Abstract:Sleep and memory are the basic function of the brain. A large number of studies from both humans and animals experiments have offered a substantive body of evidence supporting that sleep contributes crucially to memory consolidation. The processes of memory consolidation in hippocampus and cortex during sleep was reviewed and the primary cellular and molecular mechanism were briefly introduced.

Abstract:Methylation of histone by protein arginine methyltransferases (PRMTs) plays an important role in gene regulation. PRMT1- and PRMT4-catalyzed methyl-arginine is involved in transcription activation, while PRMT5- and PRMT6-catalyzed methyl-arginine is associated with transcription repression. Histone arginine methylation can be dynamically regulated in vivo, and methyl-arginine is demethylated by “arginine demethylase”. Here, the most recent progresses in the methylation studies of histone arginine were summarized.

Abstract:Tetraspanins belongs to the transmembrane 4 superfamily(TM4SF). They can be as a bridge to connect the proteins outside or inside the cell membrane. A tetraspanins web is formed by the tetraspnins-proteins complex, and the web is believed to involve in fundamental functions of immunity system, and consequnently, signaling between cells and inside cells, regulating cell activation and adhesion, participating in the identification and infection of some virus. As a family of conservative transmembrane proteins, tetraspanins play multiplex roles in invertebrate. It was described how tetraspanin microdomains might have functions in the immune system, and how they contact with virus. In addition, the important role of tetraspanins in the innate immune system of invertebrate were discussed.

Abstract:As tremendous genomic data avalanches, exploring biological mechanism by data analysis and theory methods has become important for theoretical biology research. This method is significant for the study of complex gene functions and gene networks. Bowers used higher order logic relationships to decipher protein network organization, which is a systemic method called logic analysis of phylogenetic profiles (LAPP). LAPP is a data modeling and different from traditional computational methods. This computational approach identifies logic relationships of the elements (or components) in complex networks through the logic analysis of their expression data. The method can be used to infer functional relationships of two associated proteins to one another. It is important for discovering the new function mechanism of the protein. The clusters of orthologous groups (COGs) involved in a gene network usually are large groups and therefore LAPP is also an approach for complex gene logic networks. After the establishment of the gene logic network, it is convenient for the regulation of gene through the network. The method can used in many fields, such as species evolution, oncologic diagnosis and so on. LAPP was systematically described and analyzed and recent developments in methodologies and applications were highlighted. Some opinions of them were also given.

Abstract:STIM1 is recognized as an ER Ca²⁺ sensor of calcium release-activated calcium (CRAC) channel that is constructed by membrane protein Orai1. However, this regulatory system may also be regulated by other proteins. Reticulocalbin 2 (RCN2) was purified and identified from STIM1-Orai1 complex. Confocal microscopy revealed that RCN2 co-localized with STIM1 in ER before and after Ca²⁺ store depletion. Single cell [Ca²⁺]_i measurements of RCN2 EF hands mutant showed slight influence on SOC electrophysiological characters. Furthermore, a novel collar form aggregation of RCN2 surrounding STIM1 clusters suggested that RCN2 potentially plays a role of structure maintenance in STIM1 clustering.

Abstract:Accurate prediction of the translation initiation site (TIS) is an important issue for prokaryotic genome annotation. However, it is still a challenge for the existing methods to predict the TIS in the genomes over a wide variety of GC content. Besides, the existing methods have not yet undergone a comprehensive evaluation, leaving prediction reliability as a largely open problem. A new algorithm MED-StartPlus, a tool that predicts TIS in prokaryotic genomes with a wide variety of GC content was presented. It makes several efforts to model the nucleotide composition bias, the regulatory motifs upstream of the TIS, the sequence patterns around the TIS, and the operon structure. Tests on hundreds of reliable data sets, with TISs confirmed by experiments or having annotated functions, show that the new method achieves a totally high accuracy of TIS prediction. Compared with existing TIS predictors, the method reports a totally higher performance, especially for genomes that are GC-rich or have complex initiation mechanisms. The potential application of the method to improve the TIS annotation deposited in the public database was also proposed.

Abstract:To investigate the mechanism of PI3K signaling-mediated hepatoma cell growth, specific PI3K inhibitor LY294002 were used to treat hepatocellular carcinoma cell line (SMMC-7721). LY294002 could inhibit cell proliferation of SMMC-7721. RT-PCR and Western blotting results showed that inhibition of PI3K signaling increased the protein expression of p27, but not mRNA expression. The protein expression of p27 could be inhibited by transfecting p27 SiRNA plasmid in LY294002-treated cells. And the knock-down of p27 protein expression could partly block the cell growth-inhibition induced by LY294002. Chx treatment experiment revealed that LY294002 prolonged the half-life of p27 protein, which increased its stability. In LY294002-treated cells, not only the mRNA expression of Skp2 (which is a critical molecule mediating the degradation of p27 protein) were reduced, but also the half-life of Skp2 protein were shorten. However, the activity alteration of Akt (an important downstream effector of PI3K signaling) by transfecting Akt constitutively active mutant and Akt dominant negative plasmid, did not influence the expression of p27. Taken together, these findings indicated that PI3K signaling regulated cell growth through modulating the degradation of p27 protein via Skp2 in SMMC-7721, however which was Akt independent.

