从《达达》看电影的青年文化性表达

电影《达达》通过一个少女的故事向我们展示了女性成长过程中特殊的生命体验。在主人公达达的世界里,生命的孤独与归依的需要成为推动个体成长的主要动因。导演试图用一个在逻辑上并不缜密的故事强调生命存在的特定状态。在简单的呈现背后,存在的是冷静、客观且深刻的社会思考,对于女性的成长、两性关系以及青春等主题的探讨,而这一切都围绕着青年人的生活进行。电影主题阐释的意义在于能够给予观者一个相对自由的语境去感受和体验关于青春的荒诞与真诚、勇敢与退缩。在主题阐释的过程中,观众感受到一种强烈的激进、标新立异、崇尚感性体验、对现实持批判态度以及理想主义的、边缘化的青年文化性表达。

达达

前情回顾 自被小黑救后，达达对小黑的态度大有改观。他们一行人行至深山，落入食人红鬼族的陷阱中，在牢房中，达达发现关在他们隔壁的竟是李白，这时，食人族进来把李白带了出去……

达达

小凤霞的哥哥在小店遇难后，她加入到未来之泉探寻中，并拿出了李白先生的卷轴——一张美女图，大家决定去长安一探究竟。

达达地带

脖子 我的颈椎病终于达到了一个新的层次，到早上终于爬不起来啦!! 醒来后最想说的话就是“救命呀!”哪怕动一动，脖子后面的肌肉就全体抽筋，实在太痛苦啦!好怀念脖子还睦康的时候，我却是那么不善待它，悔呀!

ISAR MOTION COMPENSATION USING ROPE

Motion compensation is a key step for inverse synthetic aperture radar (ISAR) imaging. Many algorithms have been proposed. The rank one phase estimation (ROPE) algorithm is a good estimator for phase error widely used in SAR. The ROPE algorithm is used in ISAR phase compensation and the concrete implementation steps are presented. Subsequently, the performance of ROPE is analyzed. For ISAR data that fit the ROPE algorithm model, an excellent compensation effect can be obtained with high computation efficiency. Finally, ISAR real data are processed with ROPE and its imaging result is compared with that obtained by the modified Doppler centroid tracking (MDCT) method, which is a robust and good estimator in ISAR phase compensation.

Generation of Antibodies Against DMRT1 and DMRT4 of Oreochromis aurea and Analysis of Their Expression Profile in Oreochromis aurea Tissues

Sex determination is composed of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing （Doublesex/Mab-3 DNA-binding motif） gene, is highly conserved across species. Vertebrate DMRT1 （DM-related transcription factor 1） expression occurs predominantly in the testis. Here, however, isolated two distinct DM-domain cDNA from Oreochromis aurea ovary and testis have been named DMRT4 （DM-related transcription factor 4） and DMRT1 by BLAST, respectively. Despite high homology in the DM-domain there is little similarity outside the DM-domain.To better understand the structure, function, and possible roles of DMRT4 and DMRT1 as potential candidates for sex differentiation and sex determination, the intact regions encoding DMRT4 and DMRT1 obtained by PCR were sub-cloned into the vector pMAL-c2x and introduced into the Escherichia coli TB1 cell for efficient fusion expression. After purification and cleavage, DMRT4 and DMRT1 proteins were used to immunize adult rabbits following standard protocols. Consequently, it was found by using Western blot analysis that polyclonal antibodies against DMRT4 and DMRT1 had high specificity. The relative expression levels of DMRT4 and DMRT1 mRNA were determined by fluorescent Real-time RT-PCR in female and male Oreochromis aurea with 13-actin as the internal standard. DMRT1 was expressed only in testis, whereas DMRT4 was over expressed in the ovary, but in both female and male, a slight expression in the brain was also detected. Statistical analysis showed that in the brain, mean DMRT4 mRNA levels in female were significantly higher than in male. Meanwhile, the expression of DMRT4 and DMRT1 protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. It was found that DMRT4 was exclusively expressed in the ovary and DMRT1 in the testis. Study on DMRT4 and DMRT1 expression facilitated the elucidation of their roles and the understanding of sex differentiation of fish.

