The ontogeny and distribution of gastrin- and serotonin-immunoreactive (IR) cell in the proventriculus of chicks (Gallus gallus domestica, n = 60) in different growth periods was examined immunohistochemically using antisera specific to gastrin and serotonin. Gastrin and serotonin-IR cells were detected in chick proventriculus. Gastrin-IR cells were first evident after 12 days of incubation in lamina epithelialis and compound glands, while serotonin-IR cells were observed only in compound glands at that same time. Gastrin-IR and serotonin-IR cells increased in frequency on incubation day 14 and 16, respectively. Towards the end of incubation, gastrin- and serotonin-IR cell numbers decreased. In adult chicken, both IR cells were present but not lower numbers. The observations demonstrate the presence of gastrin- and serotonin-IR cells in the proventriculus of developing chicks in temporally changing frequencies.
Animals ; Chick Embryo/*metabolism ; Endocrine Cells/cytology/metabolism ; Gastrins/*metabolism ; Gene Expression Regulation, Developmental/physiology ; Proventriculus/*embryology/*metabolism ; Serotonin/*metabolism

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca2+, suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca2+/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca2+ plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca2+/phospholipiddependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.
Amine Oxidoreductases/*metabolism ; Animals ; Cell Line ; Chick Embryo ; Female ; Hamsters ; Mice ; Phosphorylation ; Protein Binding ; Protein Kinase C/antagonists & inhibitors/*metabolism

OBJECTIVEConotruncal defects (CTD) is a common type of cyanotic congenital heart defects. It was shown that CTD might be produced by ablation of cardiac neural crest during early stage embryos in experimental studies. There were many kinds of genes involved and cardiac alpha-actin (CAA) was one of them. The purpose of this study was to investigate the spatial expression of CAA gene during embryonic heart development, and to explore its role in the pathogenesis of congenital heart defects.

METHODSExperimental chicken embryos were subjected to cardiac neural crest ablation by using electric stimulation at 36-40 hr incubation stage. The embryos and organs of experimental and normal control groups during different developing periods were taken out, RNA was extracted by Trizol, and then reverse transcription was done. The dynamic changes of CAA mRNA expression at different development stages of embryos were assayed by fluorescent real-time PCR. The embryos and tissues at different stages were taken out, and paraffin sections were made. The localizations of CAA antibody expression in the developmental embryos were detected by immunohistochemical analysis (peroxidase-DAB coloration).

RESULTS(1) The expression of CAA gene was detected at early embryo development, and increased subsequently to a stable level. Its expression was mainly limited to heart site, and could be increased along with the maturation of the cardiac muscle. There was no expression or little trace expression in liver, brain, and stomach. (2) There was a significantly low level of CAA gene expression on days 2-7 of chicken embryos whose cardiac neural crest were ablated in comparison with normal controls (P = 0.013). The level of CAA gene expression was also down-regulated on days 7, 9 and 15 of embryonic cardiac tissues (P = 0.029).

CONCLUSIONCAA gene is closely associated with heart development, its expression was adjusted by cardiac neural crest, and its dysfunction may be an important factor leading to congenital heart defects.

Actins ; genetics ; metabolism ; Animals ; Chick Embryo ; Gene Expression Regulation, Developmental ; Heart ; embryology ; Heart Defects, Congenital ; embryology ; Neural Crest ; embryology

The resistance effect on Newcastle disease virus (NDV) and Infectious Bursal Disease Virus(IBDV) in vitro of a new antimicrobial substance (AS), which produced by a Bacillus subtilis strain named B. subtilis fmbJ. Results showed that the TD50 and TD0 value of this AS on Chicken Embryo Fibroblasts cell (CEF) were 128.95mg/L and 25.79mg/L, respectively. This AS could strongly inhibit the cytopathic effects of cell induced by NDV as well as IBDV, and increase the survival rate of cell remarkably. This AS could inhibit the function of NDV and IBDV, and it could defend against the infection and inhibit multiplication of NDV and IBDV, and the effect was the same as the antiviral medicine Ribavirin. It had lower toxicity to CEF cell, therefore we would study it further that it was as antiviral medicine.
Animals ; Antiviral Agents ; metabolism ; toxicity ; Bacillus subtilis ; metabolism ; Chick Embryo ; cytology ; Fibroblasts ; cytology ; drug effects ; Infectious bursal disease virus ; drug effects ; Newcastle disease virus ; drug effects

No abstract available.
Animal ; Cattle ; Chick Embryo ; Comparative Study ; Human ; Methylation ; Protein Methyltransferases/metabolism* ; Proteins/metabolism* ; Rats ; Species Specificity ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S.

