Abstract:Human papillomaviruses are small DNA viruses and over 118 types have been identified. They often infect host through mucosal surface or skin. Some of them (types 6 and 11) cause benign tumors such as condylomas whereas others (16, 18 etc) cause malignant tumors such as cervical cancers. Currently, there are two kinds of HPV preventive vaccine have been approved to market abroad. This article reviews progress in the field of human papillomaviruses (HPVs) biology, mechanism of carcinogenesis by persistent infection of high risk HPVs, and the development of prophylactic and therapeutic viral vaccines.

Abstract:Green Fluorescent Protein (GFP) and its mutants, as reporter genes, are widely applied in the regulation of gene expression, spatial location of protein, interaction between biological molecules, transgenic animal, evaluation of drug effect, and study of drug action mechanism, infinitely promoting the development of modern biology. With the constant progress of optoelectronic information techniques, Optical molecular imaging techniques, based on the fluorescent report genes, will lead to the real time visualization of molecular and cellular events in the different levels such as cell, cell network, tissue, organ, and body, and thus play an important role in the early diagnosis of dread disease as well as drug discovery and development.

Abstract:Red fluorescent proteins (RFPs) produced from a number of Anthozoa species have been subjected to a series of in vitro molecular evolution, resulting in various emission spectra ranging from 570 nm to 655 nm and thus providing powerful tools for cellular imaging or even body imaging. This article briefly reviewed the optical properties, structures and mutagenesis of RFPs and their applications.

Abstract:Epigenetic changes are important etiological factors of human tumor. The integrity of the genome is frequently challenged by the damage of DNA. However, the highly condensed structure of chromatin imposes significant obstacles on the repair processes. Eukaryotes have developed intricate mechanisms to overcome this repressive barrier imposed by chromatin. Covalent histone modifications and ATP-dependent chromatin remodeling play important roles in the process of DNA repair. Recent advances of the epigenetic regulations in the repair process were summarized. New findings in the cellular responses to DNA double strand breaks and how histone modifications and chromatin remodeling contributes to DNA double strand break repair were introduced. Future challenges in this field are also discussed.

Abstract:MicroRNAs (miRNAs) are another interest of small, non-coding RNAs, which regulate gene expression at post-transcriptional level in a sequence-specific manner. Recent researches demonstrate that miRNAs play important roles in innate immune response at various phases in vertebrates. In order to eliminate pathogens such as virus, miRNAs are crucial molecules in signaling of innate immune, and also in directly interfering in virus replication, therefore, miRNA may work as one important aspect of classical innate immune response against pathogenic microorganism. Meanwhile, pathogenic microorganism, especially viruses, can encode miRNA or regulate the miRNAs expression in host cells to disturb the expression of many immune associated genes directly and/or indirectly, so that they can escape from immune attacking. So, pathogenic microorganism and their hosts might fight with each other at miRNA level immediately after infection in the earliest phase.

Abstract:Influenza A virus(IAV) infection is a major cause of respiratory disease in human and many kinds of animals, however, either seasonal“flu” outbreaks or periodic world-wide pandemics caused by IAV is mainly attributed to strategies exploited by IAV for evading antiviral immune responses of the host. There is growing evidence that IAV has evolved highly sophisticated strategies to overcome the antiviral signaling, such as antigen variation and encoding accessory proteins(NS1 and PB1-F2). A better understanding of the strategies exploited by viruses for evading the immunity is helpful for us to reveal the pathogenesis of IAV infection and discover targets for the development of antiviral reagents directed against IAV. Therefore，the latest progress on strategies exploited by IAV for evading antiviral immunity are reviewed.

Abstract:Metabolic characteristics of 39 human brain tumor tissues, including 15 astrocytomas, 13 fibroblastic meningiomas and 11 transitional meningiomas from 39 individual patients, have been studied using high resolution magic-angle spinning (HRMAS) ¹H NMR spectroscopy in conjunction with principal component analysis (PCA). With rich metabolite information, ¹H NMR spectra showed that the tumor-tissue metabonome was dominated by lipids, lactate, myo-inositol, creatine, choline metabolites such as choline, phosphocholine and glycerophosphocholine, amino acids such as alanine, glutamate, glutamine, taurine, N-acetyl-aspartate and glutathione. PCA of the tumor NMR spectra clearly showed metabonomic differences between low-grade astrocytomas and meningiomas whereas such differences were more moderate between fibroblastic and transitional meningiomas. Compared with meningiomas, the low-grade astrocytomas had higher levels of glycerophosphocholine, phosphocholine, myo-inositol and creatine but lower levels of alanine, glutamate, glutamine, glutathione and taurine. The N-acetyl-aspartate level was low but detectable in low-grade astrocytomas whereas it was not detectable in meningiomas. It is concluded that tissue metabonomics technology consisting of HRMAS ¹H NMR spectroscopy and multivariate data analysis (MVDA) offers a useful tool (1) for distinguishing different types of brain tumors, (2) for providing the metabolic information for human brain tumors, which are potentially useful for understanding biochemistry of tumor progression.

