A simple and effective method for the duplex PCR detection was developed by using sequences of exogenous gene(RDV MP-)and endogenous gene(Zein)as templates for PCR amplification.The results of routine PCR amplification for RDV MP-gene in transgenic maize suggested that RDV MP-gene can stably inheritate in transgenic plants and their progenies;The duplex PCR detection of all negative and part positive samples that obtained by routine PCR amplification confirmed that above negative results were exact,also showed that the quality of extracted DNA can meet the need of PCR amplification.The error ratio of negative samples was 1.4%.The method used in this study was simple and credible and can be used to detect transgenic plants and their products.

Objective: To investigate the killing activity of cytokine-induced killer (CIK) cells after incubation with autologous tumor cell lysate-pulsed dendritic cells (Ag-DC) and to evaluate the immune functions of patients, the clinical efficacy and side effect of Ag-DC in combination with CIK on hepatocellular carcinoma. Methods: Peripheral blood mononuclear cells were isolated from 24 patients with hepatocellular carcinoma, and cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4 to produce dendritic cells (DCs). The DCs were pulsed with autologous tumor cell lysate. T lymphocytes from PBMC were cultured with interferon-Y (IFN-γ), IL-2, CD3-moAb, and IL-1α to prepare CIK. The killing effect of CIK on SMMC-7721 was investigated after CIK was incubated with Ag-DC. After immunotherapy with Ag-DC and CIK, immunolog-ic and clinical responses of the 24 patients were evaluated. Results: The killing effect of CIK was remarkably improved after CIK was incubated with Ag-DC. The immunotherapy with DC and CIK alleviated symptoms and improved the immune functions of the patients. Except for transient fever and chill, no remarkable adverse events were observed. Conclusion: Ag-DC in combination with CIK shows short-term efficacy on hepatocellular carcinoma through inducing specific anti-tumor immunity and can be an effective adjuvant therapy for hepatocellular carcinoma.

OBJECTIVETo observe the clinical effects of needle-pricking therapy, a newly medical and minimally invasive technique, for functional retrograde ejaculation and to explore its mechanism. Methods Thirty-six patients with functional retrograde ejaculation were randomly divided into an observation group(19 cases) and a control group(17 cases) In the observation group,needle-pricking therapy was used at Guanyuan(CV 4) and bilateral sacral plexus nerve,lumbar 1 nerve and greater occipital nerve stimulating points,once a week. In the control group, midodrine tablets were prescribed orally,three times a day. All the treatment was given for 9 weeks. The clinical effects of the two groups were observed, and the levels of luteinizing hormone(LH), testosterone(Tes) and estra4 diol(E2) were compared between the two groups.

RESULTSThe total effective rate of the observation group was, 89. 47%(17/19), which was better than 47.06% (8/17) of the control group(P<0. 05). The LH and Tes were obviously increased and E2 was decreased compared with those before treatment in the observation group(all P< 0. 01). Tes was raised(P<0. 05) and E2 was apparently declined in the control group(P<0. 01). After treatment, the differences of serum LH and Tes were statistically significant between the two groups(both P<0. 01).

CONCLUSIONNeedle-pricking therapy has advantages for functional retrograde ejaculation probably in that stimulating lumbosacral nerves can strengthen the function of pelvic floor muscles and urethral expansion muscle and regulate sexual gland axis.

Acupuncture Points ; Acupuncture Therapy ; Adult ; Ejaculation ; Humans ; Male ; Middle Aged ; Needles ; Sexual Dysfunction, Physiological ; physiopathology ; therapy ; Treatment Outcome

