Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.

Adult ; Cytokines ; Humans ; Lymphocyte Subsets ; Lymphohistiocytosis, Hemophagocytic ; Lymphoma ; T-Lymphocyte Subsets ; Th1 Cells ; Th2 Cells

OBJECTIVETo discuss the mechanisms of electroacupuncture at "Neiguan" (PC 6) on p38 MAPK signaling pathway in rats with cardiac hypertrophy.

METHODSForty SD rats were randomly divided into four groups: a normal group, a model group, a model plus p38 MAPK inhibitor group, a model plus electroacupuncture group, ten rats in each group. The model rats were established by subcutaneous injection 3 mg/(kg x d) of Isoprenaline Hydrochloride; model plus p38 MAPK inhibitor group were injected 0.3 mg/(kg x d) of specific inhibitor SB 203580; model plus electroacupuncture group was treated by electroacupuncture at "Neiguan"(PC 6) with continuous-wave, 2 Hz and 1 mA for 20 minutes, once a day for 14 days. There was no treatment in other two groups. The contents of TNF-alpha and IL-1beta in heart tissue were detected by radioimmunoassay and the p38 MAPK, p-p38 MAPK by western blot.

RESULTSCompared with normal group, the contents of TNF-alpha, IL-1beta, p38 MAPK, p-p38 MAPK were significantly increased in model group (all P < 0.01). The contents of TNFalpha, IL-1beta, p38 MAPK, p-p38 MAPK were significantly decreased in model plus p38 MAPK inhibitor group and model plus electroacupuncture group, compared with model group, all P < 0.05; compared with normal group, P < 0.05, P < 0.01; but no significant difference between model plus p38 MAPK inhibitor group and model plus electroacupuncture group (P > 0.05).

CONCLUSIONElectroacupuncture at "Neiguan" (PC 6) can prevent the phosphorylation of p38 MAPK of myocardial hypertrophy, and the mechanism maybe adjust p38 MAPK signaling pathway by inhibiting the expression of TNF-alpha and IL-1beta.

Acupuncture Points ; Animals ; Cardiomegaly ; metabolism ; therapy ; Electroacupuncture ; Interleukin-1beta ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

METHODSPIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.

RESULTSWe successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.

CONCLUSIONThe construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.

Animals ; Cell Line ; Genetic Vectors ; Lentivirus ; genetics ; Male ; Mice ; Plasmids ; Protein Inhibitors of Activated STAT ; genetics ; Spermatocytes ; cytology ; Transfection

Hypertension is a common complication after renal transplantation. Among post-transplantation patients died of cardiovascular diseases, about 41% have hypertension. Hypertension is an independent risk factor for kidney transplant failure. Post-transplantation hypertension can be caused by many factors, including the use of immunosuppressants. When the blood pressure exceeds 130/90 mmHg in a kidney transplant recipient, it is reasonable to provide active medical intervention. In summary, prevention and treatment of hypertension is important to prolong the survival of kidney transplant recipients.
Humans ; Hypertension ; diagnosis ; etiology ; prevention & control ; therapy ; Kidney Transplantation ; Postoperative Complications ; diagnosis ; etiology ; prevention & control ; therapy

Kidney transplantation has become an important method in treating advanced renal failure. Immunosuppressants play a key tool in this progress. It is important to understand the goal, mechanism, and adverse effects of immunosuppressive therapy, so as to appropriately use these drugs in post-transplantation patients on a customized basis.
Aftercare ; Humans ; Immunosuppressive Agents ; administration & dosage ; adverse effects ; therapeutic use ; Kidney Transplantation ; Long-Term Care

OBJECTIVEIt is controversial whether unilateral interruption of the arteria iliaca interna distal end affects penile hemodynamics and erectile function. The purpose of this study was to prospectively evaluate this influence by detecting the blood flow of the penile artery before and after renal transplantation.

METHODSThirty-three patients with chronic renal failure (CRF) on maintenance hemodialysis (MHD) received renal transplantation, the grafts revascularized by end-to-end anastomosis to the right internal iliac artery. Six months before and after the surgery, we obtained the IIEF scores of the patients, recorded their penile blood flow on color Doppler ultrasonography and the levels of serum creatinine, hemoglobin and serum cholesterol, and analyzed post-transplantation immunosuppressive medication.

RESULTSThe patients ranged in age from 21 to 55 years, of whom 36% had erectile dysfunction (ED) during MHD, and 33% after renal transplantation. A total of 67% of the renal transplant recipients (RTR) complained of unchanged and 15% deteriorated ED, while 18% admitted improved erectile function. The patients showed a significantly stronger sexual desire after the transplantation than before it (6.2 +/- 1.6 vs 8.9 +/- 0.9, P < 0.01). There was a significant decrease in peak systolic velocity (PSV) in the cavernous arteries after transplantation as compared with pre-transplantation (P < 0.01). Penile arterial blood flow insufficiency was found in none of the RTRs.

