Abstract:MicroRNAs(miRNAs), small noncoding RNAs, are essential for posttranscriptional gene regulation and have important roles in a wide range of biological processes, including development, growth and differentiation. Thousands of miRNAs have been identified in animals, plants and microoaganisms. miRNAs regulate their target genes by mRNA degradation or translation suppression. About 30% of genes in an organism are subject to miRNA regulation. miRNA expression and function are regulated by transcriptional factors, epigenetics, single nucleotide polymorphisms, RNA editing and so on. Additionally, the success in knocking out a specific mouse miRNA gene has provided a valuable model for studying miRNA function.

Abstract:Techniques for functional brain imaging are critical to analyze the information processing of brain and to reveal the advanced functions in brain. These techniques are the hot topics of international research. Great success has been obtained with neuroimaging techniques in the fields of neuroscience research and clinical diagnosis. Existing brain functional imaging such as magnetic resonance imaging (fMRI), positron emission tomography (PET), electroencephalogram (EEG), magnetoencephalography (MEG) and so on, have been successfully used to study brain function. However, these methods have some limitations unavoidably in the temporal or spatial resolution at present. Comparatively, the optical imaging technologies of brain function show their unique charms. Laser speckle imaging (LSI) and intrinsic optical signals imaging (IOSI) stand out because they offer a superior combination of spatial sampling, spatial resolution and temporal resolution; on the other hand, they have no need to use exogenous contrast agents. Great developments also have been obtained in both techniques and applications of brain optical imaging, and they have become powerful tools for in vivo studying functional architecture and pathophysiology in cerebral cortex by monitoring hemodynamics. However, the two optical imaging techniques are confronted with some challenges.

Abstract:Many Novel approaches of epigenetic reprogramming of somatic cells have been reported. However, ethical issues caused by somatic nuclear transfer have triggered the development of alternative strategies for reprogramming somatic cells. Recently, many new advances have been acquired for reprogramming somatic cells, which could reverse differentiated somatic cells to a totipotent embryonic state, such as fusion of potential stem cells with somatic cells, incubation of cells in potential cell-free extraction and introduction of defined pluripotency factors into somatic cells. The epigenetic modification in these reprogramming processes, including germ cells and early embryoes, somatic nuclear transfer and other approaches for reprogramming of somatic cells were reviewed. Studies of epigenetics will be benefit for understanding the precise mechanism and improving the efficiency of somatic nuclear reprogramming, which will be eventually applied in the basic study and practice.

Abstract:Lewy body (LB) in the substantia nigra pars compacta (SNpc) is one of the cardinal pathological features in sporadic Parkinson's disease (sPD). Both pathogeny and pathomechanism of LB have been set forth. However, genetic, postmortem and experimental evidences demonstrate that impaired proteasomes and concomitant LB could result in the concept of process of aggresomes, by which aggregation reaction of abnormal proteins is mainly proposed as molecular crowding of non-fibrillar proteins followed by fibrillation of aggregated proteins, and in which dysfunction of proteasomes, loss of endoplasmic reticulum-associated degradation (ERAD), aggregates, aggresomes and LB are attractive occurrences in sPD. This suggests that dysfunction of proteasome and concomitant formation of LB in sPD is basically associated with cellular process of signal transduction involved in a variety of proteins.

