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

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

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

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

Hepatocyte growth factor (HGF) is one of major growth factors in the bone marrow microenvironments with which the proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells were closely contacted. However, its roles in the regulation of proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells remain unclear. This study was aimed to investigate the effect of HGF on biological characteristics of bone marrow-derived mesenchymal stem cells. Expression of c-Met, the receptor for HGF was detected by immunohistochemistry assay, cell proliferation was determined by MTT, activity of ALP was quantitatively assayed, cell migration and anoikis-induced MSC apoptosis were analyzed. The results showed that HGF not influenced the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Treatment of bone marrow-derived mesenchymal stem cells with recombinant human hepatocyte growth factor resulted in inhibition of anoikis-induced apoptosis. HGF significantly stimulated the migration of bone marrow-derived mesenchymal stem cells. Both PI-3 kinase and MAPK kinase were proved to be involved in HGF-induced migration. It is concluded that HGF/c-Met signal regulates the apoptosis and migration of bone marrow-derived mesenchymal stem cells.
Anoikis ; drug effects ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Hepatocyte Growth Factor ; pharmacology ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Proto-Oncogene Proteins c-met ; biosynthesis

The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.
Apoptosis ; Cell Division ; Humans ; Multiple Myeloma ; drug therapy ; pathology ; Receptor, Notch1 ; Receptors, Cell Surface ; physiology ; Signal Transduction ; Transcription Factors ; physiology