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BACKGROUND The birth prevalence of orofacial clefts (OFCs) has been widely studied, but results are considerable varied, and epidemiological studies in southern China are few in numbers. To address this gap, we carried out a register-based study to estimate the birth prevalence of OFCs in Bao'an district, Shenzhen, China. METHODS Data of perinatal infants born between 2003 and 2017 were extracted from Shenzhen Maternal and Child Health Management System. The overall OFCs birth prevalence with 95% confidence interval (CI) as well as subgroup analysis based on selected demographic factors was conducted. Cochran-Armitage trend tests were applied to evaluate the time trend by 5-year intervals. RESULTS The overall birth prevalence of OFCs, cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO) was 1.30 (95% CI 1.21-1.39), 1.00 (95% CI 0.92-1.08), and 0.30 (95% CI 0.25-0.34) per 1,000 births, respectively. An overall declining tendency was observed in the OFCs (from 1.83 to 1.04 per 1,000 births), specifically CL/P (from 1.53 to 0.69 per 1,000 births) birth prevalence over 5-year intervals, with statistical significance (p < 0.01). Subgroup analysis revealed that the CL/P and CPO birth prevalence was differed by infant gender, household registration, maternal age, and parity. CONCLUSION Our findings had firstly reported the birth prevalence of OFCs in Bao'an district, and might help other researchers to plan more comprehensive public health strategies to reduce the occurrence of OFCs in further generation.

Objective Kasabach-Merritt syndrome is a rare disease that mainly occurs in infants and adolescents. It usually manifests as disseminated intravascular coagulation and severe bleeding, and is associated with high mortality. However, its low incidence and clinical rarity in adults mean that there is currently no well-verified treatment regimen for this disease. We report on an effective novel therapeutic regimen in a patient with Kasabach-Merritt syndrome. Methods A woman with Kasabach-Merritt syndrome presented with a recurrent subcutaneous mass and disseminated intravascular coagulation, and was treated with prednisone, vincristine and thalidomide. Results This treatment regimen successfully resolved the patient’s symptoms, with tumor regression. The patient remained disease-free after 6 years of follow-up. Conclusions Prednisone combined with vincristine and thalidomide may be an effective treatment for Kasabach-Merritt syndrome, but further studies are needed to verify the use of this regimen.

Calcyclin-binding protein (CACYBP) is a multi-ligand protein implicated in the progression of various human cancers. However, its function in hepatocellular carcinoma (HCC) remains unknown. Methods: The expression of CACYBP and RNF41 (RING finger protein 41) in HCC cancer and adjacent non-tumor tissues was detected by immunohistochemistry. CCK-8 assays, colony formation assays, flow cytometry detection and xenograft models were used to evaluate the impact of CACYBP expression on HCC cell growth, apoptosis and cell cycle regulation. Immunoprecipitation and ubiquitination assays were performed to determine how RNF41 regulates CACYBP. The regulatory mechanism of RNF41-CACYBP signaling axis on P27Kip1 was investigated by western blotting and immunofluorescence. Results: CACYBP was highly expressed and associated with poor prognosis in HCC. CACYBP expression was required for HCC cell growth in vitro and in vivo. Moreover, we identified RNF41 as a specific binding partner of CACYBP at exogenous and endogenous levels. RNF41 recruited CACYBP by its C-terminal substrate binding domain, subsequently ubiquitinating CACYBP and promoting its degradation in both proteasome- and lysosome-dependent pathways. In HCC tissues, RNF41 expression was reduced and conferred a negative correlation with CACYBP expression. Mechanistically, CACYBP overexpression stimulated the Ser10, Thr157 and Thr198 phosphorylation of P27Kip1 and its cytoplasmic retention, and RNF41 co-expression attenuated this phenomenon. CACYBP depletion led to decreased levels of cyclin D1, cyclin A2, CDK2 and CDK4, causing a typical cell cycle arrest at G1/S phase and increasing apoptosis in HCC cells. P27Kip1-S10D but not P27Kip1-S10A reconstitution rescued partially the cell cycle function and apoptotic feature after CACYBP depletion. Conclusion: Our findings provide novel insights into the functional role and regulatory mechanism of CACYBP in HCC.