Abstract:In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG₂ to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG₂ cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG₂/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG₂/GFP-Daxx cells). After incubation with hydrogen peroxide (H₂O₂) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG₂/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG₂ cells and observed that the hydrogen peroxide decreased the viability of HepG₂ cells in concentration-dependent pattern. The IC₅₀ values in three groups (Normal cells, HepG₂/GFP cells and HepG₂/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG₂/GFP-Daxx cells as compared to the other two groups. HepG₂/GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG₂ cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.

Abstract:The attenuated Chinese equine infectious anemia virus (EIAV) vaccine is the first lentiviral vaccine that provides solid protective immunities to vaccinated horses. To investigate properties of EIAV vaccine, especially the relationship between its replication and the immunity, viral plasma loads of an EIAV vaccine strain EIAV_FDDV in immune suppressed horses were detected. Three horses, which were immunized with EIAV_FDDV for 16 months, were treated with dexamethasone for 14 days to suppress their immunities. Reduced immune response in these animals was confirmed by significantly declined lymphocyte proliferation rate detected after 10 days of the drug treatment. The plasma viral loads of EIAV_FDDV, which was indicated by the genomic RNA copy numbers, in horses before and after the treatment of dexamethasone were monitored by real time RT-PCR. Results revealed that the viral plasma loads in two of three immune-suppressed horses were kept a steady low level around 10^{3～104 copies/ml. The load was increased by 10 folds in the third horse, but was still among the standard levels for EIAV_FDDV vaccinated horses. As a positive control, the viral copy number of an asymptomatic carrier of EIAV virulent strain EIAV_Liao was jumped nearly 25 000-fold higher after being treated with dexamethasone. The typical clinical symptoms of EIA, characterized by febrile episodes and thrombocytopenia, were also appeared in this horse. These results clearly indicate that it is the unique biological feature of the attenuated EIAV vaccine, but not the immunity, resulted in EIAV_FDDV remaining in low levels in the body harmlessly. In addition, the steady low level of viremia and the inability to cause clinical symptoms of EIAV_FDDV in immune-suppressed hosts further demonstrated the safety of attenuated Chinese EIAV vaccines. The data provide a new sight for studies on the immunity to lentiviruses.}

Abstract:Human tumor suppressor gene beclin 1 regulates cell growth through autophagy. The mRNA expression of beclin 1 was reported to be down-regulated in breast cancer with high frequency of loss of heterozygosity (LOH). However, there was no report about the expression levels or the regulatory mechanisms of beclin 1 in gastric and colorectal cancer. Both the mRNA and protein expression levels of beclin 1 was detected in the tissues of gastric and colorectal cancers, as well as the aberrant DNA methylation and LOH related to the expression of beclin 1. By comparing with normal tissues adjacent to the tissue of these tumors, it was found that beclin 1 mRNA expression levels were significantly decreased in gastric tumor tissue. Furtherly by explorating the 5′ region of beclin 1 gene sequence, a large and dense CpG island was discovered and meanwhile methylations in the promoter and the intron 2 regions of beclin 1 were found in both gastric and colorectal tumors. And LOH was found in gastric tumors. These findings suggested that aberrant DNA methylation, as well as LOH, were involved in the regulation of beclin 1 expression in gastric and colorectal cancer.

Abstract:Haematological traits, which consist of mainly three components: leukocyte traits, erythrocyte traits and platelet traits, play extremely important role in disease resistance. But knowledge of the genetic background controlling variability of these immune traits is very limited, especially in swine. 18 haematological traits including white blood cell count (WBC), neutrophilic granulocyte count (GRAN) and percentage (GR%), lymphocyte count (LYMF) and percentage (LY%), monocytes count (MONO) and percentage (MO%), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concerntration (MCHC), red blood cell volume distribution width (RDW), blood platelet counts (PLT), mean platelet volume (MPV), platelet distribution width (PDW), and plateletocrit (PCT) were measured in a pig resource population consisting of 368 piglets of three breeds (Landrace, Large White and Songliao Black Pig) distributed in 16 boar families, after inoculation with the swine fever vaccine at 21 days of old. A partial genome scan for mapping quantitative trait loci (QTL) for these traits was performed using 35 microsatellite markers on chromosomes 2, 7, and 8. Using variance component analysis based on a linear mixed model and likelihood ratio test based on a chi-square distribution with 2 degrees of freedom, 22 significant QTL (P < 5%) were identified: 9 on chromosome 2 affecting WBC, GRAN, MCV, HGB, MCHC, PLT, MPV, PDW and PCT, respectively; 7 on chromosome 7 affecting WBC, GRAN, MCHC, HGB, PLT, MCV and RDW, respectively; 6 on chromosome 8 affecting GR%, LY%, MCHC, PLT, PCT and MCV, respectively. In order to avoid the increase of false positive rate caused by multiple tests, the FDR (false discovery rate) control approach was adopted to further test these 22 QTL. The results indicated that 14 of them were significant at FDR < 5% level, of which 9 were significant at FDR<1% level.