ObjectiveTo investigate the imaging characteristic of DW1,tumor cell apoptosis and proliferation of nasopharyngeal carcinoma (NPC) xenograft transfected hNIS gene after 131Ⅰ treatment.MethodsThe NPC xenograft models were established with CNE-2-hNIS cells(experimental group) and CNE-2 cells(control group) respectively.After i.p.injections 131Ⅰ in the experimental group and control group,the changes of xenograft tumor volume and ADC value were observed in 3,6,12,18,24 days by MR scan.The correlations of changes of ADC with pathological TUNEL,Caspase-3 immunohistochemistry of apoptosis,and Ki-67 proliferation detection were further investigated.Independent samples t-test were compared between the two groups and Pearson linear correlation analysis was used.ResultsThe tumor volume of the experimental group was significantly reduced compared with that of the control group after 131 Ⅰ treatment (t values:8.27-19.46,P ＜0.05 ).DW-MRI showed that in the 3th day after 131Ⅰ treatment,ADC values increased in the experimental group,and ADC values reached the peak in the 6th and 12th day after 131Ⅰ treatment,then ADC values were decreased. ADC values after treatment was positively correlated with apoptotic index ( r =0.72,P ＜ 0.05 ) and caspase-3 positive rate ( r =0.65,P ＜ 0.05) and there was a negative correlation with Ki-67 proliferation index ( r =- 0.71,P ＜ 0.05 ).ConclusionADC values can reflect growth inhibition of NPC xenograft transfected hNIS gene after 131 I treatment.It is possible that DWMRI techniques can be used in early non-invasively monitor the therapy effect of 131Ⅰ treatment of NPC xenografts transfected hNIS gene.

The aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO_2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline (CTC) staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after 1h incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae.The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid)at 3' end and 45 nucleotides at 5' end that annealed to sites upstream or downstream of the genomic target sequence to be deleted.After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS-1,the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.Once correctly integrated into the genome,the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS-1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure.The expression of the Cre recombinase finally resulted in the removal of the marker gene,leaving behind a single loxP site at the chromosomal locus.The diploid mutant YS-1-sfa1 was generated,which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS-1.

Objective To investigate the clinicopathological features,diagnosis,treatment,prognosis and causative agent of a case of eumycetoma on the submaxilla.Methods A case of eumycetoma diagnosed in our department was assessed for its clinical and pathological features as well as mycologic and molecular identification.Related literature was reviewed.Results The patient was primarily characterized by swelling of the submaxilla,with multiple sinuses draining many black granules.Pathologic examination revealed a pyogenic granulomatous inflammation,and a number of lotus rhizome node-like hypha were observed in tissue samples through PAS staining.Sequence analysis of multiple loci of the isolates,including ITS 1,ITS2 and D1/D2,showed that it was mostly similar to Madurella mycetomatis with a homology of 97%.Conclusion This is a case ofeumycetoma on the submaxilla induced by a novel species of Madurella.

OBJECTIVETo investigate the correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 as well as the effect of EGFR-cDNA transfection on the expression of bcl-2 and Bax in U87 cells.

METHODSThe correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 was studied by confocal microscopy and Western blot. The expression of bcl-2 and Bax in EGFR-cDNA transfected and untransfected glioblastoma cell lines was studied by Western blot.

RESULTSConfocal microscopic images showed that bcl-2 protein was localized in the periphery of the nuclear matrix and Bax in the nuclear matrix. A 26 kDa bcl-2 band and a specific band of Bax at about 66 000 were detected in nuclear matrix proteins by western blot. The expression of bcl-2 was lower but that of Bax was higher in EGFR-cDNA transfected cells than the control.

CONCLUSIONBcl-2 and Bax, being nuclear matrix associated proteins, are probably involved in the EGFR-cDNA induced malignant conversion of glioblastoma cells by introducing EGFR cDNA into the tumor cells.

Apoptosis ; Cell Line, Tumor ; Glioblastoma ; pathology ; Humans ; Nuclear Matrix ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; physiology ; Receptor, Epidermal Growth Factor ; genetics ; physiology ; Transfection ; bcl-2-Associated X Protein ; analysis ; physiology