The relationship between the serum transforming growth factor (TGF)-β level and HBsAg loss has not been clearly elaborated in patients with chronic hepatitis B (CHB).Two cohorts of patients with CHB were studied. Cohort A: A total of 207 hepatitis B e antigen (HBeAg)-negative CHB patients who finished ≥1 year nucleos(t)ide analogue monotherapy and sequentially received PEGylated interferon treatment for less than 96 weeks were included. Cohort B: Forty HBeAg-positive patients who initially received entecavir therapy for at least 96 weeks were included. Their viral markers and serum TGF-β levels were measured at different time points during therapy.The levels of serum TGF-β and HBsAg (0-24 W) were significantly lower in the patients who had HBsAg0.05 IU/mL at 48 weeks than in patients who did not in cohort A. We got the same results when we further divided the patients into subgroups according to the initial HBsAg cut-off values (1000 IU/mL, 100 IU/mL, 50 IU/mL) in cohort A. However, HBeAg seroconversion did not lead to the downregulation of TGF-β levels. The levels of serum TGF-β were significantly correlated with HBsAg quantitation in cohort A (12-24 W) but not in cohort B (0-48 W). The levels of TGF-β at week 12 could be used as an early index to predict a functional cure (AUC=0.818) as well as the levels of HBsAg itself (AUC=0.882) in HBeAg-negative chronic hepatitis B patients treated with PEGylated interferon.The levels of serum TGF-β were significantly associated with HBsAg loss but not with HBeAg seroconversion and could be used as an early index to predict a functional cure in CHB patients treated with PEGylated interferon.

To evaluate preoperative anxiety and postoperative quality of life in patients with periodontal mucogingival surgery, and provide a theoretical basis for preventing preoperative anxiety and improving postoperative quality of life in mucogingival surgery.According to the inclusion and exclusion criteria, 26 patients with mucogingival surgery were randomly selected, including 13 cases undergoing free gingival graft and 13 cases undergoing subepithelial connective tissue graft. All patients were asked to answer the following questionnaires which included self-rating anxiety scale (SAS), modified dental anxiety scale (MDAS), pain evaluation using visual pain scale (VAS), clinical performance evaluation (swelling, bleeding, nausea, oral odor), and oral function evaluation (chewing, speaking, sleeping, working). Data analysis was performed using SPSS 18.0 software package.The preoperative SAS score was 44.33±11.99, 4 patients had anxiety, accounting for 15.38%. The preoperative MDSA score was 9.85±2.41, 4 patients had anxiety, accounting for 15.38%. The VAS values at 1 day, 3 days, 5 days, 7 days, and 10 days after surgery were moderate pain (4.54±1.32), mild pain (3.31±1.31), mild pain (2.00±1.14), and painless( 0.70±0.72), painless (0.08±0.27). The VAS values at 1 day, 3 days, and 5 days after FGG were greater than those after CTG (P0.05).The most common discomforts after mucogingival surgery were swelling, bleeding, disturbance in chewing and speech. Swelling, disturbance in chewing and speech persisted until 7 days after surgery, and bleeding continued until 5 days after surgery. The postoperative discomfort of FGG was significantly higher than that of CTG.Four had preoperative anxiety prior to mucogingival surgery. The main clinical symptoms after surgery were moderate to mild pain, swelling, bleeding, disturbance in chewing and speech within 1-7 days after surgery. The effect of CTG on the quality of life of patients was significantly less than that of FGG.

Background A growing body of evidence suggests that E2Fs, by regulating gene expression related to cell cycle progression and other cellular processes, play a pivotal role in human cancer. However, the distinct roles of each E2F in the development and treatment of hepatocellular carcinoma (HCC) remain unknown. In the present study, the mRNA expression and prognostic value of different E2Fs in HCC are analyzed. Materials and methods Transcriptional and survival data related to E2F expression in patients with HCC were obtained through ONCOMINE and UALCAN databases. Survival analysis plots were drawn with Kaplan–Meier Plotter. The sequence alteration data for E2Fs were obtained from The Cancer Genome Atlas and c-BioPortal. Gene functional enrichment analyses were performed in Database for Annotation, Visualization and Integrated Discovery. Results The mRNA expression levels of E2F1–E2F8 were all significantly upregulated in HCC patients, and high expression of each E2F was obviously related to poor prognosis. Similarly, the expression of E2Fs showed prognostic prediction value in HCC patients with different cancer stages and pathological grades. Moreover, the mutation rate of E2Fs was relatively high in HCC patients, and the DNA sequence alterations primarily occurred in E2F5, E2F3, and E2F6, which were associated with worse overall survival and disease-free survival in HCC patients. Network analysis confirmed that the expression levels of cell cycle-related genes were mostly affected by E2F mutations. Conclusion High expression of individual E2Fs was associated with poor prognosis in all liver cancer patients. E2Fs may be exploited as good prognostic targets for comprehensive management of HCC patients, but this notion should be further evaluated in clinical studies.

