Expression level of augmenter of liver regeneration in patients with hepatic failure and hepatocellular carcinoma

BACKGROUND: Augmenter of liver regeneration (ALR) is an important polypeptide in the process of liver regeneration. This study aimed to determine the expression level of ALR in different liver diseases and its significance. METHODS: We prepared murine polyclonal antibody against ALR protein from Balb/C mice and purified the IgG fraction, which specifically combined to ALR protein as shown by Western blotting. Serum ALR levels in patients with hepatocellular carcinoma (HCC), hepatic failure (HF), chronic hepatitis B, and healthy persons were compared by ELISA. ALR mRNA expression levels in liver tissues in some of these patients were also compared by real-time RT-PCR. Immunohistochemical analysis was carried out on HF and HCC liver tissues. RESULTS: Different serum ALR levels foreshowed completely different prognoses in 18 HF patients. Higher ALR levels were noted in 6 improved patients (1613.5+/-369.6 pmol/ml) than in 12 deteriorating patients (462.3+/-235.8 pmol/m1). Similar levels were found in 20 HCC patients (917.9+/-332. 7 pmol/m1), 24 chronic hepatitis B patients (969.2+/-332.5 pmol/ml) and 10 healthy persons (806.9+/-240.8 pmol/ml). ALR mRNA levels in HCC liver tissues [10E6.24 (1.74x10(6)) copies/mu l] were much higher than in those of HF patients receiving orthotopic liver transplantation [10E3.45 (2.82x10(3)) copies/mu l] or in healthy liver tissues [10E4.31 (2.04x10(4)) copies/mu l]. In immunohistochemical analysis, positive immunostaining in HCC liver tissue was more intense than that in HF liver tissue. CONCLUSION: Serum ALR level is helpful in estimating the survival time of patients with HF, and ALR may play an important role in hepatocarcinogenesis.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Effect of the Flanking Sequence Architecture of Translation Initiation AUG Codon on Gene Expression Level in Rice

The relationship between the codon usage bias, gene expression level and the AUG context(from -20 to +6 positions relative to the initiator AUG codon) was examined in 541unigene sequences of rice. A significant correlation for CAI values (codon adaptationindex) was observed at five nucleotide positions (-19, -18, -9, -4, +5), eight (-19, -18,-14, -9, -6, -4, -1, +5) for CPP (codon preference parameter), and seven (-18, -16, -15,-9, -7, -1, +6) for mRNA abundance in the flanking sequence of the initiator AUG codonrespectively, but a significantly positive correlation for both CAI and CPP at twopositions (-4 and +5), indicating that both those positions are evolutionally under thenatural selection constraint at the translational level. By site-directed mutagenesis atseven specific positions (-18, -16, -15, -9, -7, -1 and +6) for allergenic protein thathad the highest mRNA abundance in this study, its expression level decreased dramatically63.3 and 72.5% respectively, indicating the importance of those 7 positions for geneexpression. A highly positive correlation (r=0.625, P<0.01) between AUGCAI and GCcontent in the flanking sequence of the initiator AUG codon showed a more effectivehigher GC content on translation initiation efficiency. The strong preference for G orC at those 8 positions (-6, -5, -3, -2, -1, +4, +5 and +6) in the AUG context suggestedthat an important factor in modulation of the translation efficiency, as well assynonymous codon usage bias, particularly in highly expressed genes.

High patatin like phospholipase domain containing 8 expression as a biomarker for poor prognosis of colorectal cancer

BACKGROUND Patatin like phospholipase domain containing 8(PNPLA8)has been shown to play a significant role in various cancer entities.Previous studies have focused on its roles as an antioxidant and in lipid peroxidation.However,the role of PNPLA8 in colorectal cancer(CRC)progression is unclear.AIM To explore the prognostic effects of PNPLA8 expression in CRC.METHODS A retrospective cohort containing 751 consecutive CRC patients was enrolled.PNPLA8 expression in tumor samples was evaluated by immunohistochemistry staining and semi-quantitated with immunoreactive scores.CRC patients were divided into high and low PNPLA8 expression groups based on the cut-off va-lues,which were calculated by X-tile software.The prognostic value of PNPLA8 was identified using univariate and multivariate Cox regression analysis.The over-all survival(OS)rates of CRC patients in the study cohort were compared with Kaplan-Meier analysis and Log-rank test.RESULTS PNPLA8 expression was significantly associated with distant metastases in our cohort(P=0.048).CRC patients with high PNPLA8 expression indicated poor OS(median OS=35.3,P=0.005).CRC patients with a higher PNPLA8 expression at either stage I and II or stage III and IV had statistically significant shorter OS.For patients with left-sided colon and rectal cancer,the survival curves of two PN-PLA8-expression groups showed statistically significant differences.Multivariate analysis also confirmed that high PNPLA8 expression was an independent prog-nostic factor for overall survival(hazard ratio HR=1.328,95%CI:1.016-1.734,P=0.038).

