coronavirus NTPase/helicase belongs to a distinct class of 5' to 3' viral helicases.

The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds double-stranded DNA, dependent on the presence of a 5' single-stranded overhang, indicating a 5'o 3' polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT24 binding over 10 times more strongly than dA24 as observed by the apparent Km.

[High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding].

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.