Wastewater treatment plants in large urban agglomerations are worldwide among the main sources of emerging contaminants in aquatic systems. The present study is a first comprehensive characterization of potentially harmful emerging contaminants in sewage of a Chinese urban area. The study area Haikou City is located at the coast of tropical Hainan Island. A GC/MS based non-target screening approach for the identification of a wider range of lipophilic to semi-polar organic sewage constituents was applied. Pollution originating from sewage was tracked in the urban coastal waters by using selected source-specific indicators. A set of sewage constituents with a high structural diversity was identified comprising both, industrial and household chemicals. Ingredients of personal care products, pesticides and pharmaceuticals were found, that were rarely reported from other regions of the world. This observation might be attributed to different environmental legislation and consumption habits in China. Exceptionally high concentrations of the illegal drug ketamine with up to 1100 ng L−1 were detected, suggesting its high popularity and consumption rate. Noteworthy, sewage constituents reported here for the first time were the pharmaceuticals marmesin, oxolamine and ansimar also with high concentrations of up to 1200 ng L−1. Elevated concentrations of sewage indicators in the main city area suggest the overflow of the sewage system, in particular in the period of a typhoon event. Tracing the sewage contamination in the surface water system of the coastal urban area of Haikou revealed a lower level of pollution. The concentration ranges point to no acute risks for the coastal ecosystem due to sewage inputs from the city area. Generally, the detected spectrum of municipal emerging contaminants has to be considered for the testing and technical improvement of wastewater treatment techniques, for regular monitoring of the surface water quality and for environmental impact assessment in China. Furthermore, the compound spectrum reported here is a good reference point for the composition of municipal sewage in other emerging economies.

Anillin (ANLN) is an actin-binding protein essential for assembly of cleavage furrow during cytokinesis. Although reportedly overexpressed in various human cancers, its role in hepatocellular carcinoma (HCC) is unclear. To address this issue, we confirmed that in 436 liver samples obtained from surgically removed HCC tissues, higher ANLN expression was detected in tumor tissues than in adjacent non-tumor tissues of HCC as measured by immunohistochemistry, quantitative real-time PCR and western blotting. Correlation and Kaplan-Meier analysis revealed that patients with higher ANLN expression were associated with worse clinical outcomes and a shorter survival time, respectively. Moreover, ANLN inhibition resulted in growth restraint, reduced colony formation, and a lower sphere number in suspension culture. Mechanistically, ANLN deficiency induced an increasing number of multinucleated cells along with the activation of apoptosis signaling and DNA damage checkpoints. Furthermore, HBV infection increased ANLN expression by inhibiting the expression of microRNA (miR)-15a and miR-16-1, both of which were identified as ANLN upstream repressors by targeting its 3' untranslated region. Thus, we conclude that ANLN promotes tumor growth by ways of decreased apoptosis and DNA damage. Expression level of ANLN significantly influences the survival probability of HCC patients and may represent a promising prognostic biomarker.

Transforming acidic coiled-coil protein 3 (TACC3) is essential for cell mitosis and transcriptional functions. In the present study, we first demonstrated that both TACC3 protein and mRNA levels were elevated in HCC tissue samples compared with non-cancerous tissue biopsies according to western blot analyses, immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) assays. Moreover, high TACC3 expression was positively correlated with poor overall survival (OS) and disease-free survival (DFS) (p < 0.001). Using HCC cell lines, we then demonstrated that either TACC3 knockdown or treatment with the potential TACC3 inhibitor KHS101 suppressed cell growth and sphere formation as well as the expression of stem cell transcription factors, including Bmi1, c-Myc and Nanog. Silencing TACC3 may suppress the Wnt/β-catenin and PI3K/AKT signaling pathways, which regulate cancer stem cell-like characteristics. Taken together, these data suggest that TACC3 is enriched in HCC and that TACC3 down-regulation inhibits the proliferation, clonogenicity, and cancer stem cell-like phenotype of HCC cells. KHS101, a TACC3 inhibitor, may serve as a novel therapeutic agent for HCC patients with tumors characterized by high TACC3 expression.

To investigate the relationship between relapse and the levels of the residual amount of HBV DNA in serum at cessation in chronic hepatitis B patients meeting 2008 Asian Pacific Association for the Study of the Liver (APASL) nucleos(t)ide analogs (NAs) cessation criteria.A total of 72 chronic hepatitis B patients who took NAs and had reached 2008 APASL cessation criteria entered the study. Patients were followed up for 6 months or longer after antiviral therapy was stopped. Serum HBV DNA level at cessation was detected by a highly sensitive polymerase chain reaction assay with detection limitation of 2 IU/mL.Of all the 72 patients, 42 patients (65.3%) relapsed after NA cessation. The detectable rate of the trace amount of HBV DNA at cessation was 41.7% by highly sensitive polymerase chain reaction reagents. The detectable rate of patients with consolidation treatment duration of18 months was higher than that with consolidation duration of ≥18 months (47.5% vs. 15.4%, P=0.034), and the detectable rate of patients with HBeAg seroconversion within 6 months of treatment was lower than that of ≥6 months (25.0% vs. 61.5%, P=0.036). The residual amount of HBV DNA and detectable rate at cessation showed significant differences between relapsed and nonrelapsed patients (130.4±420.90 vs 44.6±155.16 IU/mL, P=0.004; 55.3% vs. 16.0%, P=0.001). The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840.Residual amount of HBV DNA in serum at NA cessation is associated with HBV relapse. The cutoff value predicting relapse was 2.24 IU/mL, with a sensitivity of 0.553 and specificity of 0.840.