Natural Variation in the Sequence of SNAC1 and Its Expression Level Polymorphism in Rice Germplasms under Drought Stress

Water is a major limiting factor for food production and many countries fail to produce sufficient food for their population due to severe water scarcity （Jury and Vaux, 2005）. Rice is the main staple food worldwide. More than 50% of rice in the world is rain-fed and drought causes severe reduction in rice grain yield in rain-fed environments （Venuprasad et al., 2007; Zhang, 2007; Sandhu et al., 2014）. Therefore, enhancing drought resistance （DR） of rice is important for food security. However, DR is a complex trait, which is controlled by a large number of loci with small effect and is also affected by different genetic background, genotype-by-environment interaction and other stresses such as heat （Hu and Xiong, 2014）.

Expression of insulin-like growth factor-1 mRNA and protein level of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkeys

BACKGROUND： Insulin-like growth factor-I（IGF-1）, as one of the important members of growth factor family, participants in the regulation of many physiological functions and behaviors, having very strong neuroprotective effect. However, the expression of IGF-1 following cerebral ischemia/reperfusion is still disputed. OBJECTIVE： To observe the expression of IGF-1 and protein of corpora striata in ischemic side at the early stage of middle cerebral artery ischemia/reperfusion in rhesus monkey. DESIGN ： A completely randomized grouping design, controlled animal experiment SETTING ： Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University MATERIALS： ① Totally 17 rhesus monkeys , of either gender, aged 4 to 5 years, were enrolled . Seven rhesus monkeys observed with gene chip were randomly divided into 2 groups： sham operation group （n=3） and ischemia/reperfusion group 〈n=4〉. Ten rhesus monkeys observed with in situ hybridization and immunohistochemistry method were randomly divided into 2 groups： sham operation group 〈n=3 〉and ischemia/reperfusion group （n=7）. Rhesus monkeys observed under microscope were divided into 2 groups： sham operation group （n=6） and ischamia/reperfusion group （n=-11）.②Materials used in the experiment： cresyl violet （Sigma Company, America）; immunohistochemical reagent kit （ Huamei Bio-engineering Company）; In situ hybridization reagent kit （Boshide Bio-engineering Co.Ltd, Wuhan）; 12 800 dots chip （Boxing Company, Shanghai）. METHODS ： This experiment was carried out at the Institute of Cerebrovascular Disease, Affiliated Hospital of Medical College of Qingdao University from January 2001 to December 2003.① The onset area of middle cerebral artery was blocked for 2 hours, middle cerebral artery ischemia/reperfusion models were created.② After ischemia/reperfusion for 24 hours, cerebral tissue sections of rhesus monkeys were prepared and stained with cresyl violet. Image analysis was performed with 5001W image analysis software. Morphological change of corpora striata of operative side was observed in the rhesus monkeys between two groups. Total RNA was extracted from cerebral tissue. ③ Detection of gene chip： Cy3-duTP and Cy5-duTP were used to respectively perform reverse transcription labeling. The sample was reversely transcribed into cDNA, then hybridized with cDNA of cerebral tissue. Genes with the separate absolute value of cy3 and cy5〉800, cY3/cy5 〉 2（high expression） or 〈 0.5 （low expression） were found out. Those were genes with differential expression. ④ The expressions of IGF-1 mRNA and protein level of corpora striata in ischemic side of rhe- sus monkeys were detected between sham operation group and ischemia/reperfusion group at 9 and 24 hours after ischemia/reperfusion with in situ hybridization method and immunohistochemical method. Brown granules were IGF-1 protein positive cells. ⑤ Analysis of variance was used in the difference comparison of measurement data among groups. MAIN OUTCOME MEASURES ： ① Change of morphological structure of corpora striata at ischemic side in rhesus monkeys. ② Change of cerebral gene expression profiles at ischemia/reperfusion in rhesus monkeys between two groups.③ Expression of IGF-1 mRNA and protein level of corpora striata at ischemia/reperfu- sion in rhesus monkeys between two groups. RESULTS ： ① Pathological change ： Obvious pathological change of cerebral infarction appeared in the ischemia and reperfusion group, while there was no such pathological change in the sham operation group.② Change of gene expression profile ： There were 4480 genes with difference expression in the ischemia/reperfusion group and sham-operation group, in which, 260 genes had high expression and their absolute value was over 800, and 63 genes had low expression, cy3/cy5 of IGF-1 was 0.379, being relative low ex- pression. ③ IGF-1 mRNA and protein positive cell counts in corpora striata at cerebral ischemic side[IGF-1 mRNA： 〈9.72±1.18）,（9.11 ±0.76）,（14.77±0.60） counts/field：lGF-1 protein： （15.11 ±1.83）,（15.39±0.78）, （34.62±0.97）counts/field, P 〈 0.05-0.01]. CONCLUSION： IGF-1 mRNA and protein are lowly expressed in middle cerebral artery of rhesus monkeys at ischemia/reperfusion.

