Identification of consensus sequence from matrix attachment regions and functional analysis of its activity in stably transfected Chinese hamster ovary cells.

Matrix attachment regions (MARs) can enhance transgene expression levels and maintain stability. However, the consensus sequence from MARs and its functional analysis remains to be examined. Here, we assessed a possible consensus sequence from MARs and assessed its activity in stably transfected Chinese hamster ovary (CHO) cells. First, we analyzed the effects of 10 MARs on transfected CHO cells and then analyzed the consensus motifs from these MARs using a bioinformatics method. The consensus sequence was synthesized and cloned upstream or downstream of the eukaryotic vector. The constructs were transfected into CHO cells and the expression levels and stability of enhanced green fluorescent protein were detected by flow cytometry. The results indicated that eight of the ten MARs increased transgene expression in transfected CHO cells. Three consensus motifs were found after bioinformatics analyses. The consensus sequence tandemly enhanced transgene expression when it was inserted into the eukaryotic expression vector; the effect of the addition upstream was stronger than that downstream. Thus, we found a MAR consensus sequence that may regulate the MAR-mediated increase in transgene expression.

Molecular epidemiology investigation of beta-thalassemia in Zhongshan City, Guangdong Province, People's Republic of China.

Beta-THAlassemia (beta-thal) is one of the most common genetic diseases in southern China. In order to obtain detailed epidemiology data to be used for primary prevention programs, we have analyzed 2,055 independent subjects living in Zhongshan City, Guangdong Province, People's Republic of China (P.R. China), by using hematological biochemical screening and DNA technology. The results indicate a higher prevalence (3.07%) of beta-thal and coinheritance of alpha- and beta-thal (0.49%) than previously reported for Guandong Province. Ten beta-thal mutations were found in 63 independent chromosomes. The four most common mutations [codons 41/42 (-TCTT), IVS-II-654 (C>T), -28 (A>G), codon 17 (A>T)] accounted for 90.46% of the total. The uncommon mutations profile was different from that of other cities in Guangdong Province and the rare mutation IVS-II-2 (-T), once reported in Hong Kong, was also detected. This study will contribute to the development of prevention strategies in the region, allowing better genetic counseling and prenatal diagnosis of beta-thal.

A chimeric HS4 insulator-scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most commonly used expression systems for the production of recombinant proteins but low levels of transgene expression and transgene silencing are frequently encountered. Epigenetic regulatory elements such as the chicken ß-globin locus control region hypersensitive site 4 (HS4) and scaffold/matrix attachment regions (S/MARs) have positive effects on transgene expression. In this study, a chimeric HS4-SAR was cloned upstream or downstream of an enhanced green fluorescent protein (eGFP) expression cassette in a eukaryotic vector, and the resulting vectors were transfected into CHO cells. eGFP was detected by flow cytometry. Real-time quantitative PCR (qPCR) was used to determine copy numbers of the stably transfected cells. And fluorescence in situ hybridization (FISH) was used to detect the status of vector in the host cell chromosome. The results showed that HS4-SAR positioned downstream of the expression cassette could enhance eGFP expression by 4.83-fold compared with the control vector. There may not be a relationship between transgene copy number and gene expression level. HS4-SAR did not appear to alter the integration of the transgene into the host cell chromosome or its position in the chromosome. We found a synthetic chimeric HS4-SAR positively increased transgene expression in CHO cells.

Episomal Lentiviral Vector-Mediated miR-145 Overexpression Inhibits Proliferation and Induces Apoptosis of Human Esophageal Carcinomas Cells.

BACKGROUND: Gene therapy is a promising approach for the treatment of various cancers. However, most viral vectors used for this purpose carry risks, including potential integration into the host genome. OBJECTIVE: We addressed this issue in the present study by constructing an episomal lentiviral vector using the .-interferon matrix attachment region to express the microRNA -145(miR-145), and examining the effect of miR-145 overexpression on human esophageal carcinomas (EC) cells. Some recent relevant patents are also discussed. METHOD: Expression levels of miR-145 and the marker protein enhanced green fluorescent protein (EGFP) in infected ECA109 and EC9706 human esophageal carcinoma cells were detected by quantitative PCR and flow cytometry, respectively. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry, respectively. Plasmid rescue experiments and fluorescence in situ hybridization were used to determine the episomal status of the transfected vector. RESULTS: We found that EGFP and miR-145 were highly expressed in EC cells, and miR-145 overexpression inhibited cell proliferation and induced apoptosis. Moreover, the lentiviral vector did not integrate into the host genome, but was maintained episomally at lower copy numbers. CONCLUSION: Taken together, our results demonstrate that miR-145-expressing episomal lentiviral vectors are a promising tool for gene therapy in the treatment of EC.