With the development of the society, the power system flow calculation becomes more important. Another new method for the polar coordinates Newton Raphson power flow calculation was proposed according to the power system operating characteristics. This research eliminated the vacant of the Jacobian matrix completely, reduced the amount of computer memory requirements greatly and also canceled the inversion completely.

Converter transformer is one of the most important equipments of UHVDC power transmission system, its faults directly affects the safe and stable operation of DC system. The valve winding of converter transformer needs to withstand the voltage that consist of DC voltage and serious distortion of AC voltages (harmonic bound) which are similar to the AC sine waveform compound voltage, so in the design and manufacture of the valve side of converter transformer, bushing is much more complex and difficult than alternating current casing.Valve bushing is weak spot of converter transformer insulation. Through the anatomy of failure bushing, the reason of bushing failure is analyzed. Without changing body size of the converter transformer, the bushing of new structure is developed and the old bushing is replaced to improve the reliability of equipment operation.

When the HVDC works in mono-polar operation or bipolar unbalanced operation, DC flows through the neutral point and causes DC bias magnetic of transformer around HVDC grounding electrode, which influences the operation security of transformers. Beside highly saturated core, it also causes the distortion of exciting current, increasing harmonics and leakage magnetic flux, and thus the losses, which in turn results in the structures of transformer overheating and insulation aging. Based on the principle of transformer DC bias magnetic and UMEC of transformer in PSCAD/EMTDC, electromagnetic transient simulation platform of transformer DC bias magnetic is built. Considering the effect of DC bias magnetic on different structure of transformer, three kinds of typical transformer that has been widely applied are selected including three-phase three-limb transformer, three-phase five-limb transformer, three-phase bank transformer. After comparing the differences between these three kinds of transformer, related parameters are set such as the core structure and mode of winding connection and so on.

In this research, a converter, suitable for the convert station of the HVDC light technology, is proposed and optimized. The converter mainly consists of AC chopper, rectifier transformer, low-pass filters and smoothing reactor. This system can restrain the disturbance of the input power and output the power with high quality. This research is going to analyze the working principle of the converter and then optimize the output wave through filters. Circuit’s simulation is carried out by MATLAB to verify the reliability and feasibility of this converter topology.