Abstract:At present, the molecular mechanism underlying microgravity-induced muscular atrophy is still unknown, and gravisensing is the key point in this process. In order to answer these questions a research project of Caenorhabditis elegans (C. elegans) in spaceflight was carried out, which had been reported in this journal before. An environment of simulated microgravity on ground was established, and its major effects on body-wall muscles of C. elegans in the structures and functions were examined, which further confirmed the results from spaceflight studies, and comparing between these two different treatments was benefit for valuing the validity of simulated microgravity. Firstly, the survival rate and movement ability of C. elegans were observed, and five important muscle-related genes and three proteins were measured after 14 days 19.5 h rotation. The animals displayed reduced rates of movement with a lower ratio (height/width) in crawl trace wave in simulated microgravity, indicating a functional defect. In morphological observation deceased muscle fiber size in myosin immunofluorescence and duller dense-body staining were found in microgravity group, suggesting muscular atrophy had happened in C. elegans. Meantime the result of Western blotting showed the quantity of myosin A decreased significantly in simulated microgravity group, further confirming muscular atrophy. In genes transcription, it was noted that dys-1 increased significantly in body-wall muscles, while hlh-1, unc-54, myo-3 and egl-19 mRNA levels declined after rotation. This study provided evidence that dys-1 are involved in the transduction of mechanical information in skeletal muscle, potentially play a vital role in gravisensing. Genes of hlh-1, unc-54, myo-3 and egl-19 induced the muscular atrophy in simulated microgravity from the structures and functions ways respectively. Data of this study consolidated the results in our spaceflight researches. On the other hand, it is implied that simulated microgravity is an effective ways for improving the quality of space studies.

Abstract:Linoleic acid (C18∶2n-6) and α-linolenic acid (C18∶3n-3) are found widely in fungi, plants and some lower animals. However, they can not be synthesized in mammals due to lack of △12 and ω-3 fatty acid desaturases. To enable endogenous production of essential fatty acids in mammalian cells, here the stable expression of a Caenorhabditis elegans gene FAT-2 encoding △12 fatty acid desaturase in CHO cells was reported. First, the FAT-2 coding sequence was cloned by RT-PCR. To facilitate high level synthesis of heterogeneous protein, the codon usage of the fatty acid desaturase genes was optimized according to the codon preference of mouse by site-directed mutagenesis, 2 synonymous mutations were introduced into FAT-2 gene by overlapping PCR. The codon-modified gene was finally fused to pBudCE4.1 vector (Invitrogen) under the control of CMV promoter. The expression vector pBudCE-FAT2 was linearized with NheⅠ, and then transfected CHO cells, the cells were under Zeocin selection for nine days and then propagated, then the transfected cells were harvested. The genome and total RNA were isolated for PCR and Norhern blot ananlysis. The results revealed that FAT-2 gene has been integrated into the genome of CHO cells and expressed properly. Fatty acids of total cellular lipids were analyzed by gas chromatography. The results indicate that the expression and function of △-12 fatty acid desaturase resulted in accumulation of linoleic acid. The levels of linoleic acid in transgenic cells were 2.4-fold higher than those in wild-type cells. The moderate linoleic acid in CHO cells was derived from cell culture media uptaken by cell membrane. The results demonstrate that a heterogenous desaturase gene can function well in mammalian cells and prove that transgenic approach is an efficient strategy for changing fatty acid composition of mammals.

