To investigate the morphogenetic process of human adenovirus type 41 (HAdV-41), 293TE cells were infected with purified wild-type HAdV-41, and ultrathin sections of infected cells were prepared and observed under a transmission electron microscope. Results showed that HAdV-41 entered host cells mainly through three ways: non-clathrin-coated pit, clathrin-coated pit, and direct penetration of plasma membrane. In addition, cell microvilli might help HAdV-41 enter cells. After entering into cells, HAdV-41 virus particles could be found in vacuoles or lysosomes or be in a free state in cytoplasm. Only free virus particles could be found near nuclear pores (NP), suggesting that the virus needed to escape from lysosomes for effective infection and viral nucleoprotein entered the nucleus through NP. Progeny viruses were as-sembled in the nucleus. Three types of inclusion bodies, which were termed as fibrillous inclusion body, condense inclusion body, and stripped condense inclusion body, were involved in HAdV-41 morphogenesis. In the late phase of viral replication, the membrane integrity of the infected cells was lost and viral particles were released extracellularly. This study reveals the partial process of HAdV-41 morphogenesis and provides more biological information on HAdV-41.
Adenovirus Infections, Human ; virology ; Adenoviruses, Human ; genetics ; growth & development ; physiology ; ultrastructure ; Cell Membrane ; virology ; Cell Nucleus ; virology ; Humans ; Virus Release ; Virus Replication

Humans ; Parvoviridae Infections ; virology ; Parvovirus B19, Human ; genetics ; metabolism ; physiology

Microscopy, Electron, Transmission ; methods ; Negative Staining ; methods ; Viruses ; ultrastructure

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P > 0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P < 0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.
Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Humans ; Monocytes ; cytology ; enzymology ; Phosphotransferases (Alcohol Group Acceptor) ; drug effects ; metabolism ; Receptors, Granulocyte Colony-Stimulating Factor ; biosynthesis ; genetics ; Recombinant Proteins

OBJECTIVETo establish a TaqMan-based Real-time PCR assay with internal control for the detection of parvovirus B19 DNA in human serum.

METHODSTwo DNA fragments in length of 113 bp each were artificially synthesized, cloned into T vector and used as standard DNA or internal control, respectively. One pair of primers and two probes were included in the Real-time PCR. The probes were labeled with different fluoresceins and could bind B19 DNA or internal control, respectively. Precision and specificity of the method were evaluated. Specimens of human serum were examined by this assay to find B19 DNA.

RESULTSThe standard curve was constructed using the quantified standard B19 DNA. The Real-time PCR method was established. It was stable according to precision evaluation by the intra- and inter-assay and specific without any evident cross-reaction with human hepatitis B virus (HBV). Among 160 samples of human serum, B19 DNA was detected in 2 with a concentration of 2.1 x 10(5) Geq/ml and 3.6 x 10(3) Geq/ml, respectively.

CONCLUSIONThe Real-time PCR for B19 DNA detection was developed successfully, in which the internal control was helpful to exclude false-negative results.

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.
Adenovirus E1B Proteins ; genetics ; metabolism ; Adenoviruses, Human ; genetics ; Cell Line, Tumor ; Gene Expression ; Humans ; Immunohistochemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Animals ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

The study was to understand the impact on the proliferation and cytotoxicity of donor's T cells during mobilization with rhG-CSF. The peripheral blood mononuclear cells (PBMNC) were collected from 15 donors before mobilization and on fifth day of mobilization with rhG-CSF. After the PBMNC were activated with 500 ng/ml of CD3 monoclonal antibody and 500 microg/ml of rhIL-2 for 96 hours, the activated T cells were collected for testing proliferation, cytotoxicity, Fas expression, perforin and Fas ligand (FasL) mRNA expression, the IFN-gamma concentration in the culture medium of the activated T cells was determined by radioimmunoassay. The results showed that the proliferation activity of T lymphocytes and the cytotoxicity of T cells activated with CD3 monoclonal antibody and rhIL-2 were reduced markedly after mobilization with rhG-CSF (P < 0.05). The Fas molecule expression in the activated T cells was very high both before and after mobilization with rhG-CSF (P > 0.10). The activated T cells expressed perforin mRNA and didn't express FasL mRNA both before and after mobilization with rhG-CSF. The concentration of IFN-gamma in the culture medium of the activated T cells decreased significantly after mobilization with rhG-CSF (P < 0.01). It is concluded that activity of proliferation and cytotoxicity of donor's T cells is impaired after mobilization with rhG-CSF.
Adolescent ; Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Fas Ligand Protein ; biosynthesis ; genetics ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; pharmacology ; Hematopoietic Stem Cell Mobilization ; methods ; Humans ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; T-Lymphocytes ; cytology ; drug effects ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; fas Receptor ; biosynthesis ; genetics

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.
Base Sequence ; Cell Line, Transformed ; cytology ; physiology ; Chromosomes, Human, Pair 22 ; genetics ; Chromosomes, Human, Pair 9 ; genetics ; Fusion Proteins, bcr-abl ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; Genes, abl ; genetics ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; metabolism ; pathology ; Models, Genetic ; Molecular Sequence Data ; Proto-Oncogene Proteins c-bcr ; genetics ; Transfection ; Translocation, Genetic ; Tumor Cells, Cultured

Sphingosine 1-phosphate (S1P), which can be impacted by different growth factors through sphingosine kinase (SphK), is a bioactive lipid produced by metabolism of sphingolipid with various biological responses. Recombinant human granulocyte-colony-stimulating factor (rhG-CSF) have been used widely in peripheral blood stem/progenitor cell mobilization. This study was aimed to investigate the effects of rhG-CSF on S1P concentration in plasma of donors. The peripheral blood mononuclear cells and blood plasma were collected from 8 peripheral blood progenitor cell donors before mobilization and on the fifth day of mobilization with rhG-CSF. The SphK mRNA expression of blood mononuclear cells were detected by RT-PCR. The changes of S1P concentration were assayed with SphK enzyme catalyzed reaction. The results showed that both kinds blood mononuclear cells collected before and after rhG-CSF mobilization expressed SphK mRNA. The S1P concentration is low in donor's plasma both before and after mobilization with rhG-CSF, and there was no markedly change of S1P concentration in plasma before and after mobilization (P > 0.05). In conclusion, mobilization with rhG-CSF does not impact S1P concentration in donors' plasma.
Blood Donors ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Hematopoietic Stem Cell Mobilization ; Humans ; Lysophospholipids ; blood ; RNA, Messenger ; blood ; Recombinant Proteins ; Sphingosine ; analogs & derivatives ; blood