Long-term hematopoietic stem cells (HSCs) are capable of self-renewal and differentiation into all mature hematopoietic lineages. This process is regulated by transcription factors interact with co-factors to orchestrate chromatin structure and facilitate gene expression. To generate a compendium of factors that establish the epigenetic code in HSCs, we have undertaken the first large-scale in vivo reverse genetic screen targeting chromatin factors. We have designed and injected antisense morpholinos to knockdown expression of 488 zebrafish orthologs of conserved human chromatin factors. The resultant morphants were analyzed by whole embryo in situ hybridization at 36 hours post fertilization for expression of two HSC marker genes, c-myb and runx1, which are expressed in the developing blood stem cells. Morphants were categorized into five groups based on HSC marker expression, ranging from no change to mild, intermediate, or strong reduction in expression or an increase in expression. 29 morpholinos caused a complete or near complete knockdown of HSC marker expression, while 4 were found to increase HSC marker expression. As ubiquitous knockdown of chromatin factors could interfere with vascular development and the establishment of proper arterial identity, a crucial upstream event for HSC formation, we subsequently analyzed morphants with the most robust HSC phenotypes using two vascular markers: kdr for overall vasculogenesis and ephrinb2a for arterial formation. We found that of the 29 morpholinos that caused reduced marker expression, only 9 showed reduced overall vascular or arterial marker staining, suggesting that the majority of morphants with HSC phenotypes are specific to HSC formation. For the 4 morphants with increased HSC marker expression, vasculature appeared normal. These factors likely function as potent negative regulators of HSC development. Several genes known to be essential for HSC self-renewal and maintenance were identified in the screen. For example, knockdown of Mll or Dot1, which are also present in leukemia fusion proteins, fail to specify HSCs, as indicated by a nearly complete reduction in expression of the HSC markers in embryos tested. Of the remaining hits, many represent factors with no previous function ascribed in hematopoiesis. By incorporating protein interaction data, we have defined a handful of complexes necessary for HSC specification, including the SWI/SNF, ISWI, SET1/MLL, CBP/P300/HBO1/NuA4, HDAC/NuRD, and Polycomb complexes. As chromatin factors associated with the same complex likely share target binding sites, we analyzed 34 published ChIP-seq datasets in K562 erythroleukemia cells of chromatin factors tested in the screen, including hits from our screen: SIN3A, CHD4, HDAC1, TAF1, and JARID1C associated with the HDAC/NuRD complex and RNF2, SUZ12, CBX2, and CBX8 from the Polycomb complexes. We ranked triplet combinations of these factors together with all other groups of three factors based on the percent overlap of target genes. The HDAC/NuRD and PRC1/2 complex combinations predicted from our screen fell within the top 20% of all possible combinations of 3 factors, suggesting that our screen has identified chromatin factors that function in distinct complexes to regulate hematopoietic development. Our work has been compiled into a web-based database that will be made publicly available upon publication. Within this database, users can search by gene names and aliases, chromatin domain names and human or zebrafish genes. All experimental data, including experimental design, materials, protocols, images, and all further analyses of the 33 most robust morphants is included. Our large-scale genetic analysis of chromatin factors involved in HSC development provides a comprehensive view of the programs involved in epigenetic regulation of the blood program, offering new avenues to pursue in the study of histone modifications in HSCs and for therapeutic alternatives for patients with blood disorders and leukemia. Disclosures: Zon: FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Access distributed power will take great influence to distribution network's safe and economic operation. This paper established a distribution network planning economic model and analyzed the advantages and disadvantages of various planning algorithms, and propose an adaptive parallel genetic algorithm. This algorithm can overcome premature phenomenon of standard genetic algorithm, and the ability is better to seek the global optimal solution.

Due to the lack of fossil fuels, people are paying more and more attention to renewable energy. Wind energy is one of the important renewable energy. Unpredictability and volatility of the wind source make the output power unstable, so we need to control the active Power. This paper uses fuzzy control method, and the simulation results show that fuzzy control method mentioned in this paper is better than the conventional PI control for Wind power, the nonlinear system. Based on the analysis of pitch control theory and control process, we design fuzzy pitch controller and its model. We simulates gust wind speed imitates, wind turbine control and verifies the effects of the blur pitch control in a constant speed and constant frequency wind power generation system. According to the results of the simulation, we know the pitch controller of fuzzy logic has a better effect on the active control of the generator of the wind power generation system.

Aiming at maximum wind power tracking control problem of wind power generation system below the rated wind speed, this paper presents an improved MPPT control strategy by using turbulent part of the wind speed as a search signal to find the maximum power point. By using the Matlab/Simulink simulation of the wind power generation system below the rated wind speed, this paper proves the effectiveness of this control strategy. The simulation results show that improved MPPT control strategy can well control the wind turbine speed to track the wind speed changes to maintain optimum tip speed ratio and the maximum power coefficient.

This paper establishes the wind power system simulation model in Simulink/ Matlab environment. By adjusting the speed of variable speed wind turbine, the simulation model can keep running at the best operation condition, and then achieve maximum power transfer. In this process, this paper use PI controller to track and control the speed of wind turbine. Simulation results show that selecting the appropriate PI parameters can effectively track the speed and increase the efficiency of wind power generation system.

CD200 and its receptor CD200R are both type-1 membrane glycoproteins, which are members of the immunoglobulin superfamily (IgSF). Besides the inhibitory effect on macrophages, CD200/CD200R also play an important role in regulating the regulatory T cells, allergicreaction, autoimmune diseases, allograft, neurological diseases and other autoimmune-related diseases, etc.To investigate the role of CD200 and its receptor in the graft versus host disease (GVHD).Experimental samples were divided into aGVHD group, non-aGVHD group, cGVHD group and non-cGVHD group, the healthy persons were used as normal controls. Firstly, the expression levels of CD200 and CD200R on CD19+ cell, CD3+ cell and dendritic cell (CD19- CD14- CD1c+) surfaces in each group were detected by using flow cytometry, so as to determine whether there were expression differences among each groups. Then, the mRNA levels of each groups were tested by using real-time quantitative polymerase chain reaction for finding the differences of mRNA expression level among each group. Finally, the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1 antibody for 48 hours, and the interleukin-10 level in the co-culture system was tested by using enzyme linked immunosorbent assay for verifying the function of CD200/CD200R.The CD200 expression level on CD19+ cell surface in the aGVHD group and non-aGVHD group was both lower than that in healthy control group; that in the non-aGVHD group was higher than that in the aGVHD group. The CD200 expression level on CD19+ CD200+ cells in the non-cGVHD group were higher than that in the cGVHD group and healthy control group. There were no significant differences of CD200 and CD200R expression levels on CD3 cells and dendritic cells among all groups. The CD200 mRNA expression levels in the aGVHD group and cGVHD group were both lower than the healthy control group. The CD200 mRNA expression level was lower in the aGVHD group than in the non-aGVHD group, and was lower in the cGVHD group than in the non-cGVHD group. There was no significant difference of the CD200R mRNA expression level among all groups. After the peripheral blood mononuclear cells of the patients and healthy controls were co-cultured with anti-CD200R1 antibody for 48 hours, the interleukin-10 concentration decreased with the increasing of anti-CD200R1 antibody concentrations in the co-culture system.The CD200/CD200R may play a role in the pathogenesis of GVHD after allo-HSCT.