Effect of TGIF1 expression on epithelial cadherin and Twist1 protein expression in breast cancer cells

Objective: To observe the effect of the expression of transforming growth factor β-homologous inducible factor 1 (TGIF1) on the expression of epithelial cadherin (E-cadherin) and human Twist-related protein 1 (Twist1) in breast cancer cells. Methods: Total of twenty-four 6-week-old female SPF Balb/c mice were randomly divided into a control group, a TGIF1-silencing group, a TGIF1-normal group, and a TGIF1-overexpression group. In the TGIF1-silencing group, 4T1 breast cancer cells were interfered by lentivirus shRNA (H) lentiviral particles (sc-36659-v) to construct a breast cancer model. TGIF1-normal group used breast cancer cells (4T1) to construct a mouse model of breast cancer. And the TGIF1-overexpression group used 4T1 breast cancer cells with TGIF1 overexpression to establish a mouse model of breast cancer. Determination of TGIF1, E-cadherin and Twist1 protein levels in breast tumor tissue of mice in each group. Results: The tumor volume of mice in the TGIF1-overexpression group was significantly larger than that in the TGIF1-normal group and the TGIF1-silencing group, and the differences between the groups were statistically significant (P <0.05).The expression levels of TGIF1 and Twist1 protein in TGIF1-normal group, TGIF1-silencing group, and TGIF1-overexpression group were significantly higher than those in control group, and E-cadherin was significantly lower than that in control group. The differences between groups were statistically significant (P <0.05).The expression level of TGIF1 and Twist1 protein in TGIF1-silencing group was significantly lower than that in TGIF1-normal group, and E-cadherin was significantly higher than that in TGIF1-normal group (P < 0.05).The expression levels of TGIF1 and Twist1 proteins in the TGIF1-overexpression group were significantly higher than those in the TGIF1-normal group, and E-cadherin was significantly lower than that in the TGIF1-normal group. The differences between the groups were statistically significant (P <0.05). Pearson correlation analysis showed that the expression level of TGIF1 in breast cancer tissue was significantly negatively correlated with the expression level of E-cadherin protein, and was significantly positively correlated with the level of Twist1 protein (P <0.05). Conclusion: TGIF1 can affect the metastasis and invasion of breast cancer by regulating E-cadherin and Twist1 to interfere with the EMT pathway, which deserves further study.

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.

Molecular cloning, characterization, and antioxidant function of catalase in Lymantria dispar asiatic (Lepidoptera: Lymantriidae) under avermectin stress

The critical antioxidant catalase(CAT)breaks down hydrogen peroxide induced by environmental stresses.Here we cloned full length catalase cDNA from Lymantria dispar asiatic(LdCAT).Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa,theoretical pI of 5.00,aliphatic index of 29.92,grand average of hydropathicity of 0.764,and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis,LdCAT was most similar to CAT from Spodoptera litura and S.exigua.Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested,verifying the ubiquity of LdCAT in L.disapr.Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90,and in the cuticula,rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration.The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L.disapr defensing against pesticide stress and may be a good target for controlling this pest.

