Abstract:Lipid droplets (LDs) were originally described in milk by van Leeuwenhoeck in 1674. Although over 300 years have passed since this original observation, many fundamental questions about LDs remain unaddressed. Until recently, very few of the surface proteins had been characterized and the functions of LDs, aside from their presumed role as neutral lipid storage depots, remain unknown. To better understand LDs, we and others have recently carried out several proteomic studies using a combination of purified LDs and mass spectrometry (MS) protein identification. Many of the proteins which have been newly identified by this approach can be divided into two functional categories: lipid synthetic/metabolic enzymes and membrane trafficking proteins. Importantly, different laboratories investigating a variety of different cell types have found similar results. These findings strongly suggest that LDs are a complex, metabolically active organelle that may participate in lipid synthesis and trafficking. This review will describe progress in identifying these proteins and will also discuss the potential implications for the function of animal LDs. For detailed information on other aspects of LD biology, please read the review from Denis Murphy.

Abstract:Insulin is widely used as an effective therapeutic drug for the treatments of type 1 and type 2 diabetes mellitus. The side effect of insulin in type 2 diabetes is that it may cause hypoglycemia. Another common used prescription drug for type 2 diabetes is gliclazide. This drug is as effective as insulin, but it may cause the side effect in enteron. It is extremely important to develop a novel safe and effective therapeutic drug for type 2 diabetes. Recently, research from pharmaceutical companies and many institutions demonstrated that glucagon-like peptide-1 (GLP-1) is an excellent therapeutic target for type 2 diabetes. GLP-1 can promote the secretion of insulin and enhance its sensitivity. Furthermore, treatment of type 2 diabetes patients with GLP-1 does not lead to hypoglycemia. More recent studies showed that GLP-1 is effective for both type 1 and type 2 diabetes mellitus. The latest research status of glucagon-like peptide-1 and its receptors is reviewed.

Abstract:High mobility group proteins (HMGs) are a set of chromatin related proteins named according to their high electrophoretic mobility on SDS-PAGE. The HMG protein family is subdivided into three subfamilies: the HMGB subfamily, the HMGA subfamily and the HMGN subfamily. Each of the subfamilies has a characteristic functional motif and these motifs are the main sites of interaction between the HMG proteins and the DNA or chromatin targets. The HMG proteins have many functions and all of them work as “architectural” factors by either distorting, bending or modifying the structure of DNA complexed with transcription factors or with histones.

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 erythrokeratoderma variabilis (EKV) ， hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Using Site-directed mutagenesis (SDM) to construct two dominant hearing impairment associated Cx31 mutant expression plasmids, R180X-pEGFP-N1 and E183K- pEGFP-N1. A number of functional analysis were described to investigate the effect of two dominant hearing impairment-associated Cx31 mutants, R180X and E183K, on connexon trafficking. The majority of the mutant R180X and E183K were cytoplasmic, they can not traffic to normal intracellular localization and can not form functional gap junction channels. But not observed with wild-type Cx31.This may provide an insight into the pathological mechanism of Cx31 mutants.

Abstract:Large conductance voltage and Ca2+-activated K+ channels (BK channels) ， which are ubiquitously expressed among mammalian tissues, play a variety of physiological roles. The inactivating BK channels have not been extensively studied in primary tissues and rarely do they exhibit inactivation. An inactivating BK current (BKi) was identified in rat small dorsal root ganglia (DRG) neurons . The inactivating currents are sensitive to the specific BK channel blocker charybdotoxin (ChTX) and the effect of ChTX can be removed partially when washed with normal external solution. The inactivation of BKi can be removed by external treatment of papain.

