Nipah virus (NiV) and related viruses form a distinct henipavirus genus within the Paramyxoviridae family. NiV continues to spillover into the humans causing deadly outbreaks with increasing human-bat interaction. NiV encodes the large protein (L) and phosphoprotein (P) to form the viral RNA polymerase machinery. Their sequences show limited homologies to those of non-henipavirus paramyxoviruses. We report two cryo-electron microscopy (cryo-EM) structures of the Nipah virus (NiV) polymerase L-P complex, expressed and purified in either its full-length or truncated form. The structures resolve the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyl transferase (PRNTase) domains of the L protein, as well as a tetrameric P protein bundle bound to the L-RdRp domain. L-protein C-terminal regions are unresolved, indicating flexibility. Two PRNTase domain zinc-binding sites, conserved in most Mononegavirales, are confirmed essential for NiV polymerase activity. The structures further reveal anchoring of the P protein bundle and P protein X domain (XD) linkers on L, via an interaction pattern distinct among Paramyxoviridae. These interactions facilitate binding of a P protein XD linker in the nucleotide entry channel and distinct positioning of other XD linkers. We show that the disruption of the L-P interactions reduces NiV polymerase activity. The reported structures should facilitate rational antiviral-drug discovery and provide a guide for the functional study of NiV polymerase.
Nipah Virus/chemistry* ; Cryoelectron Microscopy ; Viral Proteins/genetics* ; RNA-Dependent RNA Polymerase/genetics* ; Phosphoproteins/genetics* ; Humans ; Models, Molecular ; Protein Binding

Influenza C virus is an important respiratory pathogen not only infecting people, but also pigs, dogs, and other animals. Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication. However, there is no commercial monoclonal antibody (MAb) targeting influenza C virus polymerase, which hampers the development of relevant research to some extent. In order to prepare MAb targeting the polymerase basic protein 2 (PB2) of influenza C virus, influenza C virus RNA-dependent RNA polymerase (RdRp, consists of PB1, PB2 and P3) was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A (huANP32A-Flag) from 293T cells based on the interaction between huANP32A and influenza virus RdRp. The purified RdRp was used as antigen to immunize BALB/c mice. Six positive hybridoma cell lines (7B11-5, 8A4-5, 13D9-6, 8D4-1, 8D4-3, 9F9-4) that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting. The subtypes of MAb 7B11-5, 8A4-5, 8D4-1 and 8D4-3 antibody were identified as IgG1, the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3, respectively. All the light chains of the MAbs were κ chain. A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites. The titer of mouse ascites antibody was determined to be 1:64 000. Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2; laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits. Moreover, ICV RdRp was highly enriched by ANP32A. The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection, structural analysis and mechanism study of influenza C virus.
Animals ; Antibodies, Monoclonal/metabolism* ; Ascites ; Humans ; Influenzavirus C/metabolism* ; Mice ; Nuclear Proteins/metabolism* ; RNA-Binding Proteins ; RNA-Dependent RNA Polymerase/genetics* ; Viral Proteins/metabolism* ; Virus Replication

Calbindin-D9k (CaBP-9k) is a cytosolic calcium-binding protein expressed in tissues in the intestine, uterus, placenta, kidney, pituitary gland and bone. Its exact function is unknown, but it is considered to regulate intracytoplasmic concentration and transport of free ions (Ca2+). CaBP-9k protein is involved in intestinal calcium absorption in the intestine and in the regulation of myometrial activity by intracellular calcium in the uterus. Renal CaBP-9k protein is expressed at the site of calcium re-absorption in the kidney and expressed in distal convoluted tubules, where it is thought to facilitate calcium re-absorption. Expression of the CaBP-9k gene has been explored in most mammalians except in a canine model. Presently, we elucidated the expression of CaBP-9k mRNA and protein in the duodenum, kidney and uterus in a canine model involving two adult (2.5-year-old) female beagles. To collect tissues, the dogs were euthanized and then the abdominal cavity was exposed by midline incision. The proximal duodenum, cortex of kidney and uterine horn were collected. Expression of CaBP-9k mRNA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. CaBP-9k protein expression and localization were ascertained by Western blot analysis and immunohistochemistry, respectively. CaBP-9k mRNA was detected in the duodenum, but not in the kidney and uterus. Its protein was expressed only in the enterocytes of the duodenum. Taken together, the results indicate that CaBP-9k mRNA and protein are highly expressed in the enterocytes of the duodenum of a canine model, consistent with findings in other mammalian species.
Animals ; Blotting, Western/veterinary ; Calcium-Binding Protein, Vitamin D-Dependent/*biosynthesis/genetics ; Dogs/*physiology ; Duodenum/*physiology ; Female ; Immunohistochemistry/veterinary ; Kidney/*physiology ; RNA, Messenger/biosynthesis/genetics ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Transcription, Genetic ; Uterus/*physiology

Biomarkers, Tumor ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; CpG Islands ; Cyclin-Dependent Kinase Inhibitor p57 ; genetics ; metabolism ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Humans ; In Situ Hybridization ; Liver ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis.

