Abstract:Drosophila melanogaster is an ideal model system for understanding the molecular mechanisms of circadian rhythm. The genetic amenability of Drosophila has led to the identification of more than ten clock genes and a set of clock-related genes, including clock input and clock-regulated genes. These clock genes and their products consist of two interlocked transcriptional/translational feedback loop, which regulate circadian rhythms of behavior and physiology in Drosophila. The working principles of the Drosophila core clock are also seen in mammals.

Abstract:In multicellular organisms protein glycosylation is a key post-translational modifications' event. Glycans of glycoproteins are not merely markers to characterize each cell type but are more aggressively involved in numerous biological phenomena, such as cell development, differentiation, implantation, morphogenesis, tumor metastasis and microbe infection. Glycomics is defined to analyse mostly the whole set of glycans of glycoproteins produced in a single organism. Several analysis techniques for separation and identification of glycoproteins and glycans are outlined, and the progress in these techniques is discussed.

Abstract:In the early 1990's, Iijima et al. separated a protein, which has the activity of antifungal to suppress the growth of C.albicans, from the hemp musca larva blood lymph. So far, more than 150 kinds of peptides which are characteristic of antifungal have been discovered. With the number of antifungal peptides discovered increasing, researchers have been studying the mechanism of antifungal peptides. The way the antifungal peptides function is mainly as follows: preventing and damaging the synthesis of cell wall; interacting with the membrane to form pores; causing the important inclusions to release; interacting with the important organelles such as mitochondrion and nucleic acid macromolecules in the fungi cell, leading to the death.

Abstract:Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Connexin31 (Cx31) is an important member of connexin β family. Mutations in Cx31 are associated with erythrokeratodermia variabilis(EKV), hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Assembly, intracellular transport, plaque assembly and stability and channel conductivity of Cx31 are finely regulated and likely involve proteins that interact with Cx31. However, little is known about the Cx31 interaction proteins. A proteomics approach was applied to screen Cx31 binding proteins using HT1080 cells stably expressing a myc-tagged Cx31. Immunoprecipitation followed by peptide sequence analysis identified association of actin and Cx31. Interaction between actin and Cx31 is further confirmed by coimmunoprecipitation and immuno-colocalization. Pharmacological disruption of actin polymerization inhibits the plasma membrane localization of Cx31. The results suggested the actin cytoskeleton may play an essential role in regulating Cx31 trafficking via direct association.

Abstract:The processes of implantation and placentation involve various cell events, such as degradation and remodeling of extracellular matrix, cellular proliferation, apoptosis, and differentiation. Evidence indicates that members of the matrix metalloproteinase (MMP) family play crucial roles in these processes. In the present study, the expression and localization of MMP-26/endometase/matrilysin-2 in human cytotrophoblast cells as well as its regulation by activin A were investigated by using methods of reverse transcriptase-polymerase chain reaction, immunohischemistry and fluorescence immunohistochemistry. The results showed that MMP-26 was strongly localized in villous trophoblast cells, and weakly in some of the villous mesenchyma. The mRNA level of MMP-26 in chorionic villi was high in early pregnancy, and was down-regulated from gestational week 10 on, reaching a very low level at week 26. However, its mRNA expression was significantly up-regulated in the term placenta. In the primary cultured cytotrophoblast cells, the mRNA expression of MMP-26 maintained a stable level during gestational week 6~12. Moreover, the expression of MMP-26 in the cytotrophoblast cells was stimulated by activin A in a dose-dependent manner. All the data indicated that MMP-26 may be involved in trophoblast invasion during early pregnancy and placenta detachment at the time of delivery. There may exist autocrine/paracrine regulation of MMP-26 expression in cytotrophoblast cells.

Abstract:Syntaxin 1A (Syn1A) and Munc18a play essential roles in vesicular trafficking and exocytosis. The molecular mechanism underlying the sorting of these two proteins to their physiological sites of action remains poorly understood. Here the localization of syntaxin1A (Syn1A) and Munc18a was analyzed in baby hamster kidney (BHK-21) cells and human embryonic kidney (HEK293) cells. The rat Syn1A gene was fused to the gene encoding the enhanced green fluorescent protein (EGFP). Munc18a was labeled with the red fluorescence protein (TDimer2) at its C terminal. The proteins were expressed by transient transfection in either BHK-21 or HEK293 cells. Under fluorescence microscopy, it was shown that Syn1A was shown to be transported to the plasma membrane. While Munc18a exhibited mainly cytosolic distribution when expressed alone. However, upon coexpression with Syn1A, Munc18a is translocated to the plasma membrane. In addition, a N-terminal truncated mutant Syn1A failed to localize at the plasma membrane, suggesting that the cytoplasmic domain of Syn1A is important for its sorting and localization.