Abstract:To investigate the expression change and location of transforming growth factor-beta1(TGF- β1) at uterus and placenta in the course of pregnancy and the effects of interferon gamma (IFNγ) on its expression, the expression of TGF-β1 was examined in uterus and placenta in different stages of gestation by methods of RT-PCR, Western blot, and immunohistochemistry respectively. And RT-PCR was used to analyze the expression of TGF-β1 mRNA after injecting different dose of IFNγ. Experimental results indicated that the expression of TGF-β1 mRNA and TGF-β1 protein were detected in uterus and placenta in every stage of gestation. The analysis of semie-quantitative RT-PCR that showed the expression of TGF-β1 mRNA was increasing gradually at uterus during the early stage of gestation (D1~D9). The expression of TGF-β1 in pre-implantation (D4) period was lower than that in implantation (D6) period and post-implantation (D9) period. The expression in post-implantation (D9) period is highest at uterus during gestation. With the process of gestation, the expression of TGF-β1 decreased significantly at uterus during mid-stage (D15) and late stage (D19) of gestation. The expression of TGF-β1 at placenta kept increasing in the course of pregnancy. The expression of TGF-β1 at mid-stage placenta (D15) was significantly higher (P<0.01) compared with that at early stage placenta (D9) and the expression of TGF-β1 at late-stage placenta D19) was significantly higher (P<0.01) compared with that at mid-stage placenta (D15). The analysis of immunohistochemistry indicated TGF-β1 mostly expressed at decidua during early stage of gestation and at trophoblast during mid-stage and late stage of gestation. After injecting exceeding normal concentration IFNγ by vaginal muscular, the expression of TGF-β1 was downregulated at uterus and placenta of pregnant rat.The results suggested that the expression of TGF-β1 was dynamic and spatio-temporal characteristics in uterus and placenta during gestation. IFNγ affected the expression of TGF-β1 at uterus and placenta in the course of pregnancy.

Abstract:Human breast cancer cell line MCF-7 and its sub-clone LM-MCF-7 with high metastatic potential supply a cell model for studying molecular mechanisms of tumor metastasis. The two cell lines were used to screen tumor metastasis-associated genes by cDNA microarray, displaying differential gene expression profile. Total RNA of the two cells was extracted and the cDNAs were labeled with Cy5-dCTP and Cy3-dCTP, respectively. Then the cDNAs were hybridized on the gene chips containing 21 329 kinds of human genes. The signals were examined by GenePix Pro 4.0 software. The two labeling nucleic fluorescent dyes were exchanged each other so that the experiments were repeated twice. The results shown that 67 kinds of genes were markedly different between the two cells, in which 41 were up-regulated and 26 down-regulated in LM-MCF-7 cells. Some of results were confirmed by real-time PCR. The above genes are associated with functions, such as cell signal transduction, transcript regulation, transportation, response to stress, metabolism, development, movement, cell cycle, differentiation, apoptosis, conglutination, and so on. According to the reports 35 of 67 genes were involved in tumor, in which 9 were related to metastasis of breast tumor, and 6 may be involved in tumor invasion and metastasis. Based on the results, apoptosis was investigated in function in LM-MCF-7 and MCF-7 cells. It was found that the ability of antiapoptosis in LM-MCF-7 cells was higher than that in MCF-7 cells. The candidate genes associated with tumor metastasis will be further demonstrated in function.

Abstract:Extracellular fatty acid-binding protein (Ex-FABP) is another family member of fatty acid-binding protein, it regulates the metabolism of fatty acid, muscle fiber, bone and so on in chicken. Three single nucleotide polymorphisms(SNPs) were detected in 5′ regulatory region of Ex-FABP gene using PCR-SSCP and DNA sequencing in crossbreed chickens. The changes of the bonding-sites of transcription factor are due to a single point mutation T → C(－1011) and a C insertion mutation in genotype BB based on genotype AA, then results in a cap site lack, and increase four sites of a Nkx-2, a AhR/Ar and tow CF1. The relationship between SNPs and chicken meat traits was analyzed, the results showed that there was significant correlation (P<0.01) between genotype BB mutant and abdominal fat weight. The results of this study indicated that Ex-FABP gene is the main candidate gene that regulates abdominal fat traits in chicken.