METHODSLoVo cells were infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1alpha and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1alpha and p21WAF1/CIP1 protein expression. Hoechst 33342 flourescein staining was performed to observe the ratio of apoptotic LoVo cells.

RESULTSThe expression levels of HIF-1alpha mRNA and protein increased after infection with Ad-HIF-1alpha- Ala564-Ala803, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 (16.2%) than in the control cells (5.5%, P=0.00).

CONCLUSIONHIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.

Adenoviridae ; genetics ; Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Colonic Neoplasms ; genetics ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Mutagenesis, Site-Directed ; Mutation ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo investigate the expressions of the aging gene P16(ink4a) and anti-aging gene HST2 in benign prostatic hyperplasia (BPH).

METHODSTwenty-three BPH and eighteen normal prostate specimens were collected and total RNA was extracted, followed by the reverse transcriptase polymerase chain reaction (RT-PCR). The expressions of P16(ink4a) was detected by semi-quantitative analysis in BPH and normal prostate tissues.

RESULTSP16(ink4a) mRNA, rather than HST2, was expressed in the BPH and normal prostate tissues. Semi-quantitative analysis showed that the P16(ink4a) mRNA expression in the normal prostate tissues (0.4868 +/- 0.545 was significantly higher than in the BPH tissues (0.2783 +/- 0.0268, with a statistical difference in between (P < 0. 05).

CONCLUSIONP16(ink4a) might play an important role in the pathogenesis of BPH and is probably one of the factors of cell aging escape.

Adult ; Aged ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Fibroblast Growth Factor 6 ; genetics ; Gene Expression Profiling ; Humans ; Male ; Pilot Projects ; Prostatic Hyperplasia ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the role of cyclin D1 and p27(kip1) in the occurrence and development of conventional renal cell carcinoma(CRCC).

METHODSRT-PCR and Western-blot were used to detect mRNA and protein contents of cyclin D1 and p27(kip1) in 25 CRCCs and 10 normal renal tissue distant to tumor. Immunohistochemistry was used to investigate the expression of cyclin D1 and p27(kip1) in pathological tissue sections of 76 CRCCs. The relationship between those index and clinicopathological parameters was analyzed statistically.

RESULTIn CRCC, the expression of cyclin D1 was higher than that of the control group. The higher cyclin D1 content was related to big tumor size (P<0.05); The expression of p27(kip1) was lower than that of the control group, and the lower p27(kip1) was related to higher nuclear grade and TNM stage (P<0.01). The 5-year cumulative survival rate of the p27(kip1) high expression group is longer than that of the low group (P<0.01).

CONCLUSIONThe excessive expression of cyclin D1 and lower expression of p27(kip1) play an important role in the carcinogenesis of CRCC. The lower expression of p27(kip1) may affect the progression of the tumors. The detection of p27(kip1) may be as a reference marker in the prognosis of CRCC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p27 ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The study was purposed to investigate the effect of arsenic trioxide (As(2)O(3))- induced p16 gene demethylation by a sensitive and specific PCR-based method (nested-methylation specific PCR, n-MSP) and DNA sequencing for rapid analysis of the promoter demethylation status, and to explore the possible mechanism of the p16 gene demethylation in human multiple myeloma U266 cells induced by As(2)O(3). The methylation status of the p16 gene in U266 cell line before and after treatment with As(2)O(3) was detected by the nested-methylation specific PCR and DNA sequencing, the mRNA of p16, DNA methyltransferase (DNMT 1, DNMT3A and 3B) gene were determined by RT-PCR, and the induced growth inhibition of U266 cell was assayed by growth curve, MTT and CFU; the DNA content of U266 cells was analyzed by flow cytometry after being exposed to As(2)O(3). The results showed that (1) all cytosines in CpG dinucleotides in untreated U266 cell not were changed, while all cytosines in treated U266 cells with As(2)O(3) had been converted to thymidine. (2) p16 gene was not expressed in U266 cell line after methylation. As compared with the beta-actin, the expression of U266 cell p16 gene mRNA was increased to (0.22 +/- 0.10), (0.59 +/- 0.11), (0.68 +/- 0.09) after exposed to 0.5 micromol/L, 1.0 micromol/L and 2.0 micromol/L As(2)O(3) for 72 hours respectively. (3) As(2)O(3) could significantly down-regulate DNA methyltransferase 1 (DNMT 1), DNMT3A and DNMT3B gene at mRNA level in a dose-dependent manner. (4) U266 cells line grew slowly and arrested at G(0) - G(1) phase after treatment with three different concentrations of As(2)O(3). It is concluded that As(2)O(3) can activate and up-regulate the expression of p16 gene which inhibits the proliferation of U266 cell through inducing the G(0) - G(1) arrest by demethylation or/and by inhibiting DNMT 1, DNMT3A and 3B gene.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Base Sequence ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; biosynthesis ; genetics ; DNA Methylation ; drug effects ; Genes, p16 ; Humans ; Molecular Sequence Data ; Multiple Myeloma ; genetics ; metabolism ; pathology ; Oxides ; pharmacology ; Polymerase Chain Reaction ; methods ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Transcription, Genetic ; drug effects