Abstract:To characterize a mimotop, peptide mimic to epitope on lipopolysaccharide (LPS) which was named as 13L and was expected to induce humoral immune response to LPS and a protective immunity , peptide 13L was synthesized and conjugated to Blue Carrier (BC) as immunogen. Balb/c mice were immunized with peptide 13L - BC conjugate and BC only as control. Anti-LPS antibodies of mice immunized with peptide 13L-BC were detected by ELISA using peptide 13L-BSA as coating antigen. Survival time of mice challenged with S. typhi, and dynamics of MAP/mmHg in mice injected with LPS were observed. The results showed that specific antibodies against S. typhi-LPS 7261 and E. coli-LPS 2630 were elicited in mice immunized with peptide 13L-BC conjugate, and could be boosted by inactive bacterial and LPS. The main isotype of anti-LPS antibodies were IgG2a, IgG2b and IgM, but less IgG1 and IgG3, The survival time of mice infected with S. typhi were (12±1.3) days and (5.3± 0.4) days(P < 0.01) in group immunized with peptide 13L conjugate and with BC, respectively. And the peptide 13L-BC can also elicit protective immunity against endotoxic shock by LPS. The dynamics of MAP/mmHg observed by carotid Artery catheterization showed a significant difference between mice immunized with peptide 13L-BC and mice immunized with BC only in 1, 2,3 and 4 hours after injection of LPS 2630(P < 0.05, P < 0.01, P < 0.05 and P < 0.01), and injection of LPS 7261(P > 0.05, P < 0.05, P < 0.05 and P < 0.01). These results suggested that the peptide 13L can mimic the antigenicity of LPS epitopes to induce secondary antibody response like as thymus-dependent antigen, and also can elicit a protective immunity of mice from infection with G- bacteria and endotoxic shock. This peptide mimics could be a new vaccine candidate of LPS.

Abstract:Four mutants from Oryza sativa mitochondria tRNA^Trp toward B． subtilis tRNA^Trp were constructed and transcribed in vitro with T7 RNA polymerase. The kinetic parameters (K_cat/K_M) of B． subtilis tryptophanyl-tRNA synthetase(TrpRS) and human TrpRS were determined with four mutant-type tRNA^Trps. Results showed that for reaction with B． subtilis TrpRS, C2/G71 and C4/G69 mutations each induced a comparable 40-fold and 53- fold of activity to Oryza sativa mitochondria tRNA^Trp respectively. Notably, when the C2/G71 and C4/G69 mutations were introduced together into B． subtilis tRNA^Trp, a 140-fold of reaction rate resulted, the catalytic efficiency was 34 percent as that of wild-type B． subtilis tRNA^Trp, but these four mutants resulted in a weak aminoacylation efficiency by human TrpRS, and the change was little. Clearly, the results indicate that C2/G71 and C4/G69 bases in the acceptor stem are important species-specific elements of Oryza sativa mitochondria tRNA^Trp, since which are significant to the aminoacryl activity.

Abstract:Osteonecrosis is a kind of common condition which affects severely health and life quality of human being . Without specific treatment 80% of clinically diagnosed cases will progress, and most will eventually require arthroplasty. The goal is therefore to diagnose and treat the condition in its earliest stage. Because the treatment of osteonecrosis is determined in large part by the stage of the disease, it is important to use a reliable and effective method of classification and staging. It is well known the treatment on early diagnosis. The traditional diagnosis methods for osteonecrosis are X-ray, CT, MRI and biopsy, which are defect in early diagnosis and side effect. Photoacoustic tomography is an emerging imaging technique with great potential for a wide range of animal tissues and organ imaging application. This new approach to medical imaging relies upon irradiating the tissue with low energy, nanosecond pulses of laser light at a wavelength in the visible or near-infrared (NIR). Broadband (～30MHz) ultrasonic thermoelastic waves are excited throughout the irradiated volume at optically absorbing subsurface features and propagate to the surface of the tissue. It is proposed and investigated to establish the feasibility of using photoacoustic tomography to detect the early osteonecrosis of human femoral head, and compared with X-ray photography. The osteonecrosis model of rabbit had been performed. On the other hand, the specimen of human femoral head complicated with necrosis had also been researched. The results show the subchondral osteolysis and local cortical bone destructed in animal model in the tenth week. The early osteonecrosis pathology of rabbit model was the same as human femoral head. The reconstructed photoacoustic images were identical with the scale and shape of the specimen. The images had satisfied contrast and resolution. The spatial resolution of the images reaches 0.3 mm. It is feasible that the technique of the photoacoustic tomography is used in the detection of early osteonecrosis. A new diagnosis method for the early stage osteonecrosis will be demonstrated.

Abstract:MicroRNAs are a class of important post-transcriptional regulators of gene expression in animals and plants. The intensive studies on differential expression and regulatory roles of microRNAs call for sensitive and specific method to detect trace amount of these small size, high sequence homological microRNAs. Here, a simple and reliable method for the quantification of microRNAs was presented. The hybridization products of target microRNAs with biotin-labeled capture probe and oligonucleotides-functioned gold nanoparticles probe were immobilized onto the surface of streptavidin-coated microplate, and the absorbance signals of gold nanoparticles were amplified by silver enhancement. Distribution of miR-122a/miR-128 in mouse brain and liver tissue were detected by this method, and then synthetic miRNA122a was quantified. Results show a lower detect limit of 10 fmol/L with a linear dynamic range from 10 pmol/L to 10 fmol/L and a high specificity to discriminate one single oligonucleotide mismatch of the target microRNAs.