Polymerase chain reaction was employed to amplify the whole HBV CP region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced and the selected clones were compared to look for the difference.The sequencing results suggested that each sequence of selected clones was different and there were HBV quasispecies groups in patients. There were hot deletion region and point mutation near the TATA like box of CP gene. To address whether the mutations were responsible for the transcriptional activity, the wild type(wt) and the mutants of HBV CP genes were subcloned into pcDNA3 1( ) vectors, respectively. The reverse oriented clones were digested with KpnI and XhoI, and cloned into the KpnI and XhoI sites of the chloramphenicol acetyltransferase (CAT) expressing vector (pCAT3 basic).The recombinant CAT plasmids were transfected into HepG2 cells using lipofectamine PLUS reagent, and the CAT expression which indirectly represented the transcriptional activity of HBV CP lying upstream of CAT gene was detected with a CAT ELISA kit. The restriction enzyme digesting results indicated that the recombinant CAT plasmids were successfully constructed, and the transfection tests indicated that the transcriptional activity of the mutants with deletion or substitute point mutation of TATA like box were reduced in comparison with that of CPwt. The HBV CP gene heterogeneity downregulated the transcriptional activity to some extent.

In nature, honeybees are the most important pollinators. They play a vital role in both protecting the diversity of natural ecosystems, and maintaining the yield-improving effects of agroecosystems. But in recent years, epidemic disease in bees has caused huge losses. Black Queen Cell Virus (BQCV) is a bee pathogen that was first reported in 1955. It mainly infects bee larvae and pupae, making their bodies turn dark and black, and causing a massive decrease in the bee population. More specifically, the virus makes the exterior of the cell walls in the larvae and pupae turn black. BQCV is a seasonal epidemic, spread by means horizontal and vertical transmission, and is often unapparent. BQCV not only infects a variety of bee species, but also spiders, centipedes and other arthropods. It can also be coinfected with other honeybee viruses. In recent years, research has shown that the Nosema intestinal parasite plays an important role in BQCV transmission and bees carrying Nosema that become infected with BQCV have increased mortality. Here we summarize current research on the incidence, prevalence, geographical distribution and transmission of BQCV.
Animals ; Bees ; virology ; Dicistroviridae ; classification ; genetics ; isolation & purification ; physiology ; Insect Viruses ; classification ; genetics ; isolation & purification ; physiology

Honeybee pupae were collected from Jilin apiaries and RNA was extracted for use as a tefnplate for amplification. Based on the complete genome sequences of black queen cell virus (BQCV) published on GenBank, we designed 10 pairs of primers to amplify genes by reverse transcription-polymerase chain reaction (RT-PCR). Using this approach, we have obtained the first complete genome sequence of a BQCV isolate in China. The genome of the isolated strain, named BQCV-JL1, is composed of 8358 nucleotides and shares between 86% and 93% homology with the complete genome sequences of the other six BQCV strains published on GenBank. ORF 1 of BQCV-JL1 is positioned between nucleot ides (nt) 546 and 4676 (4131 nt), while ORF 2 is located between nt 5750 and 8203. Between the two ORFs of BQCV-JL1 there is a short ORF, called ORF 3, between nt 4891 and 5433 (543 nt). The first functional gene ex- pression domain of the BQCV-JL1 strain is positioned between nt 546 and 5 429, encompassing both ORF 1 and ORF 3. There is an internal ribosome entry site (IRES) located before ORF 2, the last three bases of which are CCU (nt 5642-5644). These bases act as an initiation.codon facilitating the translation of ORF 2. The second functional gene expression domain of the BQCV-JL1 strain is located between nt positions 5642 and 8203. The BQCV-JL1 strain was found to share high sequence identity (93%) with the Hungary 10 genotype at the whole-genome level and analysis of the nucleotide and amino acid sequences revealed that the BQCV-JL1 strain also shows close genetic relationships with the South Korea strain, suggesting that both the BQCV-JL1 and South Korea strains may have migrated from European countries. BQCV-JL1 strain was different from the other 6 strains in dividing the nucleotides positions of QRF, which vqs because of the gene mutation.
Amino Acid Sequence ; Animals ; Bees ; China ; Genome, Viral ; Molecular Sequence Data ; Open Reading Frames ; Phylogeny ; RNA Viruses ; classification ; genetics ; isolation & purification ; Viral Proteins ; genetics

