Animals ; Biomarkers ; Hexanes ; poisoning ; toxicity ; Humans ; Neurotoxicity Syndromes ; etiology

OBJECTIVETo investigate the possible mechanism of compound Chinese sour taste herbs (CS) in preventing and ameliorating diabetic macroangiopathy by analyzing the effects of CS on the deposition of advanced glycation end products (AGEs) and gene expression of receptor for advanced glycation end products (RAGE) in the aorta tissue of rats with type 2 diabetes mellitus (T2DM).

METHODSRat model of T2DM was established by peritoneal injection of streptozotocin (STZ) and high caloric diet feeding. Experimental SD rats were divided into the normal group, the model group, the aminoguanidine (AG) group, and the CS group. At the end of the 8th and 12th week, the fasting blood glucose (FBG) was measured by glucose oxidase method; content of AGEs and collagen in aorta detected by fluorescent method and gene expression of RAGE in aorta determined by Real-time PCR method.

RESULTSFBG, AGEs and collagen contents and RAGE expression in aorta of model rats were all higher than those in the normal control group (P <0.05), while all these indices were lower in the CS group than in the model group (P<0.05).

CONCLUSIONCS could realize the goal for preventing and ameliorating diabetic macroangiopathy by way of suppressing the production of AGEs and down-regulating the gene expression of RAGE in aorta of T2DM rats.

Animals ; Aorta ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Diabetes Mellitus, Type 2 ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Glycation End Products, Advanced ; metabolism ; Male ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism

OBJECTIVETo analyze the relationship of K-ras mutation with the development of liver metastasis in colorectal cancer patients and the survival outcomes.

METHODSFrom 2003 to 2008, 300 patients who underwent colorectal cancer surgery in the Department of General Surgery of Zhongshan Hospital, Fudan University were assigned to different groups, according to the diagnosis and follow-up results. The mutation of exon 2 of K-ras was detected in primary paraffin-embedded lesions by PCR and Pyrosequencing. The association of gene mutation with the development of liver metastasis and its prognosis was studied.

RESULTSAmong 300 cases, the mutations of exon 2 were present in 120 cases(40%). The G13D mutation was more common in metachronous metastasis group than that in synchronous group(17.0% vs. 8.0%, P=0.041). Multivariable regression analysis showed that G13D mutation was an independent risk factor(HR=1.108, 95%CI:1.032-5.062, P=0.048) for metachronous metastasis. Patients with mutated K-ras had a poorer overall survival compared to those without mutated K-ras for patients without liver metastasis(median overall, 65 vs. 72 months, P=0.039), and for patients who received metastasis resection(median disease-free survival 18 vs. 24 months, P=0.048). Multivariable analysis showed that K-ras mutation was an independent risk factors of overall survival(HR=1.561, 95%CI:1.022-6.422, P=0.045) in patients without liver metastasis.

CONCLUSIONDetection of K-ras mutation may predict the development of liver metastasis and prognosis.

Aged ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Genes, ras ; genetics ; Humans ; Liver Neoplasms ; genetics ; secondary ; Male ; Mutation ; Prognosis

Acute Disease ; Adolescent ; Adult ; Aged ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Pesticides ; poisoning ; Poisoning ; epidemiology

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.
Animals ; COS Cells ; Cercopithecus aethiops ; Factor VIII ; chemistry ; genetics ; metabolism ; Genetic Vectors ; Inteins ; Leucine Zippers ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Splicing ; Trans-Splicing ; Transfection

Although two chain transfering separately could be used to overcome the volume limitation of adeno-associated virus vectors (AAV) in coagulation factor VIII (FVIII) gene delivery, it leads to chain imbalance for inefficient heavy chain secretion. In this study we aimed to improve the efficacy of two chain strategy in FVIII gene delivery through the degradation of glucose-regulated protein 78 (GRP78) known as a protein chaperone in endoplasmic reticulum (ER) by deoxynivalenol (DON) to decrease GRP78-bound FVIII heavy chain. By treating the two-chain gene transduced 293 cells with DON, the heavy chain (HC) secretion and FVIII bioactivity were observed. Data showed that 293 cells after three hours post-treatment with DON at a concentration of 500 ng mL(-1) resulted in obvious decrease the level of GRP78 but no effect on the cell proliferation. The HC secreted from DON-treated cells transfected with HC gene alone was 59 +/- 11 ng mL(-1), higher than that secreted by control cells (15 +/- 4 ng mL(-1)), and the HC secretion was further increasing to 146 +/- 34 ng mL(-1) in light chain (LC) gene co-transfected cells with an activity measured up to 0.66 +/- 0.15 U mL(-1), also greater than control cells (76 +/- 17 ng mL(-1) and 0.35 +/- 0.09 U mL(-1)). Taken together, these data suggest that DON-mediated GRP78 down-regulation could improve the efficacy of two-chain FVIII gene transfering by facilitating HC secretion, providing an experimental basis for in vivo dual-AAV application in FVIII gene delivery.
Cell Proliferation ; Down-Regulation ; Factor VIII ; chemistry ; genetics ; secretion ; Gene Transfer Techniques ; HEK293 Cells ; Heat-Shock Proteins ; metabolism ; Humans ; Transfection ; Trichothecenes ; pharmacology

BACKGROUNDTranscription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma.

METHODSSpecimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed.

RESULTSHighly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour.

CONCLUSIONSEPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.

Adult ; Aged ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; physiology ; Blotting, Western ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Male ; Middle Aged ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; physiology ; Young Adult

2600 Anthers from T0 modified cry1 Ac-transgenic rice lines of Minghui 81, an elite restoring line of commercial CMS indica hybrid rice, were cultured on SK3 media. 83 green plantlets were recovered, 43 double haploid (DH) and 40 haploid among them. Results of PCR analyzes indicated that 55 plants of 83 were harbored the cry1Ac gene, and the ratio of cry1Ac-positive against cry1Ac-negative was 2:1 (55/28). 36 putative transgenic DH plants were further confirmed by Southern blot. ELISA detection showed that Cry1Ac level in different transgenic rice plants of the same cry1Ac-DH clone was almost equal and the highest one amount to 0.25% of the total soluble protein. Pest insect-resistant bioassay at field trials demonstrated that some of the homozygous cry1Ac-transgenic rice plants not only showed high-level resistance against striped stem borer (Chilo suppressalis) but also retained elite agronomy characters. These results demonstrated that rice anther culture has a great value in rice molecular breeding.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; Blotting, Southern ; Culture Techniques ; DNA, Plant ; genetics ; Endotoxins ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hemolysin Proteins ; Immunity, Innate ; genetics ; Moths ; growth & development ; Oryza ; genetics ; growth & development ; parasitology ; Plant Structures ; genetics ; growth & development ; parasitology ; Plants, Genetically Modified

Prawn white spot syndrome is caused by the pathogen prawn white spot syndrome virus (WSSV). VP19 is a vesicle membrane protein of WSSV. HyNPV (Hybrid of AcNPV and BmNPV) constructed by the recombination of BmNPV and AcNPV is a new hybrid virus having both of their advantages. The recombinant transfer vector pBlueBicHisC-vp19 and recombinant baculovirus HyNPV-VP19 were constructed on the basis of the successful cloning of VP19. Newly-molted silkworms Bombyx mori of fifth instar were inoculated by the recombinant virus. SDS-PAGE and Western blotting analysis showed a specific band, about 21kD, which was consistent with the expectation suggesting that the WSSV-VP19 gene was successfully expressed in silkworm bodies.
Animals ; Baculoviridae ; genetics ; metabolism ; Bombyx ; genetics ; metabolism ; virology ; Genetic Vectors ; Penaeidae ; virology ; Recombinant Fusion Proteins ; genetics ; Viral Envelope Proteins ; genetics ; metabolism ; Virus Replication ; White spot syndrome virus 1 ; genetics

OBJECTIVETo investigate the clinical application value of 8.5/11.5 F transurethral seminal vesiculoscopy in the diagnosis and treatment of refractory hematospermia.

METHODSWe retrospectively analyzed 78 cases of refractory hematospermia diagnosed and treated by 8.5/11.5 F transurethral seminal vesiculoscopy from June 2012 to June 2014. The patients underwent serum PSA examination, transrectal ultrasonography, seminal vesicle ultrasonography, and pelvis CT or MRI before surgery, and all received transurethral seminal vesiculoscopy under the 8.5/11.5 F rigid ureteroscope.

RESULTSOperations were all successfully accomplished, which revealed abnormal opening of the ejaculatory duct in 5 cases, mucosal inflammatory hyperemia in the prostatic utricle and seminal vesicle in 78, dark red mucilage substance in the seminal vesicle in 34, seminal vesicle stones in 19, small polyp in the seminal vesicle in 2, and ejaculatory duct or seminal vesicle cyst in 4. All the patients received symptomatic treatment during the surgery. After surgery, hematouria was found in 13 cases, which disappeared within 2 weeks, pelvic hematoma in 1 case, which was cured by conservative treatment within 3 months, and epididymitis in 2 cases, which was controlled by anti-infection treatment. Hematospermia recurred in 3 cases during the 1-year postoperative follow-up.

CONCLUSION8.5/11.5 F transurethral seminal vesiculoscopy, with its advantages of easy operation, wide field of vision, large channel for operation, and few complications, deserves general clinical application in the diagnosis and treatment of refractory hematospermia.

Calculi ; Ejaculatory Ducts ; Endoscopy ; methods ; Epididymitis ; etiology ; Hemospermia ; diagnosis ; therapy ; Humans ; Magnetic Resonance Imaging ; Male ; Postoperative Period ; Recurrence ; Retrospective Studies ; Seminal Vesicles ; Tomography, X-Ray Computed ; Urethra