Abstract:The novel gene NGX6 is solated by tumor laboratory of Central South University on studying nasopharyngeal carcinoma. NGX6 protein includes two transmemberane regions. There are an EGF-like domain signature and three potential N-glycosylation sites in the extracellular domain of it. The short cytoplasm contains a tyrosine residue that is a potential phsophorylation site by tyrosine kinase. Previous study on NGX6 showed: NGX6 was found down-regulated in colorectal carcinomas, and expression of NGX6 was down-regulated in the tumors with metastasis and related to the clinic stages by using the in suit hybridization and tissue array techniques. Transfection of NGX6 into colorectal carcinomas cells can induce the reversion of some malignant phenotypes (data obtained from cell cycle, cell growth rate curve, soft agar colony formation, nude mice injection analysis, et al), the changes of gene/protein expression profiles and the down-regulation of expression of phosphor-EGFR. Based on these studies, in an attempt to identify the function of NGX6-induced-apoptosis, the following experiments were designed. The NGX6-transfected HT-29 cell line was used as the test, empty-vector- transfected HT-29 cell line and untransfected HT-29 cell line were used as the control. The effect of NGX6 on apoptosis was detected by FCM cells were double- stained by PI/Annexin-V; the express of NF-κB was detected by EMSA. There is no difference of apoptosis between NGX6 transfected colon carcinoma cell and NGX6 untransfected colon carcinoma cell when cells are cultivated in vitro. But apoptosis level of NGX6 transfected colon carcinoma cell of xenograft tumor in nude mice is higher than that of NGX6 untransfected colon carcinoma cell. And the express of NF-κB is inhibited in NGX6 transfected colon carcinoma cell groups. These experiments showed NGX6 gene can induce apoptosis of colon cancer and inhibit The express of NF-κB.

Abstract:Tyroserleutide (YSL) is a new anticancer polypeptide developed recently. It can induce hepatocellular carcinoma cells apoptosis and necrosis. However, the subcellular targets of YSL in the hepatocellular carcinoma cells are not known very well. Therefore, a fluorescent bioconjugate of 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (5(6)-TAMRASE)-YSL) was synthesized and purified by native polyacrylamide gel electrophoresis and capillary electrophoresis. After stability was assessed by capillary electrophoresis and fluorescent spectrophotometric assay, concentration of this conjugate was quantitated by fluorescent spectrophotometric assay. Then subcellular distribution of 5(6)-TAMRASE labeled YSL were investigated by laser scanning confocal microscopy to gain a better understanding of the anticancer mechanism of YSL in vitro. The experimental results showed that the 5(6)-TAMRASE can conjugate with YSL stably. When incubated with human hepatocellular carcinoma BEL-7402 cells, 5(6)-TAMRASE labeled YSL could entered the cells in 15 min, and mainly concentrated in the cytoplasm of BEL-7402 cells. Then the fluorescent intensity of 5(6)-TAMRASE labeled YSL which indicated the concentration of them reach the maximum until incubated with the cells for 2 h. After that it began to go down. The 5(6)-TAMRASE could also enter the hepatocellular carcinoma BEL-7402 cells, but it distributed in the whole cell and the intensities had no decrease within 3 h. The results suggest that the distribution pattern of fluorescent labeling YSL is different from that of 5(6)-TAMRASE. The subcellular distribution pattern of 5(6)-TAMRASE labeled YSL was ascribed to YSL and could represent that of YSL. In conclusion, YSL located at the cytoplasm of hepatocellular carcinoma BEL-7402 cells and distributed intensively.

