This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

AIMTo select higher thrombolytic and lower toxic single component of earthworm fibrinolytic enzymes (EFE).

METHODSEFE containing total components were obtained by affinity chromatography from Eisenia fetida. Using ion-exchange chromatography to separate three main components EfP-0-2, EfP-I-1 and EfP-I-2 from EFE, their thrombolytic activity and toxicity were compared with EFE.

RESULTSAmong these components, EfP-I-1 had higher thrombolytic activity in vitro. When 4.5 mg x kg(-1) of these components were injected, the contents of fibrinogen in rat serum were not affected, but only EfP-I-1 exhibited distinct thrombolytic activity. When 6.0 mg x kg(-1) of them were injected intravenously, the bleeding time was not evidently delayed only by EfP-I-1. The acute toxicity test showed that the LD50 of EfP-I-1 was higher than EFE by 2. 17 times.

CONCLUSIONBecause of distinct thrombolytic activity, lower toxicity in vivo, higher content in EFE and easy to purify, EfP-I-1 was adapted to be developed as a single component medicine for treating thrombus.

Amino Acid Sequence ; Animals ; Bleeding Time ; Dogs ; Electrophoresis, Polyacrylamide Gel ; Female ; Fibrinogen ; metabolism ; Fibrinolytic Agents ; chemistry ; isolation & purification ; pharmacology ; toxicity ; Lethal Dose 50 ; Male ; Mice ; Molecular Sequence Data ; Molecular Weight ; Oligochaeta ; chemistry ; enzymology ; Rats ; Rats, Wistar ; Sequence Analysis, Protein ; Spectrophotometry, Infrared ; Venous Thrombosis ; blood ; drug therapy

AIMTo study the effects of rhBNP and milrinone on the cardiac hemodynamics and renal function in anesthetized dogs.

METHODSThe actions of rhBNP given cumulatively i.v. 10, 30 and 100 ng.kg-1 for 30 min and milrinone of single dose (100 micrograms.kg-1, i.v.) on cardiac hemodynamics and renal function were studied in anesthetized open-chest dogs.

RESULTSIn anesthetized dogs (n = 7) intravenous infusion of rhBNP at 10-100 ng.kg-1, caused decreases in mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), LVdp/dtmax, pulmonary arterial pressure (PAP), left ventricular end diastolic pressure (LVEDP), total peripheral resistance (TPR) and renal vascular resistance (RVR) dose-dependently, without significant changes in cardiac output (CO), LV(dp/dt)/P, renal blood flow (RBF) and heart rate (HR), increases in urinary volume and sodium excretion. In anesthetized dogs (n = 6), there were remarkable decreases in MAP, LVEDP, PAP, TPR, RBF, RVR and urinary volume following the MIL (100 micrograms.kg-1, i.v.), with significant increases of LVSP, +/- LVdp/dtmax, HR and CO, but no marked changes in urinary volume and sodium excretion.

CONCLUSIONrhBNP reduces the pre-load and after-load in the anaesthetized dogs but showed no distinct effect on the contractility of the heart. Positive inotropic and chronotropic actions have been demonstrated after intravenous injection of milrinone 100 micrograms.kg-1 in anesthetized dogs.

Anesthesia ; Animals ; Cardiotonic Agents ; pharmacology ; Dogs ; Drug Interactions ; Female ; Heart ; drug effects ; physiology ; Hemodynamics ; Kidney ; drug effects ; physiology ; Male ; Milrinone ; pharmacology ; Natriuretic Peptide, Brain ; pharmacology ; Recombinant Proteins ; pharmacology ; Sodium ; blood ; urine ; Urine

Objective To investigate the application value of magnetic-controlled capsule endoscopy (MCE) for gastric diseases in physical examination of asymptomatic population.Methods Data of 211 asymptomatic individuals who received MCE examinations from July 2015 to December 2016 in Changhai Hospital were collected and rctrospectively analyzed.The tolerance and safety of MCE were studied by analyzing the detection rate for the focal lesions and the rate of endoscopy transfer.Results Among 211 patients,the detection rate of the gastric focal lesions was 9.5％ (20/211).The detection rate in male was higher than that in female (P＜0.05).All patients completed MCE examination successfully and no adverse event was reported.Conclusion MCE,a non-invasive endoscopic modality,is safe and better tolerated than conventional endoscopy,and can be used as a promising approach to screening the gastric diseases in asymptomatic population due to high detection rate of these diseases.

OBJECTIVECarnitine, the only carrier for fatty acid to enter mitochondria for oxidation, has significant effects on serum lipids metabolism and male fertility. This study aimed to investigate the correlation of serum carnitine levels with sex and age among healthy Chinese adults in Nanjing.