Abstract:Five field isolates of infectious bursal disease virus (IBDV) were isolated from bursae of sick young chickens by inoculating chicken embryo fibroblasts (CEF) while the morphological and physiochemical properities of these isolates were detected. Subtype of the virus isolates were analysed by cross virus neutralization. Virulence of IBDV isolates was determined with specific pathogen-free (SPF) chicken inoculation. Nucleotide sequence of genomic segment A of the isolate NB was further sequenced and analysed. All bursal suspensions produced cytopathic effects (CPE) in CEF after 2~14 passages. The physiochemical and morphological properties of IBDV isolates were in accordance with that of typical IBDV. The three CEF-adapted virus isolates JD1, JD2 and NB were divided into 3 subtypes of serotype Ⅰ IBDV except IBDV isolates HZ1 and HZ2 belonged to classical IBDV. In a pathogenicity experiment, the clinical signs and bursal atrophy resembling those of field outbreaks were produced and microscopic bursa lesions revealed that there was degeneration, necrosis and disappearance of lymphocytes in the medullary area of bursal follicles. Sequence data demonstrated that genomic A-segment of the isolate NB was composed of 3 259 nucleotides, encoding VP5 of 145 amino acid residues and the polyprotein (VP243) of 1 012 amino acid residues. Compared with serotype Ⅰ IBDV strains from GenBank, within serotype Ⅰ IBDV strains, the isolate NB has the highest identity to the polyprotein of the isolate JD1 (99.5%), VP2 of the isolates JD1, CEF94 and D78 (99.8%), VP3 of the isolate JD1 (99.2%), VP4 of the isolate JD1 (100%) and VP5 of the isolates JD1, HZ2, P2, CEF94, CT, Cu-1 and D78 (99.3%). In the VP2-predicted amino acid sequence of the isolate NB, amino acid residues at positions 253, 280 and 284 were consistent with and other published variant and classical IBDV strains, and different from very virulent IBDV. These results indicated that antigenic epitopes of IBDV are conformational and subtype forming of IBDV isolates originated from classical IBDV attenuated vaccine strains.

Abstract:Previous work indicated that there was a large difference of the sequence features of introns between highly- and lowly-transcribed yeast genes. Some potential positive transcriptional regulatory motifs in the highly-transcribed introns were extracted. It was found that these introns were very close to the upstream regions of genes, and several introns even located within 5'-UTR. These results show that introns of yeast genes may regulate the transcriptional efficiencies and cooperate with upstream regions. To understand the synergy between upstream regions and introns of highly-transcribed yeast genes, the upstream regions (800 bp upstream of translation start sites, or the regions of two adjacent genes) of these two sets of genes were retrieved, and some potential positive transcriptional motifs in the upstream of highly-transcribed genes were extracted using the statistical comparative analysis approach developed before. Most of the potential motifs extracted were supported by literature search of experimental analyses. Then, every pair of oligonucleotides, one in the upstream region and the other in the intron, was defined as an "oligonucleotide pair", i.e. a motif pair. Considering the motif pairs with the "nearest distance" not larger than 84 bp, the motif pairs, mainly tetra- and penta-nucleotide pairs, occurring in the highly-transcribed genes non-randomly were extracted and the synergistic pattern of these motif pairs including position distributions and binding transcription factors such as RAP1, ABF1 and TAF was analyzed. Some potential patterns of transcriptional synergy were observed. These results could help people to understand the mechanism of transcriptional regulation and provide evidence for biological verification.

Abstract:MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) can induce dopaminergic cell death in substantia nigra of primates and rodents. In order to investigate the effects of brain lateralization on dopamine loss and cytokine variations in mice which were treated with MPTP, male C57BL/6J mice were selected by paw preference test to constitute right pawed mice and left pawed mice. Mice were injected with 25 mg/kg MPTP i.p., for five consecutive days, and killed on day 1, day 3 and day 14 after MPTP injection. The control group was treated with saline. Striatal dopamine and DOPAC contents were measured by HPLC and striatal IL-1 β and IL-6 were quantified by ELISA. The Results showed that after MPTP treatment, dopamine contents decreased at day 1 (74.48%, P<0.001), day 3 (72.84%, P<0.001) and day 14 (63.87%, P<0.01); IL-6 levels decreased on day 1 (P<0.01), day 3 (P<0.01) and day 14 (P<0.01); while IL-1? 茁 levels only decreased on day 1 (P<0.05). Interestingly, behavioral lateralization had effect on dopamine turnover and cytokine variations. In basal condition, the significant difference was found between right pawed mice and left pawed mice in IL-6 level (P<0.05) and IL-1 β level (P<0.001). After MPTP treatment, left pawed mice showed a greater decrease of dopamine turnover (P<0.05) as compared with right pawed mice, at day 3 after treatment, left pawed animals showed a lower levels of IL-6 (P<0.05) and IL-1 β level (P<0.001) as compared with right pawed animals. Furthermore, the positive correlation between striatal IL-6 levels and striatal dopamine contents was found (P<0.001). These results indicated that MPTP-induced dopamine turnover was influenced by behavioral lateralization, possibly through an effect on brain cytokine levels.