Objective To summarize the experience in laparoscopic spleen-preserving distal panereatectomy.Methods From Nov 2003 to July 2006,six patients with distal pancreatic cystic lesions underwent laparoscopic spleen-preserving distal pancreatectomy with splenic vessels preservation. Results All operations were successful with the operative time ranging from 140～265 min and the intraoperative blood loss ranged from 350～600 ml.One case received combination resection of right adrenal adenoma,1 case with combined laparoscopic myomectomy and left ovarian teratomeetamy,1 case with combined laparoscopic myomectomy,1 case with combined laparoscopic cholecysteetomy.All patients were discharged 4 to 9 days postoperatively.The pathologic diagnosis was retained cyst in 2 cases,serous cystadenoma in 2 cases,mucious cystadenoma in 2 cases.Symptoms disappeared in all cases after operations and there was no recurrence during a follow-up period that ranged from 1 month to 31 months.Conclusions Laparoscopic spleen-preserving distal pancreatectomy with splenic vessels preservation is the most suitable procedure for the distal pancreatic benign lesions,and in experienced hands this procedure is safe and effective.

BACKGROUNDThis study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans.

METHODSMouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting.

RESULTSM. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-kappaB activation, a possible mechanism for the induction of iNOS expression.

CONCLUSIONThis study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-kappaB.

Animals ; Bacterial Proteins ; pharmacology ; Cells, Cultured ; Enzyme Induction ; Lipoproteins ; pharmacology ; Membrane Proteins ; pharmacology ; Mice ; Mycoplasma penetrans ; chemistry ; NF-kappa B ; metabolism ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; biosynthesis ; Nitric Oxide Synthase Type II ; RNA, Messenger ; analysis

BACKGROUNDDaxx has been identified as a nuclear protein that involves in apoptosis and transcriptional repression. Daxx co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Human Daxx (hDaxx) is a protein that functions as a transcriptional regulation through its interaction with some DNA-associated proteins. The aim of this study was to explore the transcriptional regulatory effect of hDaxx interacting with adenovirus (Ad) 12 E1B (Ad12E1B) 55-kDa oncoprotein.

METHODSThe co-localization of hDaxx-Ad12E1B or hDaxx-PML protein in the nucleus was observed under a confocal microscope. Interaction of hDaxx and Ad12E1B was analyzed by yeast two-hybrid assay. Direct binding of hDaxx and Ad12E1B was analyzed using coimmunoprecipitation and Western blot in vivo and in vitro. The activity of a luciferase reporter gene, which was regulated by an hDaxx modulated thymidine kinase (TK) promoter, was detected in an automat luminometer.

RESULTSAd12E1B, which co-localized with hDaxx in the nuclei of G401-CC3 cells, disrupted the co-localization of hDaxx and PML in the PML oncogenic domains (PODs). hDaxx bound directly to Ad12E1B in vivo and in vitro. hDaxx interacted with Ad12E1B along its full length. Ad12E1B enhanced transcriptional repression activity of hDaxx.

CONCLUSIONAd12E1B disrupts the co-localization of hDaxx with PML in PODs and enhances transcriptional repression activity of hDaxx.

Adaptor Proteins, Signal Transducing ; Adenovirus E1B Proteins ; physiology ; Carrier Proteins ; analysis ; genetics ; Cell Line, Tumor ; Humans ; Intracellular Signaling Peptides and Proteins ; Neoplasm Proteins ; analysis ; Nuclear Proteins ; analysis ; genetics ; Promyelocytic Leukemia Protein ; Repressor Proteins ; physiology ; Transcription Factors ; analysis ; Transcription, Genetic ; Tumor Suppressor Proteins

OBJECTIVETo investigate the value of five-repetition sit-to-stand test (5STS) in clinical evaluation of elderly patients with chronic obstructive pulmonary disease (COPD).

METHODSFifty-one patients with COPD and 20 healthy individuals were enrolled in this study. All the participants underwent 5STS, pulmonary function examination, and 6 min walking test (6MWT) and were evaluated for severity of dyspnea (by mMRC) and BODE index during the tests.