Abstract:ING1 family is a candidate for tumor suppressor, which has three splicing isoforms named p47ING1a, p33ING1b, and p24ING1c. Study of the effect of different isoforms of ING1 on HeLa cells proliferation and its molecular mechanism would help further identifying the functional relationship of ING1 isoforms, and finding important genes regulated by ING1. Cell growth curve and cell cycle analysis were used to observe the effect of ING1a, ING1b, and ING1c on HeLa cells growth, and the result indicated that they could all inhibit HeLa cells growth by arresting cell cycle at G0/G1 phase. PCR method was used to construct the PHD domain deletions of ING1a and ING1b. ING1a, ING1b, ING1c and the PHD domain deletions 1a△C and 1b△C were then overexpressed in HeLa cells. p16^INK4a, PTEN/p27^Kip1 and p53/p21^Waf1 protein levels were detected by Western blot. The result showed that ING1a, ING1b, ING1c, and 1a△C except for 1b△C induced p16^INK4a protein expression, in which ING1c had the most powerful effect. Luciferase assay identified that overexpression of pcDNA3.1(+)-1a△C facilitated p16^INK4a transcription through enhancing p16^INK4a promoter activity, while pcDNA3.1(+)-1b△C repressed the p16^INK4a promoter activity . In a word, it was found for the first time that except for the p53/p21^Waf1 pathway, three splicing isoforms of ING1 family could also inhibit HeLa cells proliferation though upregulation of p16^INK4a and PTEN, and the PHD domain deletion of ING1a enhanced p16^INK4a transcription. These findings provide new clews to further study on the mechanisms of ING1 family suppressing cancer cells growth.

Abstract:To search for nasopharyngeal carcinoma (NPC) biomarkers, laser capture microdissection (LCM) was used to purify the target cells from NPC and normal nasopharyngeal epithelial tissues (NNET). Two-dimensional gel electrophoresis (2-DE) was performed to separate the total proteins of microdissected NPC and NNEC, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the two types of tissues were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. The expression of cytokeratin 8(CK8), one of the differential proteins, in the microdissected NPC and NNEC as well as 4 NPC cell lines with different differentiated degrees and/or metastatic potentials was detected by Western blot. Immunohistochemistry was also used to detect the expression of CK8 in paraffin-embedded tissues including 63 cases of primary NPC, 28 cases of NNET and 20 cases of cervical lymphonode metastasis. In the present study, 2-DE patterns of microdissected NPC and NNEC were established, and 29 differential proteins in the above two tissues were identified, of which 15 only expressed or up-regulated in NPC and 14 only expressed or up-regulated in NNET. The expression level of differential protein CK8 between the NPC and NNET was selectively confirmed, and was found to be related to the differentiation and/or metastasis of NPC cell lines. Significant down-regulation of CK8 was observed in NPC compared with NNET, and significant up-regulation of CK8 was also observed in lymphonode metastasis compared with primary NPC. The data suggest that CK8 may be related to the differentiation and lymphonode metastasis of NPC, and may serve as molecular biomarkers for metastasis and differentiation of NPC.

Abstract:Subcellular localization is a key characteristic of protein functional research. Proteins are transported to specific compartment after they are synthesized in cells. They can take part in the cell activity and function efficiently when in correct subcellular location. Sequence homolog, protein-protein interaction information and traditional amino acid composition are combined as input parameters of support vector machine (SVM) to predict eukaryotic protein subcellular localization. The total accuracy of 5-fold cross validation is 91.8%, which is higher than other methods.

Abstract:The immunogenicity and cross-protection of Pneumococcal surface protein A (PspA) and its conjugates with polysaccharide were researched in mice injected with PspA and its conjugates respectively. The enzyme-linked immunosorbent assay (ELISA) was adopted to detect the immunogenicity of the antigen. Passive and active protection experiments against intraperitoneal challenge with Pneumococcal pneumonia were carried out to validate the protection of PspA and the conjugates with polysaccharide. The experimental results showed that the immune effects of both the conjugates and PspA were effective to against invasion of Pneumococcal pneumonia in mice (P < 0.01). And the conjugates of PspA with polysaccharide gave the best protection and survived 3 days longer than that of PspA. The conjugates with polysaccharide enhanced the higher level of both IgG and IgG2a antibody titer than that of PspA. The cross-reactivity of Western-blotting showed conjugates with polysaccharide caused immune reaction in different serological Pneumococcal pneumonia, this meant that the conjugates had some cross-protection function against different serotypes 6B, 5, 1, 23F, 19F. The conjugates of PspA with polysaccharide showed better immunocompetence and immune protection against invasion of Pneumococcal pneumonia than PapA and the capsule polysaccharide. This study demonstrated that the conjugates could enhance protection against Pneumococcal pneumonia, which should be pertinent to future efforts to develop new protein-based complex vaccines.