Abstract:It has been recently demonstrated that melatonin protects cells from calyculin A (CA)-induced neurofilament hyperphosphorylation. To further explore the mechanism of melatonin, the wild type neuroblast cells (N2awt) were treated with CA or CA and melatonin or CA and vitamin E, and detected the levels of tau phosphorylation, the activities of GSK-3 and PP-2A, as well as the antioxidant activities of melatonin and vitamin E against CA. It has been found that the antioxidant activity of melatonin and its protection on tau hyperphosphorylation at Ser199/202 and Ser396/404 and Ser422 sites are stronger than that of vitamin E, at the same time, melatonin increases the activity of PP-2A and decreases the activity of GSK-3 while vitamin E decreases the activities of PP-2A and GSK-3. These results suggest that melatonin protects neuroblast cells from CA-induced cytotoxicities not only through its antioxidant effect but also through its regulation of the phosphorylation system.

Abstract:ATPase is closely related to chilling tolerance. The EST sequence of chilling-repressed gene atpA encoding CF₁ α -subunit was obtained from Elumus sibiricus L. cv.'chuancao No.2' by reverse transcription-polymerase chain reactiom (RT-PCR) differential display. Full-length cDNA of 1 754 bp was cloned by 5 ′ RACE. The atpA has an open reading frame (ORF) of 1 518 bp that encodes a precursor protein of 505 amino acid residues. The deduced amino acid sequence exhibits 95%, 94% and 94% positional identity with atpA of wheat, rice and corn, respectively. Northern hybridization analysis on mRNA from treatment at 2 ℃ and post-treatment, in total of 13 time stages, showed that its RNA transcript was strongly inhibited after 12 h of chilling stress, whereas it was clearly higher than control during 4~8 h of chilling stress and 16~24 h after removing stress. This result provides new clue for revealing the CF₁α -subunit role to ATPase in plant defence against chilling stress

Abstract:Lymphotoxin(LT) delivers the signal of apoptosis by TNFR1, sequentially develops anti-tumor activity. Several receptor binding sites of LT were mutated randomly, then the R46 ， S106 ， L130 combined site-directed random mutation library and S106 ～ F110 region random mutation library were displayed on a filamentous phage surface. By using TNFR1 as solid phase molecule, the phage libraries were biopanned and positive mutant clones were enriched. 20 monoclonal phages were picked randomly to detect receptor binding by ELISA assay, 80% of them can bound TNFR1 specifically, and TNFR1 binding ability of 4 clones was higher than rhLT with wild type sequence. 4 mutants with high binding ability were subcloned to pET32a(+) vector, after expression in E.coli and purification process, the binding ability and the cytotoxicity in L929 mouse fibroblast cells of these mutants were examined. 3 mutants were having similar binding ability compared with monoclonal phage, the binding ability to TNFR1 and the cytotoxicity to L929 of C199 clone have been enhanced near 30% and 90% respectively. The effort to proceed molecular evolution study of lymphotoxin in vitro by phage display not only can be very useful for the following research involved in the relation between structure and function, but also provides potent tool for macromolecule drug development

Abstract:It is reported recently that truncated tau at Glu391 and Asp421 presents in the patients of Alzheimer's disease (AD) brain, and caspase-3 is involved in tau truncation at Asp421. In vitro studies show that caspase-3 can cleaved tau only at Asp421 and the proteolytic cleavage of tau at Asp421 by caspase-3 generates a truncated tau fragment namely tau1-421, which enhances filament assembly in vitro. Expression of tau1-421 in neurons promotes apoptosis. It is not known that if phosphorylated tau is also a substrate of caspase-3. To understand the question, recombinant human tau protein was purified and phosphorylated with protein kinase A (PKA), calcium/calmodulin dependent protein kinase Ⅱ (CaMK Ⅱ ) or kinases from newborn rat hippocampi extract, and then these different tau species were cleaven by caspase-3. It was demonstrated that tau phosphorylated by PKA, CaMK Ⅱ and newborn hippocampi extract is also cleaven by caspase-3. As the phosphorylated tau also can be cleaven by caspase-3, it was speculated that phosphorylation tau is still the substrate of caspase-3.

Abstract:In the salt-independent chromatography, the component, structure, density and some other characteristics of the adsorption media are improved, so the variations of feedstock ion strength have no significant effect on adsorption. The great advantage of the salt-independent is absent in the traditional chromatography methods such as the ion-exchange chromatography, the thiophilic chromatography and the hydrophobic chromatography. This kind of technologies can lower the demands on preparation of the crude feedstock, therefore the steps of desalting, diluting or adding the extra salts could be bypassed. It simplifies the process and makes it more economical, so it is compatible with large scale requirements of the separation of the proteins, especially the enzyme and the antibody. The recent development of the new ligands and methods on salt-independent chromatography are summarized.