OBJECTIVEThe aim of this study was designed to investigate the effect of TSA on human umbilical vein endothelial cells and to reveal its possible mechanisms and relationship between apoptosis and activity of telomerase reverse transcriptase.

METHODSsulforhodamine B method was employed to determine the growth rate of umbilical vein endothelial cells. The cell apoptotic rate was measured by flow cytometry (FCM). The hTERT and p21(Waf1) mRNA expression before and after TSA treatment were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The quantitative of hTERT protein expression in cells were detected by flow cytometry. After transfection, the cell telomerase activity was detected by PCR and telomeric repeat amplification protocol assay (PCR-TRAP-ELISA) and early apoptosis was measured by Annexin V/PI stain and flow cytometry.

RESULTSAfter being treated with TSA, the proliferation of umbilical vein endothelial cells was inhibited. Slight apoptosis and cell cycle arrest were detected. However, the same concentration of TSA induced serious apoptosis in HeLa cells. Up-regulation of hTERT mRNA expression was observed within 48 h after TSA treatment, but the change of p21(Waf1) expression was not significant. The umbilical vein endothelial cells hTERT protein expression level was increased within 24 h. After transfection of the dominant negative, wild type and control hTERT plasmid, a significant difference of telomerase activity in these cells was observed by PCR-TRAP-ELISA assay. WT-hTERT-transfected cells were more resistant to apoptosis induced by trichostatin A.

CONCLUSIONHuman umbilical vein endothelial cells could be resistant to apoptosis induced by high concentrate TSA, and hTERT might play an important role in this process.

Apoptosis ; drug effects ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; methods ; Flow Cytometry ; Gene Expression ; drug effects ; HeLa Cells ; Humans ; Hydroxamic Acids ; pharmacology ; Plasmids ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Telomerase ; genetics ; metabolism ; Transfection ; Umbilical Veins ; cytology

OBJECTIVETo evaluate the relationship between mammalian target of rapamycin (mTOR) signaling pathway and histone acetylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells.

METHODSHuman gastric cancer cell lines, MKN45 and SGC7901 were treated with trichostatin A, rapamycin and/or LY294002, a PI3K inhibitor. Cell viability was analyzed by methylthiazolyl tetrazolium. Cell cycle distribution was evaluated by flow cytometry. The transcription level of p21(WAF1) gene was detected by using real-time polymerase chain reaction. Proteins were detected by Western blotting.

RESULTSCell viability remarkably reduced after treatment by more than two drugs (P< 0.01). Through flow cytometry assessment, MKN45 cells were arrested in G2 phase (P< 0.05), while SGC7901 cells were in G2 or G1 phase (P< 0.05) whether treated with single or more than two drugs. The expression of p21(WAF1) mRNA was remarkably increased in the gastric cancer cells treated with conjoined drugs (P< 0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 was significantly reduced in cells treated with conjoined drugs (P< 0.01). And histone acetylation of H4/H3 was also increased in cells treated with conjoined drugs (P< 0.01).

CONCLUSIONmTOR singnaling pathway has an important relationship with histone acetylation in gastric cancer cell lines. There is a co-effect of mTOR inhibitor and histone deacetylase inhibitor on gastric cancer cells.

Acetylation ; drug effects ; Adaptor Proteins, Signal Transducing ; metabolism ; Blotting, Western ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Flow Cytometry ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Morpholines ; pharmacology ; Phosphoproteins ; metabolism ; Phosphorylation ; drug effects ; Polymerase Chain Reaction ; Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Signal Transduction ; drug effects ; physiology ; Sirolimus ; pharmacology ; Stomach Neoplasms ; metabolism ; pathology ; physiopathology ; TOR Serine-Threonine Kinases