Transmission and transformation is an important part of National Electric Power building, Based on the reality of our country, this paper proposed a comprehensive evaluation model, refer to the entire life­cycle environmental friendly, sophisticated products, independent research, cost optimization of management, etc, in order to supply reference for the decision-makers of power transmission and transformation projects.

This paper studied the impact of silicon oxide layer on photovoltaic characteristic of iron-doped amorphous carbon film/silicon heterojunction (a-C:Fe/Si). The results show that a native SiO2 layer on the silicon surface can provide a significant improvement of the a-C:Fe/Si devices’ photovoltaic performances, especially for the short circuit current and fill factor. This improvement partly may be attributed to the electron recombination process is suppressed and the interface is modified by the SiO2 film based on the open circuit voltage decay measurement.

Identification of risk factors for recurrence and metastasis of HCC is important for the prognosis of HCC surveillance in chronic HBV infection. In this article, 125 HCC patients recruited were followed up prospectively for tumor metastasis and recurrence for a median of 104 (10–130) weeks. HBV DNA level was detected by LightCycler-based real-time fluorescence quantitative polymerase chain reaction-restriction system. HBV genotypes were determined by using PCR restriction-fragment length polymorphism. BCP and PC mutations were performed by PCR and direct sequencing of amplified products. Among 125 HCC patients, 19 patients were excluded because of the lack of follow-up data and the remaining 106 patients were followed up of 2 years and entered into analysis. Sixty-nine patients had tumor metastasis or recurrence during the follow-up and the cumulative probability of HCC metastasis or recurrence was 65.1%. On multivariate analysis, genotype C and HBV DNA level were the risk factors for HCC recurrence or metastasis. The incidence of recurrence or metastasis increased with baseline HBV DNA level in a dose-response relationship ranging from 22% for HBV DNA level of less than 3 log10 copies/ml to 80% for HBV DNA level of 5 log10 copies/ml or greater (P = 0.012). Fifty-seven (74.0%) and 12 (41.4%) patients had metastasis or recurrence in patients with genotype C and B, respectively. The adjusted OR of recurrence or metastasis for genotype C compared with genotype B was 9.755 (P = 0.009). In conclusion, elevated HBV DNA level and genotype C are strong risk predictors of HCC metastasis or recurrence. J. Med. Virol. 80:591–597, 2008. © 2008 Wiley-Liss, Inc.

Eight Hepatitis B virus (HBV) genotypes (A to H) have been identified based on an intergenotype divergence of 8% or more in the entire nucleotide sequence. Subgenotypes have also been identified in different HBV genotypes. As a highly endemic area for HBV infection, the prevalence of chronic HBV infection in China is between 8 and 20% of the general population. Genotypes B and C were identified as the most common HBV strains and account for approximately 95% of Chinese patients. Further study confirmed all genotype B strains belong to subgenotype Ba. Two of genotype C subgenotypes, C1 (Cs) and C2 (Ce), were found in China and they showed different geographic distributions. Genotype A was very rarely found, while genotypes E, F, G and H have not beenreported until now. Two types of HBV C/Drecombinant viruses have been identified in west China and distinct geographic and ethnic distributions were observed. Significant differences were observed (P

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), beta-naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases -3338 to -3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at -3381/-3299. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the XRE core region (CACGCA) was deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah receptor and Arnt antibodies demonstrated that the nuclear protein was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.

