Inhibitory effects of Shuanghuanglian injection on nuclear factor-kappa B expression in mice with viral encephalitis in a time-and dose-dependent manner

Previous studies have confirmed that the anti-virus effects of Shuanghuanglian injection may be associated with nuclear factor-kappa B activity. This study observed nuclear factor-kappa B expression in mice with viral encephalitis, and showed significant decreases in nuclear factor-kappa B protein and mRNA levels following Shuanghuanglian injection. The inhibitory effect was more significant with prolonged intervention duration and increased treatment dose. These findings verify that Shuanghuanglian injection plays a therapeutic role in viral encephalitis by reducing expression of nuclear factor-kappa B in a time- and dose-dependent manner.

MicroRNA-regulated viral vectors for gene therapy

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

Fibrinogen-like protein 2 fibroleukin expression and its correlation with disease progression in murine hepatitis virus type 3-induced fulminant hepatitis and in patients with severe viral hepatitis B

AIM: To evaluate the expression of fibrinogenlike protein 2 (fgl2) and its correlation with disease progression in both mice and patients with severe viral hepatitis.METHODS: Balb/cJ or A/J mice were infected intraperitoneally (ip) with 100 PFU of murine hepatitis virus type 3 (MHV-3), liver and serum were harvested at 24, 48, and 72 h post infection for further use. Liver tissues were obtained from 23 patients with severe acute chronic (AOC) hepatitis B and 13 patients with mild chronic hepatitis B. Fourteen patients with mild chronic hepatitis B with cirrhosis and 4 liver donors served as normal controls. In addition, peripheral blood mononuclear cells (PBMC) were isolated from 30 patients (unpaired) with severe AOC hepatitis B and 10 healthy volunteers as controls. Procoagulant activity representing functional prothrombinaseactivity in PBMC and white blood cells was also assayed. A polyclonal antibody against fgl2 was used to detect the expression of both mouse and human fgl2 protein in liver samples as well as in PBMC by immunohistochemistry staining in a separate set of studies. Alanine aminotransferase (ALT) and total bilirubin (TBil) in serum were measured to assess the severity of liver injury.RESULTS: Histological changes were found in liver sections 12-24 h post MHV-3 infection in Balb/cJ mice.In association with changes in liver histology, marked elevations in serum ALT and TBil were observed. Mouse fgl2 (mfgl2) protein was detected in the endothelium of intrahepatic veins and hepatic sinusoids within the liver 24 h after MHV-3 infection. Liver tissues from the patients with severe AOC hepatitis B had classical pathological features of acute necroinflammation. Human fgl2 (hfgl2)was detected in 21 of 23 patients (91.30%)with severe AOC hepatitis B, while only 1 of 13 patients(7.69%) with mild chronic hepatitis B and cirrhosis had hfgl2 mRNA or protein expression. Twenty-eight of thirty patients (93.33%) with severe AOC hepatitis B and 1of 10 with mild chronic hepatitis B had detectable hfgl2expression in PBMC. No hfgl2 expression was found either in the liver tissue or in the PBMC from normal donors. There was a positive correlation between hfgl2expression and the severity of the liver disease as indicated by the levels of TBil. PCA significantly increased in PBMC in patients with severe AOC hepatitis B.CONCLUSION: The molecular and cellular results reported here in both mice and patients with severe viral hepatitis suggest that virus-induced hfgl2prothrombinase/fibroleukin expression and the coagulation activity associated with the encoded fgl2protein play a pivotal role in initiating severe hepatitis.The measurement of hfgl2/fibroleukin expression in PBMC may serve as a useful marker to monitor the severity of AOC hepatitis B and a target for therapeutic intervention.

Sequencing of PCR amplified HBV DNA pre-c and c regions in 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence

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Epigenetic changes in the regulation of carotenoid metabolism during honeysuckle flower development

Flower development is one of the most vital pathways in plant development, during which the epigenetic regulation of gene expression is essential. DNA methylation, the most conserved epigenetic modification, participates in gene expression regulation and transposable element silencing. Honeysuckle(Lonicera japonica) is an important medicinal plant renowned for its colorful and fragrant flowers. Honeysuckle flowers change color from white to gold as a result of carotenoid accumulation during development. However, the role of DNA methylation in flower color changes is not well understood in L. japonica. Here, we performed whole-genome bisulfite sequencing and transcriptome sequencing during flowering development in honeysuckle. The results showed that a decrease in the levels of genome-wide average DNA methylation during flower development and changes in DNA methylation were associated with the expression of demethylase genes. Moreover, many genes involved in carotenoid biosynthesis and degradation, such as Lj PSY1, LjPDS1, LjLCYE, and LjCCD4, have altered expression levels because of hypomethylation, indicating that DNA methylation plays an important role in flower color changes in honeysuckle. Taken together, our data provide epigenetic insights into flower development and color change in honeysuckles.