Cloning and characterization of geranylgeranyl diphosphate synthetase from Pinus massoniana and its correlation with resin productivity

Geranylgeranyl pyrophosphate synthetase(GGPPS) has gained increasing attention as a key enzyme in terpene analysis.We designed specific primers based on plant GGPPS homologs and used reverse transcription polymerase chain reaction(RT-PCR) to obtain and identify Pin GGPPS,a GGPPS gene sequence from Pinus massoniana,using bioinformatics tools.Quantitative PCR analysis of Pin GGPPS expression levels in roots,pine needles,immature stems,and semilignified stems from 6-month-old P.massoniana showed that expression levels of Pin GGPPS were highest in pine needles,followed by immature stems and semilignified stems,and lowest in roots.When we examined the correlation between Pin GGPPS gene expression levels and resin productivity in 20 adult plants for 28 successive days,Pin GGPPS expression levels presented a substantially linear distribution when plotted against their corresponding resin yields.In summary,we characterized the gene Pin GGPPS for the first time in P.massoniana,and established a correlation between Pin GGPPS gene expression levels and resin productivity,suggesting the importance of theory and production practice for P.massoniana.

High Level Expression of tcs Gene in E.coli

Trichosanthin is a valuable protein in medical applications. An NMR analytical of the trichosanthin solution conformation is made to study the structural-functional relationship of this protein. For preparing a sample labled with 15 N, the tcs gene was cloned into the expression vector pQE 30, and produced a high level expression of trichosanthin in the E coli strain M15. The amount of fusion TCS synthesized in E coli was about 6 4% of the total cellular protein. The fusion protein was purified by using affinity chromatography with Ni NTA resin.

3-D rheologic model of earthquake prepa-ration (III): Precursor field

On the basis of the theory of viscoelastic displacement and strain field for the three-dimensional rheologic model of earthquake preparation, this paper mainly studies the theoretical solution of precursor field for the three-dimensional rheologic model of earthquake preparation. We derive the viscoelastic analytical expressions of the ground tilt, underground water level, earth resistivity at an arbitrary point （x, y, z） in the rheologic medium, and analyzed the earth resistivity preliminarily, providing a certain theoretical basis for the precursor analysis of seismogenic process.

The effects of thyroid-stimulating hormone,estradiol and prolactin on sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels

Objective The present study investigated the sodium/iodide symporter mRNA expression in mouse lactating mammary gland cells under different iodine levels and the effects of thyroid-stimulating hormone(TSH),estradiol(E2)and prolactin(PRL)on NIS mRNA expression in mouse lactating mammary gland cells.

Expression patterns and functional divergence of homologous genes accompanied by polyploidization in cotton(Gossypium hirsutum L.)

Naturally allotetraploid cotton has been widely used as an ideal model to investigate gene expression remodeling as a consequence of polyploidization.However,the global gene pattern variation during early fiber development was unknown.In this study,through RNA-seq technology,we comprehensively investigated the expression patterns of homologous genes between allotetraploid cotton(G.hirsutum)and its diploid progenitors(G.arboreum and G.raimondii)at the fiber early development stage.In tetraploid cotton,genes showed expression level dominance(ELD)bias toward the A genome.This phenomenon was explained by the up-/downregulation of the homologs from the nondominant progenitor(D genome).Gene ontology(GO)enrichment results indicated that the ELD-A genes might be a prominent cause responsible for fiber property change through regulating the fatty acid biosynthesis/metabolism and microtubule procession,and the ELD-D genes might be involved in transcription regulation and stress inducement.In addition,the number and proportion of completely A-and D-subfunctionalized gene were similar at different fiber development stages.However,for neofunctionalization,the number and proportion of reactivated D-derived genes were greater than those of A at 3 and 5 DPA.Eventually,we found that some homologous genes belonging to several specific pathways might create novel asymmetric transcripts between two subgenomes during polyploidization and domestication process,further making the fiber property meet the human demands.Our study identified determinate pathways and their involved genes between allotetraploid cotton and their progenitors at early fiber development stages,providing new insights into the mechanism of cotton fiber evolution.