METHODSWe obtained fasting blood samples from 347 healthy Chinese adults in Nanjing (182 males, 165 females, aged from 20 to 89), determined the levels of serum free, acylcarnitine and total carnitine by high performance liquid chromatography with pre-column ultraviolet derivatization, and analyzed the correlation of serum carnitine levels with the age and sex of the subjects.

RESULTSTotal and free carnitine levels were significantly higher in the males than in the females (P < 0.05) while no statistically significant differences were found in the acylcarnitine level (P > 0.05). Among those aged between 20 to 50 years, levels of serum free and total carnitine were significantly higher in men than in women (P < 0.05) and they increased with age in females, but with no significant differences between males and females older than 50 years. The acylcarnitine level showed no obvious differences in any age and sex groups.

CONCLUSIONSerum free and total carnitine levels are correlated with sex and age in healthy adults.

Adult ; Age Factors ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; Carnitine ; blood ; Chromatography, High Pressure Liquid ; Female ; Humans ; Male ; Middle Aged ; Sex Factors ; Young Adult

BACKGROUND: Leukocyte-poor platelet-rich plasma (LP-PRP) from peripheral blood is currently used as a concentrated source of growth factors to stimulate repair at sites of soft tissue injury. Fibroblasts are primary mediators of wound healing. Thus, we aimed to assess the positive effect of LP-PRP on human fibroblast proliferation in vitro. METHODS: LP-PRP was prepared from 49 donors. The fibroblasts were seeded, and at 24 hours after seeding, 1 × 107/10 µL LP-PRP was added once to each well. The cells were harvested 10 times during study period at our planned points, and we examined cell proliferation using the water-soluble tetrazolium salt-1 assay. We collected the supernatants and measured the amount of growth factors such as platelet-derived growth factor (PDGF)-AB/BB, insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor (VEGF), which are known to be involved in wound healing processes, by multiplex assay. RESULTS: Human fibroblasts treated with LP-PRP showed a significant increase in proliferation when compared to untreated controls (p < 0.001 at days 4, 6, and 8). Multiplex cytokine assays revealed various secretion patterns. PDGF-AB/BB appeared at early time points and peaked before fibroblast proliferation. IGF-1 and TGF-β1 secretion gradually increased and peaked on days 4 and 6 post-treatment. The early VEGF concentration was lower than the concentration of other growth factors but increased along with cell proliferation. CONCLUSIONS: Platelets in LP-PRP release growth factors such as PDGF, IGF-1, TGF-β1 and VEGF, and these growth factors have a promoting effect for human fibroblast proliferation, one of the important mediators of wound healing. These results suggest that growth factors derived from LP-PRP enhance the proliferation of human fibroblast.
Cell Proliferation ; Fibroblasts* ; Humans* ; In Vitro Techniques* ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins* ; Platelet-Derived Growth Factor ; Platelet-Rich Plasma ; Soft Tissue Injuries ; Tissue Donors ; Vascular Endothelial Growth Factor A ; Wound Healing

Objective:To explore the feasibility of 400 mL Sprite Zero ? in gastric preparation for magnetically controlled capsule endoscopy (MCE) . Methods:A randomized controlled trial at the Department of Gastroenterology of Changhai Hospital, Naval Medical University from December 16th, 2019 to January 15th, 2020 was conducted. The patients and healthy volunteers who intended to receive MCE were randomly divided into the Sprite Zero ? (S) group and the water (W) group at 1∶1. For subjects in the W group, 800 mL water was taken 10 minutes before swallowing the capsule. And for subjects in the S group, 400 mL Sprite Zero ? was taken. The primary endpoint was gastric filling score and the secondary endpoint included the fullness score, gastric transit time (GTT), small bowel transit time (SBTT), completion rate (CR) for small bowel examination and the diagnostic yield. Results:A total of 102 subjects were enrolled, 52 subjects in the S group and 50 subjects in the W group. The median score of gastric filling was 4 at 0-5 min, >5-10 min and >10-15 min after taking the capsule in both groups, with less median liquid consumption in the S group than the W group (500 mL VS 950 mL, P<0.001). The S group showed lower median fullness score (7.0 scores VS 7.5 scores, P=0.030) and higher proportion of patients with GTT less than 30 minutes [69.57% (16/52) VS 27.59% (8/29), P=0.030] compared with the W group. The CR of small bowel examination in the S group was 100.00%, higher than that of the W group (89.66%, P=0.245). Conclusion:Compared with 800 mL water, 400 mL Sprite Zero ? can fully fill the stomach with more comfort. It has the potential to accelerate gastric emptying and improve the CR of small bowel examination, which is feasible for the gastric preparation.