Abstract:In order to study the effects of LDL and oxLDL on expression of PCSK9 and LDLR in THP-1 macrophages and find the relationship between them. THP-1 cells were induced to differentiate into macrophages by PMA treatment. Cells were then co-incubated with LDL or oxLDL with a concentration of 0 mg/L, 10 mg/L, 20 mg/L, 30 mg/L for 24h respectively. Cellular lipid was visualized by oil red O staining. The localization and semiquantitation of PCSK9 was confirmed by immunofluorescence assay，the expression of PCSK9 and LDLR was analyzed by RT-PCR and Western blotting. Oil red O staining showed that a number of visualized lipid droplet accumulated within THP-1 cells. The lipid droplet is big and accumulating in cells co-incubated with oxLDL but small and dispersing when co-incubated with LDL. Matured PCSK9 are expressed on the cell surface and some organelle, and increased with the increasing of LDL concentration through immunofluorescence assay. RT-PCR, Western blot showed that, in THP-1 , LDLR was downregulated while PCSK9 was upregulated especially when treated with LDL , but the treatment of low concentration of oxLDL likely had no effect on expression of LDLR and PCSK9. Together, these results reveal that PCSK9 and LDLR may co-expressed in THP-1 cells, which are unable to be influenced by oxLDL, but may be down-regulated (LDLR) or up-regulated (PCSK9) by LDL, providing primary evidence of the correlation between PCSK9 and LDLR.

Abstract:Identifying protein fold is an important issue in protein structure research. Based on the classification of SCOP1.65, 17 Globin-like proteins from four homology families ( < 25% sequence identity) are selected from Astral 1.65. The sequence alignment result, from structure alignment tool MUSTANG combined with manual inspection, has been used to generate a profile HMM of Globin-like fold. In a fold identify test on 68 057 sequences of Astral-1.65, the model identified 1 097 Globin-like proteins rightly, only 4 proteins of this fold are not correctly distinguished. The sensitivity and specificity of the profile HMM reach to 99.64% and 100%, respectively. Compared with Pfam and SUPERFAMILY which construct HMM based on merely sequence alignment, the model number is reduced from about 100 to 1, while keeping the sensitivity at the same level. The result shows that, for those proteins with same fold type but low sequence identity, a unified HMM could be constructed by introducing structure alignment to fold identify with high accuracy.

Abstract:In order to establish and analyze core-fucosylated glycoprotein profiles of human normal liver tissues, which could contribute to the finding of more aberrantly fucosylated glycoproteins related to liver diseases, based on the approach, “lectin affinity chromatography, 2-DE and MALDI-MS/MS”, the lens culinaris agglutinin (LCA) affinity glycoprotein profiles from human normal liver tissues were obtained, in which 130±3 protein spots were detected. Altogether, 90 silver-stained spots in the 2-DE map were cut out, destained and subjected to in-gel tryptic digestion, and 53 proteins were identified by MALDI-TOF MS/MS. These proteins, mainly in the middle range (pI 5～9, M 10～100 ku), were found to participate in the metabolism and play crucial roles in binding and catalytic reactions. Putative N-linked glycosylation consensus sequence, i.e., -N-X-S/T-, was applied to evaluate glycosylation of all the identified proteins. Furthermore, protein immunoprecipitation combined with lectin blot was used to further validate fucosylation of the identified possible candidate proteins, haptoglobin and alpha enolase. All the results suggested that lectin affinity chromatography and 2-DE in combination with MALDI-MS/MS enabled the identification of all the specific subsets of glycoprotein, and the database could give us a help when searching for some aberrantly core-fucosylated glycoproteins associated with some diseases.

