Expression of cocaine- and amphetamine-regulated transcript (CART) in hen ovary

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. METHODS: Small white follicles (1-2 mm in diameter) were treated for RNA isolation; Small white follicles (1-2 mm in diameter) and large white follicles (4-6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4-6 mm in diameter), small yellow follicles (6-8 mm in diameter), large yellow follicles (9-12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. RESULTS: The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6-8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4-6 mm follicles relative to follicles of other sizes (P < 0.05). CONCLUSIONS: Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.

A phage display combined with DNA affinity magnetic system can be applied to a screening of DNA binding proteins, such as transcription factors / Un fago pantalla combinado con un sistema de afinidad magnético al ADN se puede aplicar al exámen de las proteínas de unión al ADN, tales como los factores de transcripción

Here we introduce a new approach for the screening of DNA binding proteins, using a phage library based on a phage display technique. In principal, a complementary DNA (cDNA) library based on the recombinant bacteriophage T7 expressing target proteins on its capsid (phage display) is constructed. These phage particles are hybridized with a biotinylated target DNA fragment which is immobilized on the surface of streptavidin paramagnetic particle (SA-PMP). The phage particles are released from the target DNA fragment by a nuclease treatment and the recovered phages are used to the next round of hybridization. These processes are repeated three times to amplify the target phages in the population. This simple method is faster, and more systemic than other current methods (e.g. yeast one hybrid system). As a proof of this principle, we tried to isolate transcription factors which specifically bind to the promoter region of the Arabidopsis thaliana AtGST11 gene. Two obtained candidates, RING zinc finger protein and AtHB6, showed DNA binding activity to the AtGST11 promoter region. We could validate that our new application of phage display is a superior method for isolation of DNA binding proteins with a broad range of potential applications.

Molecular cloning and expression of human Gamma Interferon[IFN-Lambda] Full cDNA in Chinese Hamster Ovary [CHO] Cells

IFN-gamma is mostly secreted by activated CD4+, CD8+ T cells and NK cells. This cytokine has immunomodulatory, anti-cancer and anti-microbial effects and is important for prophylaxis, diagnosis and treatment of chronic infections and cancers. The purpose of this study was to clone the full cDNA of human IFN-gamma and express it on CHO cell line. Lymphocytes from a healthy individual were isolated and activated by phytohaemagglutinin [PHA] in vitro. After 4 hours, total RNA extracted and first cDNA str and was synthesized. cDNA was amplified with primers containing EcoRI and NotI sites. The amplified fragment and the PcDNA3.1 vector were cut by EcoRI and NotI and ligated. The construct [pcDNA3.1-IFN-gamma] was transferred into E.coli [strain: DH5 alpha] using CaCl2 method and selected by plating on a medium containing ampicillin. The construct sequence was confirmed by PCR and sequence analysis. Construct expression was achieved by performing a calcium phosphate-mediated transfection into CHO cells and followed by selection of stable drug [G418] resistant clones by limiting dilution assay [LDA]. The IFN-gamma production by transected CHO cells was measured using ELISA technique. Out of 33 grown transformed bacterial colonies, only 6 had the entire sequences of the insert and one of them was used for the transfection experiment. Out of 768 wells, 5 clones produced more than 100 ng/ml/10[6] cells of IFN-gamma. Among the 5 clones, one with the maximum production of INF-gamma [143 ng/ml/10[6] cells] was selected and used for propagation

Identification of differentially expressed cDNAs in Acanthamoeba culbertsoni after mouse brain passage

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from 5% to 70%. Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.

Characterization of recombinant antibodies developed for capturing enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli O157:H7, an emerging cause of food-borne disease with the occurrence of an estimated 20,000 illnesses and 250 deaths each year in the United States, has now been reported from several countries worldwide. Infections with this bacteria, which follows the ingestion of contaminated food by humans, causes bloody diarrhea, hemolytic uremic syndrome (HUS), and renal disease, that can have serious health implications. The source of food contamination is usually associated with animals, mainly cattle. Many cattle become infected early in life when they are exposed to an environment that is contaminated by other animals shedding the organisms in their feces. Detection of E. coli O157:H7 in feces or contaminated food samples requires tests with high sensitivity, which is increased by the use of monoclonal antibodies. However, the production of concentrated monoclonal antibodies in ascites raises animal welfare concerns, and can be expensive. In this study, single chain of variable fragment (scFv) molecules were developed from hybridoma clones that produce immunoglobulins specific for the LPS and flagella antigen of E. coli O157:H7 using phage display technology. The reactivity of the soluble scFv for their respective antigens was preserved in ELISA and by partial inhibition of bacterial agglutination with polyclonal antiserum. Furthermore, the scFv were able to capture E. coli O157:H7 bacteria demonstrating their potential use in diagnostic assays.

Construction and significance of directional expression cDNA library from human NB4 cells / 华中科技大学学报（医学）（英德文版）

Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.

Comparative nucleotide and deduced amino acid sequence of the envelope glycoprotein gene among three dengue virus type 2 strains isolated from patients with different disease severities in Maha Sarakham, northeast Thailand.

The nucleotide (nt) sequence of the envelope glycoprotein (E) gene of dengue virus type 2 was determined by the primer-extension dideoxy chain-termination method for 3 dengue virus type 2 (D2) strains which had been isolated from patients with dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), in Maha Sarakham, Northeast Thailand, in 1986-1987. Their nt sequences were essentially the same except for a single silent nt replacement in each DHF and DSS strain compared with DF strain. Therefore, these 3 strains possessed identical deduced amino acid (AA) sequences in their E protein. The result indicated that the primary structure of the E protein of D2 virus is not related to the clinical severity of the infected patients. Eleven nt replacements which resulted in 4 amino acid replacements were found to be unique to these 3 Northeast Thai strains. Sequence similarity showed that the 3 Northeast Thai strains were closest to the DSS isolate (H) followed by the DHF isolate (D) identified in Bangkok in 1980.