Vascular and non-vascular smooth muscle within the vagina mediate important physiological changes during sexual arousal in women. In this study, we have characterized alpha-adrenergic receptors (AR) in rabbit vagina by assessment of radioligand binding, contractility of isolated tissue strips and genital hemodynamics. [3H]Prazosin and [3H]RX821002 (alpha-1 and alpha-2 AR selective antagonists) bound to rabbit vaginal membrane preparations with high affinity and limited capacity. Competition binding assays using both non-selective and subtype selective ligands for AR (phentolamine, prazosin, delequamine, rauwolscine and UK14304) further confirmed the presence of alpha-1 and alpha-2 AR in vaginal tissue. In organ bath preparations of vaginal tissue strips, norepinephrine-induced contraction was attenuated by alpha-1 and alpha-2 AR antagonists (prazosin, tamsulosin, delequamine and phentolamine). In anesthetized rabbits, intravaginal injection of the alpha-1 AR selective antagonist REC 15/2615 (50 and 100 microg/kg) caused a 2 to 3-fold increase in genital tissue oxyhemoglobin (OHb) concentration. Similar increases in tissue OHb were observed with intravaginal injection of phentolamine (500 microg/kg) or a tri-mixture of vasodilators (PGE1, papaverine, phentolamine). REC 15/2615, phentolamine or the tri-mixture also enhanced the amplitude and/or duration of change in genital tissue OHb after pelvic nerve stimulation. Thus, vaginal tissue expresses functional alpha-1 and alpha-2 AR, which modulate vaginal smooth muscle contractility and genital engorgement.

There have been limited anatomic and physiological investigations of the female sexual arousal response. A broader understanding of the physiologic mechanisms of female sexual arousal function is required to improve the management of women with sexual dysfunction. Three experimental test systems have been developed to understand better the biochemical and physiological mechanisms of female sexual arousal response. An in vivo animal model was developed to record physiological and hemodynamic changes in the clitoris and vagina following pelvic nerve stimulation and administration of vasoactive agents and physiological modulators. In vitro organ baths of clitoral and vaginal tissue were utilized to investigate mechanisms involved in the regulation of smooth muscle contractility. In addition, primary cell cultures of human and animal clitoral and vaginal smooth muscle cells were developed to investigate signal transduction pathways modulating smooth muscle tone. In vivo studies revealed hemodynamic changes in vagina and clitoris in response to pelvic nerve stimulation, vasodilators and physiological modulators. Organ bath studies have demonstrated that clitoral and vaginal smooth muscle tone is affected by non-adrenergic and non-cholinergic neurotransmitters, and the presence of functional alpha 1 and alpha 2 adrenergic receptors in these tissues has been established through biochemical studies. These changes are regulated by the tone of vascular and non-vascular smooth muscle in the vagina and clitoris. Primary cell culture studies have suggested that several physiological modulators such as vasoactive intestinal polypeptide (VIP), nitric oxide (NO), and prostaglandin E (PGE) regulate vaginal smooth muscle contractility. Data from experimental models have provided a preliminary understanding of the mechanisms of the female sexual arousal response.

The goal of this study was to assess the potential cross-regulation of cyclic nucleotides in human corpus cavernosum (HCC). Incubation of primary cultures of HCC smooth muscle cells with either the NO donor sodium nitroprusside (SNP, 10 microM) or the phosphodiesterase type 5 (PDE 5) inhibitor sildenafil (50 nM) produced little or no changes in the intracellular cGMP levels. Incubation with both SNP and sildenafil produced marked increases in cGMP. Interestingly, incubation of cells with 10 microM of forskolin or PGE(1) produced significant enhancement of cGMP accumulation. These increases were not further enhanced by the addition of SNP and sildenafil. Kinetic analyses of cGMP hydrolysis by PDE 5 showed that high concentrations of cAMP reversibly inhibited the enzyme with a K(i) of 258 +/- 54 microM. The increase in cGMP levels in response to cAMP generating agents is not due to assay artifact since cAMP did not cross-react with cGMP antibody. Our data suggest that cAMP up-regulates intracellular levels of cGMP, in part, by inhibition of PDE 5. We also noted that cGMP down-regulates cAMP synthesis via a mechanism requiring G-protein coupling of adenylyl cyclase. These observations may have important implications in the utility of pharmacotherapeutic agents targeting cyclic nucleotide metabolism for the treatment of erectile dysfunction.