Polyploid Gene Expression and Regulation in Polysomic Polyploids

Polyploidization is one of the most crucial pathways in introducing speciation and broadening biodiversity, especially in the Plant Kingdom. Although the majority of studies have focused only on allopolyploid or disomic polyploids, polysomic polyploid species have occurred frequently in higher plants. Due to the occurrence of the capabilities of more copies of alleles in a locus which can have additive dosage effects and/or allelic interactions, polysomic polyploids can lead to unique gene regulations to silence or adjust the expression level to create variations in organ size, metabolic products, and abiotic stress tolerance and biotic stress resistance, etc. This review aims to comprehensively summarize the contemporary understanding and findings concerning the molecular mechanisms of gene expression as well as gene regulation in natural typed and resynthesized polysomic polyploid plants. The review investigates the molecular level of phenomena in polysomic polyploid plants such as 1) typically enlarging organ size and stabilizing meiosis, 2) increasing phytochemical content and metabolic products, 3) enhancing the ability to adapt with biotic and abiotic stress, and 4) changing in gene regulation to silence or adjust the expression levels involve in sequence elimination, methylation, gene suppression, subfunctionalization, neo-functionalization, and transposon activation.

Expression Regulation of Starch and Storage Protein Synthesis Related Genes in Rice Grains

Starch and the storage proteins are the main nutritious substances in crop grains,and their composition and content in grains play a decisive role in the grain quality of rice and other staple food crops.This review has mainly summarized the new advances in the expression regulation of starch and storage protein synthesis related genes in rice grains.Moreover,the challenges of the starch and storage protein synthesis substances in rice genetic improvement were also discussed.This review will provide important information for genetic improvement of grain quality in rice and,potentially,other staple cereals.

DIFFERENCES IN EXPRESSION AND REGULATION BETWEEN TRANSFORMED CELLS OF THE HUMAN GASTRIC CARCINOMA ONCOGENE Ha-ras AND THE UNTRANSFORMED PARENT CELLS

An Ha-ras oncogene was isolated from a cell line of gastric carcinoma called BGE-823 in order to elucidate genetic control and the influence of DNA sequences. The oncogene was cloned and identified as a single nucleotide substitution of thymine for guanine in the 12th codon through the sequencing of its first axon. We compared the differences of expression and regulation between the transformed Ha-ras cells and untransformed parent cells. Data indicated that the expression of Ha-ras in the transformed cells was five-fold higher than in the untransformed cells and that the Ha-ras gene in the former was hypersensitive toward DNase I. In addition, a nuclear protein of 35 kilodaltons bound strongly to the 2.5 Kb fragment located upstream of the 6.6 Kb Ha-ras gene and contained a CC rich region. These results suggest that there might be another mechanism of activation for the ras gene besides point mutation.

Cloning and expression of core gene cDNA of Chinese hepatitis C virus in cosmid pTM3

AIM To clone core gene cDNA of Chinesehepatitis C virus(HCV)into eukaryoticexpression vector cosmid pTM3 and to expressHCV core antigen in HepG2 cells.METHODS Core gene cDNA of HCV wasintroduced into eukaryotic expression vectorcosmid pTM3.Using vaccinia virus/bacteriophage T7 hybrid expression system,HepG2 cells were transfected with therecombinant plasmid pTM3-Q534 by lipofectin.RESULTS From the transfected bacteriaTop10F’,2 pTM3-Q534 clones containing therecombinant plasmid were identified fromrandomly selected 10 ampicillin-resistantcolonies.By reverse transcription PCR andindirect immunofluorescence technique,HCVRNA and core protein was identified in HepG2cells transfected with the recombinant plasmid.CONCLUSION The construction of arecombinant plasmid and the expression of coregene cDNA of HCV in HepG2 was successful.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells

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HBeAg gene expression with baculovirus vector in silk worm cells

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Roles and regulation of bone morphogenetic protein-7 in kidney development and diseases