Making the Bread： Insights from Newly Synthesized AIIohexaploid Wheat

Bread wheat （or common wheat, Triticum aestivum） is an allohexaploid （AABBDD, 2n = 6x = 42） that arose by hybridization between a cultivated tetraploid wheat T. turgidum （AABB, 2n = 4x = 28） and the wild goatgrass Aegilops tauschfi （DD, 2n = 2x = 14）. Polyploidization provided niches for rigorous genome modification at cytogenetic, genetic, and epigenetic levels, rendering a broader spread than its progenitors. This review summarizes the latest advances in understanding gene regulation mechanisms in newly synthesized allo- hexaploid wheat and possible correlation with polyploid growth vigor and adaptation. Cytogenetic studies reveal persistent association of whole-chromosome aneuploidy with nascent allopolyploids, in contrast to the genetic stability in common wheat. Transcriptome analysis of the euploid wheat shows that small RNAs are driving forces for homoeo-allele expression regulation via genetic and epigenetic mechanisms. The ensuing non-additively expressed genes and those with expression level dominance to the respective pro- genitor may play distinct functions in growth vigor and adaptation in nascent allohexaploid wheat. Further genetic diploidization of allohexaploid wheat is not random. Regional asymmetrical gene distribution, rather than subgenome dominance, is observed in both synthetic and natural allohexaploid wheats. The combinatorial effects of diverged genomes, subsequent selection of specific gene categories, and subgenome-specific traits are essential for the successful establishment of common wheat.

Preventive effects of the polysaccharide of Larimichthys crocea swim bladder on carbon tetrachloride(CCl_4)-induced hepatic damage

The aim of the present study was to determine the preventive effects of the polysaccharide of Larimichthys crocea swim bladder(PLCSB) on CCl4-induced hepatic damage in ICR mice.The in vitro preventive effects of PLCSB on CCl4-induced liver cytotoxic effect were evaluated in BRL 3A rat liver cells using the MTT assay.The serum levels of AST,ALT,and LDH in mice were determined using commercially available kits.The levels of IL-6,IL-12,TNF-α,and IFN-γ were determined using ELISA kits.The pathological analysis of hepatic tissues was performed with H and E staining,and the gene and protein expressions were determined by RT-PCR and Western blotting,respectively.PLCSB(20 μg·m L-1) could increase the growth of BRL 3A rat liver cells treated with CCl4.The serum levels of AST,ALT,and LDH were significantly decreased when the mice were treated with two doses of PLCSB,compared with the control mice(P < 0.05).PLCSB-treated groups also showed reduced levels of the serum pro-inflammatory cytokines IL-6,IL-12,TNF-α,and IFN-γ.PLCSB could decrease the liver weight,compared to the CCl4-treated control mice.The histopathology sections of liver tissues in the 100 mg·kg-1 PLCSB group indicated that the animals were recovered well from CCl4 damage,but the 50 mg·kg-1 PLCSB group showed necrosis to a more serious extent.The 100 mg·kg-1 PLCSB group showed significantly decreased mR NA and protein expression levels of NF-κB,i NOS,and COX-2,and increased expression of IκB-α compared with the CCl4-treated control group.In conclusion,PLCSB prevented from CCl4-induced hepatic damage in vivo.

The upregulated intestinal folate transporters direct the uptake of ligand-modified nanoparticles for enhanced oral insulin delivery

Transporters are traditionally considered to transport small molecules rather than large-sized nanoparticles due to their small pores.In this study,we demonstrate that the upregulated intestinal transporter(PCFT),which reaches a maximum of 12.3-fold expression in the intestinal epithelial cells of diabetic rats,mediates the uptake of the folic acid-grafted nanoparticles(FNP).Specifically,the upregulated PCFT could exert its function to mediate the endocytosis of FNP and efficiently stimulate the traverse of FNP across enterocytes by the lysosome-evading pathway,Golgi-targeting pathway and basolateral exocytosis,featuring a high oral insulin bioavailability of 14.4%in the diabetic rats.Conversely,in cells with relatively low PCFT expression,the positive surface charge contributes to the cellular uptake of FNP,and FNP are mainly degraded in the lysosomes.Overall,we emphasize that the upregulated intestinal transporters could direct the uptake of ligand-modified nanoparticles by mediating the endocytosis and intracellular trafficking of ligand-modified nanoparticles via the transporter-mediated pathway.This study may also theoretically provide insightful guidelines for the rational design of transporter-targeted nanoparticles to achieve efficient drug delivery in diverse diseases.