Crosstalk among the proteome, lysine phosphorylation, and acetylation in romidepsin-treated colon cancer cells.

Romidepsin (FK228) is one of the most promising histone-deacetylase inhibitors due to its potent antitumor activity, and has been used as a practical option for cancer therapy. However, FK228-induced changes in protein modifications and the crosstalk between different modifications has not been reported. To better understand the underlying mechanisms of FK228-related cancer therapy, we investigated the acetylome, phosphorylation, and crosstalk between modification datasets in colon cancer cells treated with FK228 by using stable-isotope labeling with amino acids in cell culture and affinity enrichment, followed by high-resolution liquid chromatography tandem mass spectrometry analysis. In total, 2728 protein groups, 1175 lysine-acetylation sites, and 4119 lysine-phosphorylation sites were quantified. When the quantification ratio thresholds were set to > 2.0 and < 0.5, respectively, a total of 115 and 38 lysine-acetylation sites in 85 and 32 proteins were quantified as increased and decreased targets, respectively, and 889 and 370 lysine-phosphorylation sites in 599 and 289 proteins were quantified as increased and decreased targets, respectively. Furthermore, we identified 274 proteins exhibiting both acetylation and phosphorylation modifications. These findings indicated possible involvement of these proteins in FK228-related treatment of colon cancer, and provided insight for further analysis of their biological function.

A study on the inhibitory effect of Radix Semiaquilegiae extract on human hepatoma HEPG-2 and SMMC-7721 cells.

The main objective of this paper was to investigate the extraction process of ethanol extract of Radix Semiaquilegiae, as well as its inhibitory activity on human hepatoma HepG-2 and SMMC-7721 cells, and to compare the inhibitory effects of different concentrations of ethanol extracts against these two hepatoma cells. Ethanol reflux extraction and ultrasound-assisted extraction with ethanol at room temperature were used in the extraction process, and MTT assay was mainly used in the activity experiment to perform in-vitro anti HepG-2 and SMMC-7721 cell activity screening of ethanol extract, and to calculate the cell inhibition rates of the extracts. The results showed that among the two types of extracts, ethanol reflux extract had more superior antitumour activity to that of the ultrasonic extract, but all of the extracts obtained had certain anti-cancer activities, and the anti-proliferative activity increased with the increase of concentration.

[Matrine-induced erythroid differentiation of K562 cells is associated with activation of the apoptotic pathway].

OBJECTIVE: To observe matrine-induced erythroid differentiation of K562 cells in relation to activation of the apoptotic pathway in vitro. METHODS: K562 cells were cultured in the presence or absence of matrine at different concentrations for 4 days, and the morphological and ultramicrostructural changes of the cells were observed using inverted microscopy and transmission electron microscopy, respectively. The expression of apoptosis-related protein p27kip1 was detected by immunocytochemistry. RESULTS: Compared to untreated K562 cells, the cells treated with matrine at 0.10 g/L exhibited apoptostic characteristics in the cellular morphology and ultramicrostructure, with the expression of p27kip1 protein upregulated in a concentration- and time-dependent manner. CONCLUSION: Matrine-induced erythroid differentiation of K562 cells is associated with cell apoptosis, and upregulation of p27kip1 protein expression may play a crucial role in the process of apoptosis.

[Blood motilin concentration and enteral nutrition in premature infants].

OBJECTIVE: To study changes of plasma motilin concentration and it's effect on enteral nutrition in premature infants. METHODS: The plasma motilin concentration of 72 premature infants was measured within 12 hours after birth before enteral feeding and on day 3 and 7 of life by using radioimmunoassay. Sixteen full-term neonates were enrolled as controls. RESULTS: (1) The plasma concentrations of motilin in premature infants before enteral feeding after birth and on day 3 and 7 were 198.65 +/- 58.42 ng/L, 248.83 +/- 56.00 ng/L, and 376.77 +/- 139.46 ng/L, respectively, which were significantly lower than those in the control group (300.33 +/- 67.15 ng/L, 334.26 +/- 83.81 ng/L, 510.64 +/- 179.85 ng/L) (P < 0.001 or < 0.01). There was positive correlation between the concentration and gestational age, age in day and the volume of milk. On day 7 the level of motilin was higher than the pre-enteral feeding level of the full term control group. (2) The plasma motilin concentration in feeding un-tolerated premature infants group was lower than that in the normal group, especially on day 3 of life (P < 0.05). (3) Early enteral feeding could improve the plasma motilin levels, gastrointestinal motility and nutrition tolerance in premature infants. CONCLUSIONS: The gastrointestinal functions of premature infants are adaptable to enteral nutrition. Early enteral feeding (including minimal enteral nutrition and non-nutritive sucking) can promote adaptive rapid growth and development of intestine.