The gene encoding bone morphogenetic protein-7(BMP7) is expressed in the developing kidney in embryos and also in the mature organ in adults. During kidney development, expression of BMP7 is essential to determine the final number of nephrons in and proper size of the organ. The secreted BMP7 acts on the nephron progenitor cells to exert its dual functions: To maintain and expand the progenitor population and to provide them with competence to respond to differentiation cues, each relying on distinct signaling pathways. Intriguingly, in the adult organ, BMP7 has been implicated in protection against and regeneration from injury. Exogenous administration of recombinant BMP7 to animal models of kidney diseases has shown promising effects in counteracting inflammation, apoptosis and fibrosis evoked upon injury. Although the expression pattern of BMP7 has been well described, the mechanisms by which it is regulated have remained elusive and the processes by which the secretion sites of BMP7 impinge upon its functions in kidney development and diseases have not yet been assessed. Understanding the regulatory mechanisms will pave the way towards gaining better insight into the roles of BMP7, and to achieving desired control of the gene expression as a therapeutic strategy for kidney diseases.

Identification and Expression Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Peanut (Arachis hypogaea L.)

Phosphoenolpyruvate carboxylase （PEPC） is widely distributed in plants and bacteria, and catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate. To investigate the molecular mechanisms of the regulation and control of peanut oil, with the degenerated primers and RACE-PCR approach, five PEPC genes were cloned from peanut, and designated as AhPEPC1, AhPEPC2, AhPEPC3, AhPEPC4, and AhPEPC5, respectively. The structure and phylogenetic analysis of PEPC protein indicated that AhPEPC1-4 genes encoded a typical plant-type PEPC-enzyme, and AhPEPC5 a bacterial-type. By real-time quantitative RT-PCR approach the expression pattern of each gene was detected in various tissues of normal and high oil-content peanut varieties. It was found that there was a lower expression level of AhPEPCs genes except for the AhPEPC2 in high-oil peanut than normal-oil peanut line. The results provide some fundamental information for the further investigation of plant PEPC proteins and their role in regulation of oil-content in peanut seeds.

Effect on Survivin Regulation of Transcription Level by p21^(waf1) Overexpression in HepG2 Hepatocellular Carcinoma Cells

The effect of cyclin-dependent kinase inhibitors Cip1/Wafl （p21） on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin （DOX） was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction （RQ-PCR）. Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction （RT-PCR） was used to measure the levels of E2F-1 and p300. The results showed that： （1） After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; （2） After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; （3） Overexpressed p21 resulted in GI/G0 phase arrest （F=31.59, P〈0.01）, meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls （FE2F-1=I25.28, P〈0.05; Fp300= 46.01, P〈0.01）. It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Molecular characterization and expression analysis of three homoeologous Ta14S genes encoding 14-3-3 proteins in wheat(Triticum aestivum L.)

The purpose of this study was to characterize Ta14 S homoeologs and assess their functions in wheat seed development.The genomic and c DNA sequences of three Ta14 S homoeologous genes encoding 14-3-3 proteins were isolated.Sequence analysis revealed that the three homoeologs consisted of five exons and four introns and were very highly conserved in the coding regions and in exon/intron structure,whereas the c DNA sequences were variable in the 5′ and 3′-UTR.The three genes,designated as Ta14S-2A,Ta14S-2B and Ta14S-2D,were located in homoeologous group 2 chromosomes.The polypeptide chains of the three Ta14 S genes were highly similar.These genes were most homologous to Hv14 A from barley.Real-time quantitative PCR indicated that the three Ta14 S genes were differentially expressed in different organs at different developmental stages and all exhibited greater expression in primary roots of 1-day-old germlings than in other tissues.Comparison of the expression patterns of the three homoeologous genes at different times after pollination also revealed that their expression was developmentally regulated.The transcription of Ta14S-2B was clearly higher during seed germination,whereas expressions of Ta14S-2A and Ta14S-2D were up-regulated at the beginning of seed imbibition(0–12 h),but declined thereafter.The results suggested that the three Ta14 S homoeologous genes have regulatory roles in seed development and germination.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control f gene expression in the gynogenetic fish embryogenesis.