Objective To study the impact of hypothyroidism on sperm motility in male rat.Methods According to body weight,20 male Wistar rats were randomly divided into control group and hypothyroidism group (1 ml/100 g/day,0.1％ propylthiouracil by intragastric administration for 60 days) 10 rats in eachgroup.Body weight of these rats was observed every 3 days.After the last intragastric administration,all rats were killed.The levels of thyroid hormones [total triiodothyronine (T3),total thyroxine (T4),and thyroid stimulating hormone (TSH)] were measured by radioimmunoassay.Sperm motility parameters[average path velocity(VAP),straight fine velocity (VSL),straightness (STR),amplitude of lateral head displacement (ALH),sperm density(p),curvilinear velocity (VCL),linearity(LIN),wobble (WOB),mean angular deviation (MAD) and beat cross frequency (BCF)] were measured by a WLJY-9000 color-detection system.Results Compared with the control groups[(298.20 ± 12.15) g,(344.00 ± 13.73)g],the weights of hypothyroidism group of the 30 days[(239.00 ± 15.02) g] and the 60 days [(232.67 ± 17.86)g] were significantly lower(t =7.704,11.380,all P ＜ 0.05).The levels of T3[(373.3 ± 101.3) ng/L] and T4 [(4.00 ± 0.89) × 103 ng/ml] of hypothyroidism group were significantly decreased compared with that of the control groups [(1000.0 ± 273.5)ng/L,(44.33 ± 7.84)× 103 ng/L,t =5.262,12.520,all P ＜ 0.05].Level of TSH[(5.77 ± 0.89) × 103 U/L] of hypothyroidism group was significantly increased compared with that of the control group[(1.87 ± 0.70) × 103 U/L,t =8.413,P ＜ 0.05].Values of VAP[(27.45 ± 1.59)μm/s],VSL [(21.08 ± 1.10)μm/s],STR[(70.53 ± 3.48)％] and ALH[(1.96 ± 0.26)μm] of hypothyroidism group were significantly increased compared with that of the control groups[(24.38 ± 2.59)μm/s,(17.99±2.06)μm/s,(65.93 ± 2.71)％,(1.53 ± 0.27)μm,t =2.687,2.404,2.420,3.175,all P ＜ 0.05].p[(5.07 ± 0.74)109/L] of hypothyroidism group was significantly decreased compared with that of the control group [(8.76 ± 1.01)109/L,t =6.463,P ＜ 0.05].VCL[(52.83 ± 5.56)μm/s],LIN[(38.58 ± 3.41)％],WOB[(52.64 ± 3.24)％],MAD [(64.21 ± 6.71) radian/s] and BCF [(8.93 ± 0.62) Hz] of hypothyroidism group were not significantly different compared with that of the control groups[(49.92 ± 6.43) μm/s,(36.52 ± 2.73)％,(52.49 ± 3.49)％,(62.77 ± 7.34)radia/s,(9.32 ± 0.61)Hz,t =0.805,1.089,0.037,0.341,1.033,all P ＞ 0.05].Conclusion Hypothyroidism can affect sperm activity in male rats,decrease sperm density and cause damage to the reproductive system.

OBJECTIVETo summarize the experience of urethral reconstruction using circular fasciocutaneous flap for the treatment of complex anterior urethral strictures.

METHODThe circular fasciocutaneous flap was harvested from the distal penile shaft or foreskin. Then the circular configuration was converted into longitudinal strip of skin which was rotated to ventral side to repair the exposured urethral strictures using the ventral onlay method. The surveillance protocol after urethroplasty was urination observation, regularly uroflowmetry and urethrography examination.

RESULTSFrom Nov. 2006 to Oct. 2012, 15 cases were treated. The mean age was 45 years (20-76 years) and mean follow-up period was 18 months(3 months-3 years). Stricture was caused by chronic urethritis in 4 cases, long-term urethral catheterization in 3 cases, transurethral perfusion chem other aphy in 3 cases, transurethral prostatectomy in 3 cases and hypospadias after surgery in 2 patients. The mean stricture length was 7.0 cm (3.5-12.0 cm). The overall success rate was 80.0% (12/15). Recurrence stenosis was noted in 2 cases and diverticulum formation in 1 case.

CONCLUSIONSThe penile circular fasciocutaneous flap can be used for anterior urethral stricture in nearly any length. The flap has the characteristics of hairless, adequate mobile and length, well-vascularized pedicle and easy to harvest. The onlay reconstruction provides excellent cosmetic results, less trauma, higher success rate. Therefore it should be one of the preferred techniques for complex anterior urethral stricture repair.

Follow-Up Studies ; Foreskin ; Humans ; Hypospadias ; surgery ; Male ; Penis ; surgery ; Prostatectomy ; adverse effects ; Recurrence ; Surgical Flaps ; transplantation ; Urethra ; surgery ; Urethral Stricture ; etiology ; pathology ; surgery ; Urologic Surgical Procedures, Male ; Wound Healing