Abstract:Tachyplesin is a 17-aa peptide, isolated from marine “living fossil” horseshoe crab, Tachypleus tridentatus, showing wide-spectrum antibacterial activity. However, its mechnism of killing bacterium is not very clear. The aim was to investigate the targets of tachyplesin for further studying the antibacterial molecular mechnism. In vitro bacterial inhibition method was used to determine feature of bacterial inhibition dynamic of tachyplesin. Inorganic phosphate measurement and ultraviolet absorption methods were used to observe the leakage of inorganic phosphorus and large mass molecular before and after bacterium were incubated with tachyplesin. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were adopted to investigate the morphologic and structural changes of bacterium before and after being incubated with tachyplesin. Ultroviolet absorption method and electrophoretic mobility shift assay (EMSA) were used to investigate the effect of tachyplesin on structure of genomic DNA and plasmid DNA of bacterium. Plasmid transformation method was applied to observe the effect of tachyplesin on copy and transcription of plasmid DNA. The results showed that, (1) Tachyplesin had different feature of bacterial inhibition dynamic in Gram positive and negative bacterium. In the Gram positive bacterium (E. coli K88 and E. coli F41), the antibacterial activity of tachyplesin increased sharply during the first 10 h, and was in the plateau phase during 10～30 h, and then decreased slowly. In the Gram negative bacterium (B. subtilis W B800 and S. aureus), the antibacterial activity of tachyplesin increased sharply during the first 3 h, and decreased during 3～10 h, and increased again during 10～15 h, the trend was similar to the Gram positive bacterium after 20 h. (2) Bacterium lost inorganic phosphorus and large mass molecular. Phosphorous concentration in the culture media of S. aureus was significantly higher after treated with tachyplesin, the high concentration could keep long time (30 h later). The concentration of the UV absorbing substance in E. coli K88 became higher and higher depending on tachyplesin concentration and time. At low tachyplesin concentration, the UV absorbing substance concentration kept low. (3) The structure of cell wall, cell membrane and the whole cell body were damaged to some extent after treated by tachyplesin. (4) Tachyplesin could combine with genomic DNA and plasmid DNA of bacterium, high concentration of tachyplesin probably could break down DNA, and inhibited copy and transcription of plasmid DNA. It was suggested that the antibacterial targets include at least cell wall and membrane and DNA of bacterium.

Abstract:Previous studies indicated that NGX6, an EGFR negative regulating gene, can reverse the malignant phenotype of the colon cancer and down-regulate the expression of MADD(MAP-kinase activating death domain) which is an essential protein in the JNK pathway. All these results implied that whether NGX6 inhibits tumor growth by inactivating the EGFR-mediated JNK pathway? Western blot and immunohistochemistry were performed to detect the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the colon caner cell line and in the tissue of xenografts in nude mice, aiming at deciphering the effects of NGX6 on EGFR/ K-ras/JNK/c-Jun/cyclin D1 pathway both in vitro and in vivo. The results showed that NGX6 re-expression considerably suppressed the growth of xenografts in the nude mice. Data from Western blot revealed that NGX6 significantly down-regulated the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the colon cancer cells. And the further analysis by immunohistochemistry and Western blot in vivo indicated that NGX6 decreased the expression of EGFR, K-ras, p-JNK, c-Jun and cyclin D1 in the xenografts in nude mice，which were consistent with in vitro observations. Such evidences suggest that the major mechanism of NGX6 in colon cancer is negatively regulating EGFR-mediated JNK pathway, laying the experimental basis for further elucidating the mechanism of NGX6 gene.