Differential expression of a novel colorectal cancer differentiation-related gene in colorectal cancer

AIM To investigate SBA2 expression in CRC cell lines snd surgical specimens of CRC and sutologous healthy mucosa.METHODS Reverse transcription-polymerase chain reaction (RT-PCR) was used for relative quantification of SBA2 mRNA levels in 4 human CRC cell lines with different grades of differentiation and 30 clinical samples.Normalization of the results was achieved by simultaneous amplification of β-actin as an internal control.RESULTS In the exponential range of amplification, fairly good linearity demonstrated identical amplification efficiency for SBA2 and β-actin (82%). Markedly lower levels of SBA2 mRNA were detectable in tumors, as compared with the coupled normal counterparts ( P ＜ 0.01 ). SBA2 expression was significantly (0.01 ＜ P ＜0.05) correlated with the grade of differentiation in CRC,with relatively higher levels in well-differentiated samples and lower in poorly-differentiated cases. Of the 9 cases with lymph nodes affected, 78% (7/9) had reduced SBA2 mRNA expression in contrast to 24% (5/21) in nonmetastasis samples (0.01 ＜ P ＜ 0.05 ).CONCLUSION SBA2 gene might be a promising novel biomarker of cell differentiation in colorectal cancer and its biological features need further studies.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells ( HSC).``METHODS The expressions of MMP-2, tissue inhibitor of mjatrix metalloproteinese-2 ( TIMP-2 ) and membrane type matrix metalloproteiness-l (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supematant was revealed by zymography.``RESULTS In the situation of hypoxia for 12 h, the expression of MMP-2 protein wss enhenced (hypoxiagroup positive indexes: 5.7 ± 2.0, n = 10; control: 3.2 ±1 .0. n -- 7; P＜0.05). while TIMP-2 protein wss decreased in HSC ( hypoxia group positive indexes: 2.5 ± 0.7, n =10: control: 3.6 ± 1.0, n = 7; P＜O.05), and the activity ( total A) of MMP-2 in supematant declined obviously (hypoxia group: 7.334 ± 1.922, n = 9; control: 17.277 ±7.424. n= 11; P＜0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6 h group. The highest value (Ahypoxia-Acontrol) ofthe protein and the most intense signal of mRNA were in the period of hypoxia for 6 h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12 h,the contonts (A450) of MMP-2 and TIMP-2 in supernatant were both higher then those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 _+ 0.0144, n = 16;control: 0.0219 ± 0.0098, n = 14; P＜0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 _+ 0.771,n: 14; control: 4.304 +_ 1 .083, n = 12; P＜0.05), and the expression of MT1-MMP was increased.``CONCLUSION HSC Js sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Epigenetic regulation of stemness maintenance in the neurogenic niches

In the adult mouse brain, the subventricular zone lining the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are two zones that contain neural stem cells(NSCs) with the capacity to give rise to neurons and glia during the entire life of the animal. Spatial and temporal regulation of gene expression in the NSCs populationis established and maintained by the coordinated interaction between transcription factors and epigenetic regulators which control stem cell fate. Epigenetic mechanisms are heritable alterations in genome function that do not involve changes in DNA sequence itself but that modulate gene expression, acting as mediators between the environment and the genome. At the molecular level, those epigenetic mechanisms comprise chemical modifications of DNA such as methylation, hydroxymethylation and histone modifications needed for the maintenance of NSC identity. Genomic imprinting is another normal epigenetic process leading to parentalspecific expression of a gene, known to be implicated in the control of gene dosage in the neurogenic niches. The generation of induced pluripotent stem cells from NSCs by expression of defined transcription factors, provide key insights into fundamental principles of stem cell biology. Epigenetic modifications can also occur during reprogramming of NSCs to pluripotency and a better understanding of this process will help to elucidate the mechanisms required for stem cell maintenance. This review takes advantage of recent studies from the epigenetic field to report knowledge regarding the mechanisms of stemness maintenance of neural stem cells in the neurogenic niches.

Design and Implementation of Visual Dynamic Display Software of Gene Expression Based on GTK

The paper presented an implement method for a dynamic gene expression display software based on the GTK. This method established the dynamic presentation system of gene expression which according to gene expression data from gene chip hybridize at different time, adopted a linearity combination model and Pearson correlation coefficient algorithm. The system described the gene expression changes in graphic form, the gene expression changes with time and the changes in characteristics of the gene expression, also the changes in relations of the gene expression and regulation relationships among genes. The system also provided an integrated platform for analysis on gene chips data, especially for the research on the network ofgene regulation.