Abstract:Microsatellite is a genetic marker, explored recently. In order to improve related studies on genetics of Anopheles gambiae, simple sequence repeats of the entire mosquito genome with 1~6 bp nucleotide motifs were analyzed. Abundance and distribution of microsatellites across the A.gambiae genome were analyzed and compared between various (exons, introns and intergenic) regions of all the chromosomes. About 2.14% of the mosquito genome was occupied by SSRs. Chromosome X had the maximum density of SSRs. Abundance of A repeats was similar to C repeats. AC was a little more than two times as abundant much as AG. However, AT and CG repeats were rare. For tri-and tetramer repeats, AGC, AAAC and AAAT predominated while ACG, ACT, AGG, CCG, ATGC, CCCG, ACTG, AACT, ACGT, AGAT, CCGG, ACCT and AGCT were rare. For some pentamer repeats, one was completely absent on a certain chromosome, even on several chromosomes. SSRs in exons of all chromosomes were less abundant than in introns and intergenic regions except for mono- and dimer repeats in exons of chromosome 2L. Abundance and distribution of SSRs on the two arms of each chromosome showed much in common.

Abstract:In order to comparatively analyze the core-fucosylated glycol-proteome expression profile of hepatocellular carcinoma cell lines (HCC) with different metastasis potentials, some key glycoprotein correlated with the metastasis of hepatoma were screened. Using SDS-PAGE, 2-DE followed by LCA lectin blot, differential display maps of core-fucosylated glycol-proteome were generated and analyzed, and then the glycoproteins were identified by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF-MS/MS). 1-DE lectin blot showed that MHCC97-H and MHCC97-L, which were established by authors' institute and had higher metastasis potentials than Hep3B, displayed numerous bands. From the core-fucosylated glycol-proteomics expression profiles, (55±7) spots in Hep3B (n=3), (60±6) spots in MHCC97-L(n=3), and (61±4) spots in MHCC97-H (n=3) were detected. Matched with individual 2-DE maps, the core-fucosylated glycol-proteomics expression profiles showed that there were (25±3) detected spots in Hep3B(n=3), (30±4) detected spots in MHCC97-L and (28±3) detected spots in MHCC-97H (n=3). There were 13 unmatched spots between the expression profile of Hep3B and MHCC97-L, and 9 unmatched spots between Hep3B and MHCC97-H. The 12 differential glycoproteins were identified by MALDI-TOF-MS/MS. Individually differential core-fucosylated glycoproteomics expression profiles existed in HCC with different metastasis potentials. All the results suggested that the metastasis of HCC might be correlated with these differential glycoproteins.

Abstract:The cellular processes in cells are controlled by protein-protein interactions (PPI), and comprehensive PPI maps are important to understand the complicated regulatory, metabolic and signaling pathways. Recently, new parameters for PPI prediction are under discovering. Here, a Na?ve Bayesian algorithm with the correlated Gene Ontology (GO) was used for PPI prediction. The characteristic pairs of GO terms was demonstrated by training a non-redundant PPI data set from two online budding yeast databases, and the probability about this two correlated GO terms was also obtained. The accuracy of the prediction was tested by both positive and negative control data. The approach can distinguish them properly, with a satisfied sensitivity and low false positive rate. After comparing the prediction result to the data derived from high-throughput experiments, it is proved that the method is more sensitive and efficient than other means. Furthermore, some new insightful knowledge about interactions of the proteins will be found using this prediction approach, and the prediction is very helpful to the laboratory experiments.