CONCLUSIONUnilateral interruption of the internal iliac artery decreases penile arterial blood flow, but not to such a degree as to result in ED. Unilateral interruption of the arteria iliaca interna distal end does not affect the erectile function of RTRs.

Adult ; Anastomosis, Surgical ; Humans ; Iliac Artery ; surgery ; Kidney Transplantation ; Male ; Middle Aged ; Penile Erection ; Penis ; blood supply ; Priapism ; etiology ; Prospective Studies ; Renal Artery ; surgery ; Young Adult

OBJECTIVETo study the apoptotic effect on the squamous cell carcinoma cell line TCa83 induced by recombined adenovirus vector containing TRAIL gene and CMV promoter.

METHODSThe TCa83 cell line was firstly infected with different titre of AdCMV-EGFP containing enhanced green fluorescence protein gene (EGFP) as control, and investigated the transducing rate through fluorescence to obtain the definite titre. Then TCa83 cell line was infected with AdCMV-TRAIL in proper titre, and TRAIL gene was detected by means of RT-PCR. After TCa83 cell line was infected with AdCMV-TRAIL and AdCMV-EGFP at day 1, 3, 5, 7, the activity of TCa83 cell line were evaluated by MIT and the apoptosis were detected by flow cytometer.

RESULTSProper titre was of 1,000 particles/cell, and TCa83 cell line could be infected 100% in this titre. TRAIL gene was detected by RT-PCR after infected with AdCMV-TRAIL. The activity of TCa83 decreased in both groups, but the AdCMV-TRAIL group decreased more sharply than AdCMV-EGFP group (P < 0.001). Both AdCMV-TRAIL and AdCMV-EGFP could lead to apoptosis of TCa83 cells, but the AdCMV-TRAIL, function stronger than AdCMV-EGFP. Especially there was remarkable statistic difference between two groups (P < 0.0001).

CONCLUSIONAdCMV-TRAIL could effectively decrease the activity of TCa83 cell line and induce apoptosis.

Adenoviridae ; Apoptosis ; Carcinoma, Squamous Cell ; Cell Line ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Promoter Regions, Genetic

OBJECTIVETo investigate the histological components and solubility of the bile-cast, and to study the pathological course of bile cast formation.

METHODSHE staining, bilirubin staining (Gmelin reaction), Masson's staining, alcian blue staining and fibrin staining (weigert's) were performed on the formalin-fixed paraffin-embedded section of the bile cast. Ultrastructure was examined under the scanning electron microscope. Solubility test was also conducted using chymotrypsin, heparin, trypsin solution, HCl and NaOH solution to dissolve the bile-cast.

RESULTSThe major components of the bile-cast were bilirubin crystals and collagen fibers. Between the mass of collagen fibers there was certain blood vessel structure. Necrosis bile duct structure was not found in the cast. Under the scanning electron microscope, four kinds of crystal morphologies were viewed. There were some mucoid mass and necrosis defluvium epithelial cells in the bile cast. Solubility test showed that the bile cast could be partial dissolved in NaOH solution (pH = 12.5). No dissolution was found in HCl solution (pH = 5.0), chymotrypsin solution, heparin and trypsin solution.

CONCLUSIONSCollagen fibers work as framework in the bile cast with bilirubin crystal filling between the framework. The emergence of fibroblast and blood vessels indicated the formation of bile cast might be the course of exudation and organization due to bile duct epithelium damage. Bile cast could be partially dissolved in alkaline solution, but could not be dissolved in acid solution, or in chymotrypsin, heparin and trypsin solutions.

Bile Duct Diseases ; etiology ; pathology ; Bile Ducts ; ultrastructure ; Humans ; Immunohistochemistry ; Liver Transplantation ; adverse effects ; Microscopy ; Postoperative Complications ; Staining and Labeling

Objective:To investigate the effects of family dignity intervention(FDI)on anxiety,depression,hope level and quality of life(QOL)of male infertility patients and their spouses.Methods:Using quasi-experimental design,we selected male in-fertility patients and their spouses undergoing human-assisted reproductive technology(ART)in our Center of Reproductive Medicine from June to December 2022 and divided them into an intervention group(38 couples)and a control group(40 couples).The former underwent a four-stage FDI,including ovulation promotion cycle assessment,family sharing,pre-transplantation interview and post-transplantation follow-up,while the latter received routine nursing.Using Hospital Anxiety and Depression Scale,Herth Hope Index and Fertility Quality of Life Scale,we evaluated the effects of FDI before and after transplantation.Results:After FDI,the anxiety and depression scores were significantly lower(P＜0.05)and the total scores on the hope level and all other dimensions remarkably higher in the intervention group than in the control(P＜0.05).The self-confidence of the couples in the intervention groups in ART treatment was markedly increased in comparison with that of the controls,and their scores on physical and mental health were signifi-cantly higher than those of the latter(P＜0.05).Conclusion:FDI can effectively relieve the anxiety and depression,raise the hope level and improve the quality of life of both male infertility patients and their spouses.