Characterization of ST13 Protein Expression in Human Colorectal Cancer Tissues

Objective: To characterize the expression of ST13 protein in human tissuesfor investigation of the function of colorectal cancer related gene ST13. Methods: ST13 ORF wascloned and over-expressed in E.coli. The recombinant ST13 protein was purified by affinitychromatography. ST13 monoclonal antibodies were generated and affinity purified with the recombinantprotein. Immunoblot and immunohistochemical staining were employed to analyze ST13 proteinexpression in human tissues. Results: The expression and purification of the recombinant ST13protein were confirmed by SDS-PAGE. The protein yield reached about 2.5 mg/L of induced bacterialculture with a purity of 91.3%. Three strains of hybridoma were obtained with antibody titers from10~4 to 10~5 in ascites fluids and with high specificity for ST13 protein. Immunoblot showed thatthe apparent Mr of ST13 protein in SW480 cells and human tissues estimated by SDS-PAGE mobility wasapproximately 50 000, which was about 10 000 larger than the 41 324 calculated, but theglycosylation of the protein was excluded. Computer modeling revealed the protein to be ahydrophilic molecule. Immunohistochemical staining showed that ST13 protein was evenly distributedin cytoplasm and expressed in colon, stomach, liver, and other epithelial cells. Differences in thestaining intensity of the protein were observed between normal and cancer tissues as well as amongdifferent normal or carcinoma tissues. Conclusion: ST13 protein is a cytoplasmic molecule with anapparent Mr of 50 000. The protein is expressed in colorectal and other epithelial tissues. Theexpression level of the protein is down-regulated in colorectal cancer and varies among differentnormal and/or carcinoma tissues. Comparison of cDNA sequences and protein characteristics indicatesthat ST13 protein and hsp70-interacting protein (Hip) are same proteins, raising the possibilitythat ST13 protein is involved in the development of colorectal cancer through Hsp70 molecularchaperone machinery.

Deletion of 93 bp Far-upstream Fragment of Rice Cytosolic Fructose- 1, 6-Bisphosphatase Promoter Completely Alter Its Expression Pattern

The 1 195 bp 5′ flanking region of rice ( Oryza sativa L.) cytosolic fructose_1, 6_bisphosphatase (cyFBPase) can direct tissue, cell specific expression in transgenic rice. In order to identify sequence elements responsible for the regulation of mesophyll_specific expression, the 5′ flanking regions of -1 195 bp, -1 102 bp, -768 bp, and -644 bp upstream of the translation initiation ATG codon were fused to the reporter gene encoding β_glucuronidase (GUS) and transferred to rice via particle bombardment. Analysis of the 5′ promoter deletions identified that a 93 bp fragment between -1 195 bp and -1 102 bp is essential for directing mesophyll specific expression. High constitutive expression of GUS reporter gene was found in the -768 deletion lines and another two deletion series. These results indicate the great potential utility of the promoter in rice biotechnology.

ISAR MOTION COMPENSATION USING MODIFIED DOPPLER CENTROID TRACKING METHOD

With regard to the phase compensation in inverse synthetic aperture radar (ISAR),the modified Doppler centroid tracking (MDCT) method is developed which applies the phase gradient autofocus (PGA) algorithm developed by Wahl[1]to improve the Doppler centroid tracking (DCT) method[2].When the phase compensation is performed,the proposed approach smartly eliminates the effect of the rotational phase component (RPC) on the estimation of the translational phase component (TPC) by circular shifting,windowing and iteration steps. After several iterations,the maximum likelihood estimation and compensation of the TPC of the target can be realized more effectively.The processing results of live data show that the proposed method can improve the imaging quality of ISAR significantly.