Abstract:To explore the antimicrobial mechanism of human cervical mucus, HCP-21and HCP-26 were isolated and purified from acid-soluble extracts of human cervical mucus by acid-urea polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography(RP-HPLC). Both molecules could effectively kill E. coli ML-35p determined by agarose radial diffusion assay. The N-terminal amino acid sequence of HCP-21 was PKRKAEGDAK and its molecular mass was 9 263.62 identified by amino acid sequencing and mass spectrometry analysis. HCP-21 showed 100% identity to HMGN2 (high mobility group protein N2, HMGN2) fragment 2～11 by GenBank BLAST searching, and had the same molecular mass as HMGN2. So it was certain that HCP-21 was HMGN2. HCP-26 was SLPI (secretory leukocyte protease inhibitor, SLPI) fragment by N-terminal amino acid sequencing which was SGKSFKAGVC. It was the same with SLPI fragment 26～35 by GenBank BLAST searching. Total RNA was extracted from primary culture cervical epithelial cells, and the specific primer was designed based on HMGN2 cDNA sequence. RT-PCR showed that a fragment about 270 bp was amplified, which was same to HMGN2 cDNA. It was suggested that cervical epithelial cells could express HMGN2 mRNA in physiological condition. The fusion protein GST-HMGN2 was purified by low-pressure chromatography. The antiserum of HMGN2 was prepared from the immune rabbit with the fusion protein. Cervical tissue paraffin section and cervical mucus smear were detected by immunhistochemistry. The staining results showed that HMGN2 mainly distributed in mucosa surface of cervix and existed in cervical mucus. HMGN2 constitutively expressed in cervical mucosa and mucus, so it might play an important role in innate defensive of cervical.

Abstract:Cationic colloidal gold (CCG) nanoparticles were used for labeling on the anioinic sites of living cells under two-photon fluorescence (TPF) microscope, and for delivering macromolecules into the target cells when irradiated by focused femtosecond laser pulses. 15 nm CCG nanoparticles which were made by conjugation with poly-L-Lysine, were attached on the anionic sites, especially on the membrane, of CHO-K1 cells because of their strong positive charge at physiological pH. Target cells labeled with cationic gold nanoparticles were imaged under TPF microscope, and lifetime images of the same targets were taken by time correlated single photon counting (TCSPC) technique in order to verify the fluorescence of the marker and the luminescence of the gold particles. The results shown that CCG nanoparticles first accumulated on the negatively charged sites of the membrane, then entered via endocytic pathway and attached anionic sites in plasma. A macromolecular 10 ku fluorescein isothiocyanate dextran (FITC-D) was added into the sample and the focused femtosecond laser of TPL microscope was employed to scan the target cells layer by layer. Typical laser power level used in biological imaging is about 3～5 mW. Here the laser power of scanning was below 5 mW in order to prevent photochemical damage of the fs-pulses alone and to localize effects to the nanoparticles on a nano-scale. After scanning the target cells under stack mode, macromolecular fluoresceins surrounding the cells was observed to cross the membrane and to diffuse in the cytoplasma. Comparing with the images before scanning, the two-photon fluorescence and fluorescence lifetime images revealed the delivery of FITC-D into target cells. Photothermal effects, which may be responsible for the permeabilisation, are highly localized in nanoscale and are not expected to cause damage exceeding the cell membrane. After extensive of laser scanning also cell death occurred. The ratio of the uptake of FITC-D and cellular death under different conditions were measured by flow cytometer. The results shown: with the increased scanning times or ratio of particles to cells, transfer efficiency increased first and decreased afterwards, but the ratio of cellular death went up all along.

Abstract:High levels of RNase, polysaccharides and polyphenol compounds make isolation of high quality RNA difficult. Thus it is presented an effective RNA extraction method based on the nuclease adsorbent macaloid, poly vinyl pyrrolidone, and high concentration of KAc and ethylene glycol monobutyl ether, which has successfully extracted high-quality RNA from many materials difficult to RNA isolation, such as RNase-rich rabbit liver, plant and microbial tissues rich in polysaccharides, lipids and polyphenol compounds. This method was found to be better than the ones in common use-Trizol and Guanidinium isothiocyanate, the yield of which was at least three time higher. Furtherly, small RNA was enriched from total RNA sample from rice seedling through by repeat deposit which deals with high concentration of LiCl, PEG8000 and NaCl. The small RNA gained was confirmed to be used for following molecular biological research by RT-PCR with the primers designed on osa-mir-156 sequence from rice miRNA.

Abstract:Carbohydrate chip, a wonderful tool for studying the carbohydrate-protein interactions, is developing very fast in recent years and shows a bright future. The methods of fabrication of carbohydrate multi-arrays are systematically introduced, factors that influence the obtained chip?蒺s quality are analyzed in detail, and the general requirement of plate materials and immobilization methods for design of carbohydrate chip are discussed.