OBJECTIVETo investigate the expression of annexin in human pancreatic cancer and to elucidate its role in oncogenesis of pancreatic cancer.

METHODSA pancreatic carcinoma cell line Suit-II with high-expression of annexin I gene was adopted. Three subtypes of annexin I -siRNA sequences and a non-related fragment were combined, and the eukaryotic expression vectors bearing siRNA fragments were constructed. Then they were transfected into pancreatic carcinoma cells to knock down the expression of annexin I by RNAi. After knocking down the expression of annexin I , the growth speed, cell cycling, morphological features and apoptosis of pancreatic carcinoma cells were examined by RT-PCR and MTT test.

RESULTSWhen the expression of annexin I was blocked, the growth speed of pancreatic carcinoma cells was significantly decreased, the morphological features were changed and pronounced apoptosis occurred.

CONCLUSIONAnnexin I can modulate pancreatic carcinoma cell cycle, promote the cell proliferation, increasingly stimulate the cell growth, and suppress the process of apoptosis in pancreatic carcinoma cells.

Annexin A1 ; genetics ; metabolism ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

PURPOSE: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory enzyme expressed in atherosclerotic plaques. We investigated the association of circulating Lp-PLA2 with characteristics of vulnerable coronary atherosclerotic plaques. MATERIALS AND METHODS: We recruited 113 patients with either unstable angina (UA, n=59) and stable angina (SA, n=54) by coronary angiography. Thirty-six healthy subjects served as controls. Intravascular ultrasound (IVUS) was used to evaluate the characteristics of coronary atherosclerotic plaque, and serum Lp-PLA2 concentration was measured as well. RESULTS: Lp-PLA2 concentration was significantly higher in both UA and SA patients [(396+/-36) microg/L and (321+/-39) microg/L, respectively] compared with the controls [(127+/-49) microg/L, p<0.01], and higher in UA than SA group. IVUS findings showed that remodeling index (RI) (0.91+/-0.15 vs. 0.85+/-0.11, p=0.005) and eccentricity index (EI) (0.73+/-0.16 vs. 0.65+/-0.22, p=0.039) were larger in UA than in SA group, and fibrous caps were thicker in SA than UA group [(0.91+/-0.23) mm vs. (0.63+/-0.21) mm, p=0.032]. Moreover, Lp-PLA2 correlated positively with EI (r=0.439, p<0.01) and RI (r=0.592, p<0.05) in UA group. There was an inverse relationship between Lp-PLA2 and fibrous cap thickness in both UA (r=-0.587, p<0.001) and SA (r=-0.318, p<0.05) groups. The independent risk factors in UA group were Lp-PLA2 (OR=1.055, 95% CI: 1.03-1.08, p=0.013), LDL-cholesterol (OR=0.032, 95% CI: 0.00-0.05, p=0.041) and fibrous cap thickness (OR=0.008, 95% CI: 0.00-0.45, p=0.019). Lp-PLA2 was strongly associated with both EI and fibrous cap thickness in both groups. CONCLUSION: Serum level of Lp-PLA2 is associated with both eccentricity index and fibrous cap thickness in both UA and SA groups. Elevated levels of circulating Lp-PLA2 might to be a strong risk factor and more serious for unstable angina than stable angina.
1-Alkyl-2-acetylglycerophosphocholine Esterase/*blood ; Adult ; Aged ; Aged, 80 and over ; Angina, Stable/*blood/enzymology/*pathology ; Angina, Unstable/*blood/enzymology/pathology ; Coronary Angiography ; Coronary Artery Disease/*blood/enzymology/*pathology ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo analyze the value of dynamic contrast-enhanced magnetic resonance imaging (CEMRI) for evaluating diffuse tumor infiltration of bone marrow in patients with acute leukemia (AL).

METHODSFrom January 2010 to February 2016, 80 AL patients admitted in our hospital were chosen as AL patient group, 100 healthy people were chosen as control group, and all subjects were diagnosed with MRI and CEMRI. The Emax and Slope of ilium and vertebra lumbalis were compared between AI patient and control groups. The relation of Emax and Slope with protocells % was analyzed.

RESULTSThe Emax and Slope of AL patients were significantly higher than those of control group (P<0.05). After treatment, the Emax and Slope of CR/PR patients decreased significantly (P<0.05). Multiple regression analysis showed that the protocells %=-0.5632+0.0540 Emax+0.0056 Slope. The Emax and Slope of AL patients had significant correlation with Protocells %(P<0.05).

CONCLUSIONThe results of CEMRI relate with pathological examination and treatment effect.