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene (pl) and Parasponia hemoglobin gene ( phl,) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pi and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pi gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pi gene was expressed in 3 out of 18 plants with 17% and 7.5% in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum by. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.

DEVELOPMENT OF MOVING TARGET DETECTION AND IMAGING BY AIRBORNE SAR

The detection and ima ging of moving targets based on airborne synthetic aperture radar (SAR) is a cru cial technique for the modern radar. Firstly, the mathematical model of SAR ech o signal which comes from moving targets is constructed. Based on this model, th e features of moving target imaging are introduced and the effects of target mov ement to SAR imaging are analyzed. Then the development and the status of this t echnique are reviewed in detail. Finally, some frontiers of this field are point ed out.

Cloning and Expression Patterns of a Metallothionein-like GenehtMT2 of Helianthus tuberosus

A novel cDNA sequencehtMT2, which encodes a type 2 metallothionein_like protein, was isolated from Helianthus tuberosus L. tuber cDNA library. The whole sequence is 509 bp, including an open reading frame (ORF) of 240 bp, a 5′ UTR of 62 bp and a 3′ UTR of 207 bp. Two genomic sequences covering the coding region ofhtMT2were cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe thatwere cloned by PCR reaction. Sequence analysis revealed that the genomic sequences htMTG_1 of 986 bp and htMTG_2 of 982 bp were both composed of three exons and two introns. The deduced protein consisted of 79 amino acid residues with a predicted molecular weight of 7.8 ku (kD). Amino_terminal and carboxy_terminal domains contained 8 and 7 cysteine residues respectively, separated by a central cysteine free spacer. Sequence alignment revealed that the predicted protein ofhtMT2 was homologous to type 2 metallothioneins (MTs) of plants. Southern blotting analysis indicated that htMT2was encoded by a small multi_gene family in H. tuberosus genome. Northern blotting analysis showed that htMT2 transcripts were detected in stems, leaves and leafstalks, but no transcripts were detected in roots. The expression level in stems was the highest among the above tissues. Transcripts in stems were significantly reduced by Cu 2+ treatment. Judging from the homologies between the deduced HtMT2 and other type 2 plant metallothioneins as well as responses to metal ions, we believe that[ShtMT2 encodes a new type 2 metallothionein.

Cloning and Expression of Pun1 Gene Controlling Pungency of Pepper (Capsicum spp.)

[Objective] This study was conducted to investigate cloning and expression of Pun1 gene controlling pungency of pepper. [Method] With Capsicum annuum L as a material, the cDNA sequence of Capunl gene was obtained, with a total length of 1 457 bp, coding 440 amino acids. [Result] Phylogenetic analysis showed that Capunl was closest to Pun1 of C. chinense, with a genetic distance of 0.019 3. Plant expression vector pCAM-Punl-GFP was constructed and transformed into to- bacco, and it was found that the protein coded by fusion gene Punl：：GFP was lo- cated on cell membrane. Prokaryotic expression vectors were constructed, and by SDS-PAGE and Western Blot detection, an induced protein with a molecular weight of 63 ku was obtained. It was found by real-time fluorescence quantitative expres- sion that Pun1 gene was expressed at the highest level 30 d after flowering, de- creased then, and could not be detected substantially 40 and 45 d after flowering. [Conclusion] This study provides information and reference for molecular regulation mechanism of Pun1 gene.

Expression of Exogenous Gene in Prokaryotes and Eukaryotes

[Objective] This study aimed to investigate the expression of exogenous gene in prokaryotes and eukaryotes. [Method] The expression vector pTYB2-WF harboring target gene was transformed into E. coli ER2566. IPTG was employed to induce the expression of phytase gene. The expression of phytase fusion protein was detected by SDS-PAGE, and the fusion protein was further purified. Phytase gene phyA was expressed in Pichia pastoris expression system. Yeast recombinant vector pPIC9K-phyA was constructed and transformed into P. pastoris GS115 to construct engineering strain GS115-pPIC9K-phyA. [Result] Phytase protein was ex-pressed under methanol induction. Enzyme activity assay indicated that the activity of phytase was 7.3 U/ml. P. pastoris engineering strain GS115-pPIC9K-phyA was successful y constructed. [Conclusion] Methanol yeast expression mechanisms play a certain role in molecular biology and industrial applications.