RESULTSAll the participants completed 5STS test with a good reproducibility of the time used for 3 sessions of the test (P<0.001). The mean time used by COPD patients for 5STS was significantly longer than that by healthy individuals (12.93±3.11s vs 0.72±0.71 s, P=0.002). The results of 5STS showed a significant negative correlation with those of 6MWT in the case group and control group with correlation coefficients of -0.611 and -0.682, respectively. The results of 5STS were negatively correlated with FEV1%Pre and body mass index (P<0.05) but positively with mMRC and BODE index in COPD patients (P<0.05).

CONCLUSION5STS is a simple and reproducible test to evaluate the patients' exercise capacity and the severity of COPD, and is well correlated with the current methods for clinical evaluation of COPD.

Body Mass Index ; Case-Control Studies ; Dyspnea ; Exercise Test ; Humans ; Pulmonary Disease, Chronic Obstructive ; diagnosis ; Reproducibility of Results ; Respiratory Function Tests ; Walking

OBJECTIVETo evaluate the feasibility and efficacy of laparoscopic distal pancreatectomy.

METHODSTotally 68 patients (male 23, female 45) aged 17 to 77 years, with distal pancreatic lesions, underwent laparoscopic distal pancreatectomy from November 2003 to December 2010. The clinical data were collected. Safety, feasibility and crucial technique manipulation were analyzed retrospectively.

RESULTSAll 68 operations were successful with two cases conversion to open, including 48 cases combined with splenectomy, and 18 cases with preservation of spleen. Fourteen cases received with combination resection of multi-organs, including 4 cases with cholecystectomy, 1 case resection of right adrenal adenoma and cholecystectomy, 1 case with myomectomy and left ovarian teratomectomy; 1 case with right ovarian teratomectomy, 1 case with resection of left adrenal adenoma, 1 case with resection of both adrenal adenoma, 1 case with resection of liver metastasis, 1 case with cholecystectomy and resection of liver metastasis, 1 case with resection of left adrenal adenoma and liver metastasis, 1 case with resection of left adrenal adenoma and colon and spleen, 1 case with biopsy of liver nodule. The mean operative time was (209 ± 58) minutes, the mean intraoperative blood loss was (191 ± 123) ml, and the mean postoperative hospital stay was (8 ± 4) days. The rate of overall postoperative complications was 18.1%, including an 12.1% rate of clinical pancreatic fistula. Only one case needed a reoperation, and there was no postoperative mortality.

CONCLUSIONLaparoscopic distal pancreatectomy with or without splenectomy is safe and feasible in the treatment of most distal pancreatic tumors.

Adolescent ; Adult ; Aged ; Female ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Pancreatectomy ; methods ; Retrospective Studies ; Young Adult

Objective To study the effects of environmental factors,including high temperature,high humidity and low temprtature,on the quality of whole blood.Methods Fresh blood was collected from 9 blood donors and divided into 40 parts.One group that comprised 20 samples was used to assess the effects of storage at high temperature and high humidity (0,3,6,12 and 24 h),and the other group(20 samples)was used to determine the effects of low temprtature(0,1,2 and 3 h).The serum free hemoglobin(FHb)concentration, plasma prothrombin time(PT), activated partial thromboplastin time(APTT),thrombin time(TT)and fibrinogen(FIB)were measured and analyzed.Results The high temperature(27-37℃)and humidity(60%-90%RH)had no effect on FHb, PT, APTT, TT or FIB of whole blood. The low temperature(0-13℃and -18--20℃)had some effect on the quality of whole blood.The FHb of test group was higher than that of control group.There was no significant difference in PT, APTT, TT and FIB between the two groups.Conclusion Under high temperature and humidity conditions, 24 hours of whole blood placement is feasible. Under low temperature or cold conditions,short-term whole blood placement is feasible in a tent with heating facilities.

OBJECTIVETo construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function.

METHODSThe mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast.

RESULTSThe subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function.

CONCLUSIONA subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

Cloning, Molecular ; Gene Library ; RNA, Messenger ; analysis ; Trans-Activators ; physiology ; Transcriptional Activation