Abstract:To clarify the roles of nuclear localization signal (NLS) in inhibitory effect of HSP70 on H2O2-mediated nucleolar segregation, four eukaryotic vectors pcDNA3.1(-)-HSP70^WT (wild type), pcDNA3.1(-)-HSP70^ΔNLS (truncated mutant of HSP70 without NLS), pEGFP-N1-HSP70WT, and pEGFP-N1- HSP70^ΔNLS were constructed respectively. To mimic oxidative stress in vitro, final concentration of 1.0 mmol/L hydrogen peroxide (H²O²) was added into cultured C²C¹² myogenic cells. Toluidine blue staining showed predominantly compact, centrally localized nucleoli in intact control cells, but in H²O²-treated cells, an early onset of nucleolar segregation could be found after 3 h. The nucleolar segregation could be inhibited significantly in heat shock-pretreated cells and pcDNA3.1(-)-HSP70^WT-transfected cells. Fluorescence microscopy and immunoblotting for nucleolar protein showed that HSP70^WT was localized within cytoplasm in normal cells and redistributed to nucleus and nucleolus at 1 h after treatment with H^2O2. NLS deletion mutation of HSP70 (pcDNA3.1(-)-HSP70^ΔNLS) abolished H²O²-mediated translocation of HSP70 from cytoplasm to nucleus and nucleolus, and subsequently, its inhibitory effect on H²O²-mediated nucleolar impairment. It was suggested that wild type of HSP70 can attenuate significantly nucleolar segregation induced by oxidative stress. By mediating translocation of HSP70 from cytoplasm to nucleus and nucleolus, NLS played important role in the inhibitory effect of HSP70 against nucleolar impairment induced by oxidative stress.

Abstract:It is a difficult problem in molecular biology study of nasopharyngeal carcinoma(NPC) to obtain pureness tumor cells from tiny NPC tissue.To seek a convenient and applied way to obtain pureness and high quality RNA from NPC tissue for cDNA Microarray ， the study was designed to use RNAlater technology to keep the NPC dissected tissue and obtain pureness NPC cells by microdissection and gain RNA through RNA line amplification technology. The results hinted that RNAlater technology could keep the stability of NPC tissue and obtain the pureness and high quality RNA for cDNA Microarray combined with microdissection and RNA line amplification through optimum conditions.

Abstract:With the completion of genome-sequencing projects, intentional modification of definite bacterial artificial chromosomes (BACs), which carry entire components of most eukaryotic genes, is becoming more important for the subsequent functional genomics studies. The newly optimized Red/ET recombination system was applied to the BAC modification. Mediated by the plasmid, pSC101-BAD-gbaA, and assisted by the counter-selectable/selectable system conferred by rpsL-Neo, two BACs were successfully modified. One step selectable BAC modification such as simple deletion or insertion was achieved in one week. For insertion or fusion of unselectable gene such as Cre, EFGP and LacZ as well as point mutation into targeted BACs, two rounds of Red/ET recombination was proceeded and two weeks was needed. After L-arabinose induction for transient expression of redγ/redβ/redα/recA, the pSC101-BAD-gbaA plasmid died out spontaneously from the BAC host bacteria by shifting the temperature from 30℃ to 37℃ . Thus, there was no DNA contamination in the modified BACs being used for subsequent transgenesis research. The high efficient BAC modification mediated by optimized Red/ET recombination offers a significant facility to the functional genomics investigations.

Abstract:One novel tool and new methods used to punch the graft tissues and make the receptive blocks for constructing the tissue microarrays (TMAs) were devised. Two kinds of receptive blocks of TMAs consisting 390 and 448 spots of the graft tissues respectively were made successfully by the novel tool and new methods. These two kinds of TMAs sections had no loss of tissue spot. The H&E staining TMAs slides, which were observed under the light microscope, showed that the morphologic structure of all graft tissues had not be changed by punching, and the sections were appropriated thickness with symmetrical cells morphology. The positive expressions of P53 and P16 proteins in specimens of general sections were well consistent with that in TMAs sections by immunohistochemistry. The new tool and novel methods to punch the graft tissues and make the receptive blocks for constructing TMAs, which have many advantages such as low expense, easy operation, convenience and practicality, might be popular in future of the practical application in laboratory.