Identification and characterization of new plant microRNAs using EST analysis

Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to GenBank’s plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.

Entropy function optimization for radar imaging

The convergence performance of the minimum entropy auto-focusing（MEA） algorithm for inverse synthetic aperture radar（ISAR） imaging is analyzed by simulation. The results show that a local optimal solution problem exists in the MEA algorithm. The cost function of the MEA algorithm is not a downward-convex function of multidimensional phases to be compensated. Only when the initial values of the compensated phases are chosen to be near the global minimal point of the entropy function, the MEA algorithm can converge to a global optimal solution. To study the optimal solution problem of the MEA algorithm, a new scheme of entropy function optimization for radar imaging is presented. First, the initial values of the compensated phases are estimated by using the modified Doppler centroid tracking （DCT）algorithm. Since these values are obtained according to the maximum likelihood （ML） principle, the initial phases can be located near the optimal solution values. Then, a fast MEA algorithm is used for the local searching process and the global optimal solution can be obtained. The simulation results show that this scheme can realize the global optimization of the MEA algorithm and can avoid the selection and adjustment of parameters such as iteration step lengths, threshold values, etc.

A potential oncogenic role of the commonly observed E2F5 overexpression in hepatocellular carcinoma

AIM: To explore the expression pattern of E2F5 in primary hepatocellular carcinomas (HCCs) and elucidate the roles of E2F5 in hepatocarcinogenesis. METHODS: E2F5 expression was analyzed in 120 primary HCCs and 29 normal liver tissues by immunohistochemistry analysis. E2F5-small interfering RNA was transfected into HepG2, an E2F5-overexpressed HCC cell line. After E2F5 knockdown, cell growth capacity and migrating potential were examined. RESULTS: E2F5 was significantly overexpressed in primary HCCs compared with normal liver tissues (P = 0.008). The E2F5-silenced cells showed significantly reduced proliferation (P = 0.004). On the colony formation and soft agar assays, the number of colonies was significantly reduced in E2F5-silenced cells (P = 0.004 and P = 0.009, respectively). E2F5 knockdown resulted in the accumulation of G0/G1 phase cells and a reduction of S phase cells. The number of migrating/invading cells was also reduced after E2F5 knockdown (P = 0.021). CONCLUSION: To our knowledge, this is the first evidence that E2F5 is commonly overexpressed in primary HCC and that E2F5 knockdown significantly repressed the growth of HCC cells.

Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

Downregulation of wild-type p53 protein by HER-2/neu mediated PI3K pathway activation in human breast cancer cells: its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy. Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused Gl-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.

The tumor-selective over-expression of the human Hsp 70 gene is attributed to the aberrant controls at both initiation and elongation levels of transcription

The tumor selective over-expression of the human Hsp70 gene has been well documented in human tumors,linked to the poor prognosis,being refractory to chemo-and radio-therapies as well as the advanced stage of tumorous lesions in particular.However,both the nature and details of aberrations in the control of the Hsp70 expression in tumor remain enigmatic.By comparing various upstream segments of the Hsp70 gene for each''s ability to drive the luciferase reporter genes in the context of the tumor cell lines varying in their p53 status and an immortal normal liver cell line,we demonstrated in a great detail the defects in the control mechanisms at the both initiation and elongation levels of transcription being instrumental to the tumor selective profile of its expression.Our data should not only offer new insights into our understanding of the tumor specific over-expression of the human Hsp70 gene,but also paved the way for the rational utilization of the tumor selective mechanism with the Hsp70 at the central stage fortargeting the therapeutic gene expression to human tumors.