Abstract:In an effort to find drugs for facilitating proliferation of transplanted stem cells to provide adequate new tissue, the influence of PCA from A. oxyphylla on the proliferation capacity of hADSCs in vitro was examined. Human ADSCs could differentiate into neuron-like cells in vitro, and protect PC-12 cells from apoptosis induced by serum deprivation. Cell counts showed that treatment of hADSCs with 0.5 mmol/L, 1.0 mmol/L and 1.5 mmol/L of PCA for 48 h increased the cell numbers in a dose-dependent manner. In addition, the cell numbers of hADSCs at various time points after treatment of 1.5 mmol/L PCA were increased in a time-dependent manner. Flow cytometric analysis of DNA content demonstrated the cell cycle progress from G1 phase to S phase. The most pronounced effect was seen with 1.5 mmol/L PCA, where the fraction of cells in S phase increased more than 2 folds, accompanied by a significant increase in the fraction of cells in G2/M phase and a significant decrease in the fraction of cells in G0/G1 phase. Western blot analysis revealed the elevated expression of cyclin D1 in hADSCs induced by 1.5 mmol/L PCA treatment. Furthermore, cyclin D1-siRNA transfection significantly inhibited the promotion of cell proliferation by PCA. Flow cytometric analysis of the cell surface antigens, osteogenic induction and adipogenic induction demonstrated that after PCA treatment, hADSCs retained their morphological and functional characteristics of multipotential mesenchymal progenitors. The proliferative enhancement of PCA suggests the possibility that PCA may be useful in hADSCs-mediated therapy.

Abstract:To investigate the relationship between the changes of Lewis(y) antigenic content and the drug resistance of the cells to carboplatin in ovarian cancer lines with RMG-Ⅰ-H which expresseed higher Lewis(y) antigen on cell surface stably. RMG-Ⅰ was one kind of epithelial ovarian cancer cell lines. Cell lines RMG-Ⅰ-C and RMG-Ⅰ-H were obtained from RMG-Ⅰ which were transfected with keno-plasmid and α1, 2-fucosyltransferase gene, respectively. The method of methyl thiazolyl tetrazolium (MTT) was used to determine the fractional inhibition ratio (IR) of the three cell lines which were affected by carboplatin in different concentrations. Accordingly, the IC₅₀ of the three cell lines could be calculated. Carboplatin induced apoptosis of three cell lines was observed by flow cytometry (FCM). In addition, the apoptotic ratios of the three lines that were treated by 30 mg/L and 60 mg/L carboplatin were measured by the FCM at the same time. And then, the apoptosis conditions of the three lines were observed by the methods of fluorescent staining and transmission electron microscope (TEM). The IC₅₀ of RMG-Ⅰ-H was (58.07±2.42), which was obviously higher than that of RMG-Ⅰ(28.83±3.57) and RMG-Ⅰ-C(25.71±8.24), all showed a significant statistical difference (P < 0.01) and there was no statistical difference between the latter two ones(P > 0.05). FCM analysis confirmed the cell apoptosis in the three lines. The corr-apoptotic ratios of RMG-Ⅰ-H treated with 30 mg/L and 60 mg/L carboplatin were (20.43±0.71)% and (38.11±0.33)%, respectively, which were about 49%～63% of the RMG-Ⅰ(35.87±3.84)%, (63.37±9.59)% and RMG-Ⅰ-C(34.80±3.59)%, (60.17±6.64)%, both presented a statistical difference (P < 0.05). But there was no significant statistical difference between the latter two ones (P > 0.05). The apoptotic degree of RMG-Ⅰ-H was lower than that of the other cell lines in the same carboplatin concentration with the method of inv-fluorescence microscopy and transmission electron microscopy (TEM). The drug resistance of ovarian cancer cells to carboplatin was stronger when the Lewis(y) antigenic contents on cell surface increased.

Abstract:Somatic cell nuclear transfer (SCNT) has great value in medicine, stock breeding and saving endangered animals, but the low efficiency of SCNT restricts its application. Imprinted genes regulate fetal growth and many are essential for normal development in mammals. GTL2 is imprinted in human and mouse, which act as noncoding RNAs regulating the translation of target mRNA. In order to identify the expression of GTL2 in cattle produced by natural reproduction and by SCNT, a single nucleotide polymorphisms (SNPs) in GTL2 through PCR-SSCP was identified and the GTL2 expression patterns in six organs of hybrids was analyzed by RT-PCR-SSCP. The results demonstrated that GTL2 was monoallelic expression in all six examined organs of cattle produced by natural reproduction, and showed monoallelic expression in heart and liver but biallelic expression in brain, spleen, lung and kidney of cattle produced by SCNT that died shortly after birth. The abnormal expression of GTL2 may contribute to the organ development defects and the low efficiency of SCNT.

Abstract:Based on the conservation of nucleotides at splice sites, the characteristics of base composition and base correlation in the adjacent segment sequences, the distance between alternative donor or acceptor splice sites and the content of GC and TC near splice sites, the donor and acceptor splice sites for alternative and constitutive introns are predicted by use of the method of diversity measure combined with quadratic discriminant analysis. For alternative splice sites the total prediction accuracies are 87.9% and 89.9% for donors and acceptors respectively (with the chosen threshold -2). For constitutive splice sites the total accuracy are 92.8% and 94.3% for donors and acceptors respectively (with the chosen threshold -1).