OBJECTIVETo investigate the effect of small interference RNA (siRNA) targeting nuclear factor-kappaB (NF-kappaB) on endometriosis.

METHODThe eutopic endometrium of women with endometriosis were transplanted into the nonvascular region of 8-day-old chicken embryo chorioallantocic membrane (CAM), and the effects of NF-kappaB p65 siRNA on the vascularization and endometriotic lesion formation were tested with proper controls.

RESULTSTransplantation of the endometrium onto the CAM resulted in a strong angiogenic response in the chicken tissue. The angiogenesis was significantly reduced and endometriotic lesion formation significantly suppressed with siRNA targeting NF-kappaB in comparison with the control group.

CONCLUSIONSThe NF-kappaB pathway is involved in the development of endometriotic lesions in vitro, and NF-kappaB gene silencing reduces endometriotic angiogenesis and promotes cell apoptosis in the endometriotic lesions, suggesting that NF-kappaB might be a good target for endometriosis treatment.

Animals ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; metabolism ; Endometriosis ; genetics ; physiopathology ; Female ; Humans ; NF-kappa B ; deficiency ; genetics ; Neovascularization, Pathologic ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics

Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.
Animals ; Chick Embryo ; Chickens ; Down-Regulation ; Fibroblasts ; virology ; Gene Targeting ; Lentivirus ; genetics ; metabolism ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; physiology ; Phosphoproteins ; genetics ; metabolism ; Poultry Diseases ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo study the effect of alternatively activated macrophages /mononuclear phagocytes(MNP) on breast cancer cells and explore the mechanisms for the action of tumor-associated macrophages in breast cancer.

METHODSHuman peripheral blood monocytes were isolated and cultured in vitro and divided into 3 groups, namely classically activated monocytes (CAM) which were induced by lipopolysaccharide, alternatively activated monocytes (AAM) induce by IL-4, and control cells treated with the culture medium only. After cell culture for 48-72 h, the mRNA of tumor necrosis factor-alpha (TNF-alpha), alternative monocytes activation- associated CC-chemokine 1 (AMAC-1), and beta-actin of the 3 groups were extracted for RT-PCR, or the cells were cocultured with breast cancer cell line SKBR3, or seeded in chicken chorioallantoic membrane along with SKBR3.

RESULTSTNF-alpha mRNA was significantly increased in CAM, and AMAC-1 was highly expressed in AAM. The coculture experiments showed that CAM exhibited obvious inhibitory effect on SKBR3 cells after a 3-day culture whereas AAM significantly promoted the growth of SKBR3 cells after a 5-day culture. In chicken on chorioallantoic membrane experiment, the macrophages promoted tumor angiogenesis and AAM showed the most obvious effect.

CONCLUSIONIL-4 induces high expression of AMAC-1, a molecular marker of AAM, in the macrophages, and AAM can promote the growth of SKBR3 cells and tumor angiogenesis.

Animals ; Breast Neoplasms ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Chemokines, CC ; metabolism ; Chick Embryo ; Coculture Techniques ; Humans ; Interleukin-4 ; metabolism ; Macrophage Activation ; Phagocytes ; immunology ; Tumor Necrosis Factor-alpha ; metabolism

Vertically aligned, laterally spaced nanoscale titanium nanotubes were grown on a titanium surface by anodization, and the growth of chondroprogenitors on the resulting surfaces was investigated. Surfaces bearing nanotubes of 70 to 100 nm in diameter were found to trigger the morphological transition to a cortical actin pattern and rounded cell shape (both indicative of chondrocytic differentiation), as well as the up-regulation of type II collagen and integrin beta4 protein expression through the down-regulation of Erk activity. Inhibition of Erk signaling reduced stress fiber formation and induced the transition to the cortical actin pattern in cells cultured on 30-nm-diameter nanotubes, which maintained their fibroblastoid morphologies in the absence of Erk inhibition. Collectively, these results indicate that a titanium-based nanotube surface can support chondrocytic functions among chondroprogenitors, and may therefore be useful for future cartilaginous applications.
Animals ; Apoptosis ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chick Embryo ; Chickens ; Chondrocytes/cytology/drug effects/metabolism ; Chondrogenesis/*drug effects ; Collagen Type II/metabolism ; Immunohistochemistry ; Integrin beta4/metabolism ; Mesenchymal Stem Cells/*cytology/*drug effects/metabolism ; Nanotubes/*chemistry ; Titanium/*chemistry/*pharmacology

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Chick Embryo ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Rats ; Rats, Wistar ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Swine ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Virus Replication ; genetics