Abstract:The molecular mechanism underlying muscular atrophy and gravisensing during spaceflight is still unknown. The major effects of spaceflight on body-wall muscles of Caenorhabditis elegans (C. elegans) in the structures and functions were examined, and five important muscle-related genes and three proteins were studied after nearly 15-day spaceflight. The changes for the wall-muscles were observed in situ. Decreased muscle fiber size was observed with myosin immunofluorescence and duller dense-body staining in flight samples, which suggested that muscular atrophy had happened during spaceflight. However, F-actin staining showed no differences between the spaceflight group and ground control group. Otherwise, after returning to the earth the C. elegans displayed reduced rate of movement with a lower ratio (height/width) in crawl trace wave, which indicated a functional defect. These results demonstrated that C. elegans muscular development was changed in response to microgravity, and changes also occurred at the level of gene transcription and protein translation. Expression of dys-1 increased significantly in body-wall muscles, while hlh-1, myo-3, unc-54 and egl-19 RNA levels decreased after spaceflight. Dystrophin (encoded by dys-1) is one of important components in dystrophin-glycoprotein complex (DGC). Increased dys-1 expression after flight implied that the muscular cell would accept more gravity signals by DGC in microgravity in order to keep mechanical balance within the cells. It is concluded that DGC was involved into the mechanical transduction in body-wall muscles of C. elegans when gravity varied, which potentially played a vital role in gravisensing. The changes of hlh-1, myo-3, unc-54 and egl-19 suggested that they had the effects of promoting microgravity-induced muscular atrophy in structure and function aspects. Result of Western blotting showed that the level of myosin A in spaceflight group decreased, further confirmed that atrophy happened during flight.

Abstract:During the incubation of purified α-synuclein with fructose or glucose, it was observed that the protein intrinsic fluorescence at 308 nm decreased while the fluorescence of glycated derivant at 447 nm increased. This fluorescence representing nonenzymatic glycation of α-synuclein was more rapidly formed in the presence of fructose than that of glucose. Interestingly, an energy transfer could be observed from the intrinsic fluorescence to the nonenzymatic glycating fluorescence, suggesting a near distance between Tyr residues and the nonenzymatic glycated derivant. Experiments using circular dichroism showed that the content of α-helix of nonenzymatic glycated α-synuclein was increased during the nonenzymatic glycation, especially incubated with fructose. The nonenzymatic glycated α-synuclein was in some rod-like filaments under the electronic microscope. That is to say, nonenzymatic glycation induces the conformational changes of α-synuclein which is more vulnerable to the nonenzymatic glycation of fructose. It appears that nonenzymatic glycation induces α-synuclein misfolding and probably aggregation in cell.

Abstract:An optimized two-dimensional polyacrylamide gel electrophoresis (2-DE) system for analyzing plant proteins was developed by evaluating different reagents and concentrations used in the sample extraction solutions and lysis buffers. Two main sample preparation methods, referred to as trichloroacetic acid (TCA)-acetone method and phenol extraction-ammonium acetate/methanol (phenol-NH₄Ac/methanol) precipitation method, were compared. Four ecotypes of reed plants (Phragmites communis Trin.) from the desert region of north-western China were used as experimental materials: (1) swamp reed (SR) which grows in water about 1 m deep; (2) dune reed (DR) which grows on 5～10 m high sand dunes; (3) heavy salt meadow reed (HSMR) which grows on low-lying salt flats; and (4) light salt meadow reed (LSMR) which grows in the transition area between DR and HSMR growing areas. The optimized phenol-NH4Ac/methanol precipitation method consisted of extracting leaf proteins of different ecotypes of reed with water-saturated phenol and then precipitating with a 5-fold volume of 0.1 mol/L NH4Ac in methanol, followed by dissolving in the lysis buffer. The optimized protein lysis buffer consisted of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 2% Ampholine(pH 3.5～10∶pH 5～8 = 1∶4) and 65 mmol/L DTT. The prepared protein sample (80 μg) was then separated by 2-DE gel and detected by silver staining method. This improved 2-DE system resulted in a 2-D protein profile of higher resolution and higher protein yields as analyzed by PDQuest software. Good results were also obtained when this 2-DE system was used in 2-D analysis of proteins from other plant materials, such as rice leaves, indicating that it is a suitable 2-DE system for analyzing leaf proteins of different plant species.