While the physiological effects of contractile (e.g. norepinephrine) and relaxatory (e.g. PGE1, forskolin) agents on corpus cavernosum smooth muscle tone have been characterized, the regulation of alpha adrenergic receptor mRNA expression in erectile tissue remains to be investigated. The goal of this study was to investigate the modulation of alpha-1 and alpha-2 adrenergic receptor mRNA expression in cultured human corpus cavernosum smooth muscle cells in response to increased intracellular cAMP induced by prostaglandin E1 and forskolin. Human corpus cavernosum smooth muscle cells were incubated for 24 h with or without PGE1 (5.7 µM), forskolin (10 µM) or an admixture of both. Total RNA was prepared from the cultures. Expression of alpha-1d adrenergic receptor, alpha-2a adrenergic receptor and m2 muscarinic acetylcholine receptor was determined by RNase protection assays. Loading was normalized by RNase protection of the housekeeping gene, cyclophilin A. The relative abundance of mRNAs was quantitated by scanning densitometry. Treatment of human corpus cavernosum smooth muscle cells with PGE1 or forskolin resulted in decreased mRNA expression of alpha-1d and alpha-2a adrenergic receptors and m2 muscarinic acetylcholine receptor when compared to untreated cells. Combinations of PGE1 and forskolin produced a more pronounced decrease in mRNA than either agent alone. PGE1 and forskolin increased intracellular levels of cAMP in human corpus cavernosum smooth muscle cells and combinations of both agents produced a more pronounced increase in cAMP synthesis. These results suggest that cAMP modulates the expression of alpha adrenergic receptors, one of the principal contractile receptor systems in the corpora cavernosa. These observations further support the concept that erectile function is a balance between contractile and relaxatory processes, which in turn regulate structure and function of the corpora cavernosa. International Journal of Impotence Research (2000) 12, Suppl 1, S41-S47

We investigated the pathophysiology of structurally based corporeal veno-occlusive dysfunction.We prospectively evaluated 24 impotent patients (mean age plus or minus standard error 46 +/- 3 years) who had exposure to vascular risk factors and/or disorders inducing diffuse trabecular structure alterations and who underwent penile prosthesis insertion. Preoperative indexes of veno-occlusive function (flow to maintain, venous outflow resistance and pressure decay measurements using repeat dosing pharmacocavernosometry) were correlated with postoperative erectile tissue computer assisted color histomorphometry (percent trabecular smooth muscle to total erectile tissue area). To develop further study findings and correlate histomorphometric findings with molecular biological properties molecular biological studies (ribonuclease protection analysis, reverse transcription-polymerase chain reaction assay for expression of transforming growth factor-beta 1 messenger [m] ribonucleic acid [RNA] and protein affinity labeling techniques for specific transforming growth factor-beta receptors) were performed in representative patients with high (39 to 43%), intermediate (30 to 37%) and low (13 to 29%) trabecular smooth muscle content (normal 42 to 50%).Flow to maintain, venous outflow resistance and pressure decay values significantly correlated with trabecular smooth muscle cell content (r = -0.89, 0.82 and -0.85, respectively). In the high, intermediate and low smooth muscle content subgroups flow to maintain, venous outflow resistance and pressure decay values were 1 to 5, 9 to 30 and 50 to 120 ml. per minute, 17 to 84, 3 to 9 and 1 to 2 mm. Hg/ml. per minute, and 40 to 60, 48 to 80 and 110 to 120 mm. Hg decrease in 30 seconds from 150 mm. Hg, respectively. There were no significant differences in patient age or prevalence of risk factors among the 3 subgroups. Patients representative of all 3 subgroups had transforming growth factor-beta 1 mRNA, auto-induction of transforming growth factor-beta 1 mRNA and induction and/or increased availability of all 3 types of transforming growth factor-beta receptors.The pathophysiology of structurally based corporeal veno-occlusive dysfunction is related to elevated corporeal connective tissue content. Based on our data and those in the literature corporeal fibrosis is hypothesized to develop secondary to abnormalities in the regulation of normal collagen synthesis and degradation, most likely associated with adverse influences of chronic ischemia.

Purpose: To investigate the effects of oxygen tension on prostanoid synthesis in rabbit penile corpus cavernosum tissue (RCC) in organ culture.Materials and Methods: Strips of rabbit corpus cavernosum were incubated in organ culture media under varying oxygen conditions (0 percent, 12 percent and 21 percent oxygen), in the presence or absence of acetylcholine and arachidonate stimulation. Prostanoids were measured in collected media by radioimmunoassay. Prostaglandin H synthase (PGHS) protein levels and mRNA PGHS expression were measured under both 0 percent and 21 percent oxygen conditions.Results: Basal and acetylcholine-stimulated PGI2 release was progressively diminished as a function of diminishing oxygen tension (pO2 from approximately 165 to 25 mm.Hg). The basal and stimulated production of other prostanoids, thromboxane A2, PGF2 alpha and PGE2, was also significantly inhibited under 0 percent oxygen (approximately 25 mm.Hg) conditions. However, incubation under 0 percent oxygen did not alt...

To understand the influencing factors on the death of infants born to HIV infected mothers in areas with high prevalence of HIV/AIDS in China.Based on the follow-up cohort study targeting at HIV/AIDS infected pregnant women and their babies initiated in 2004, a survey on the death status and influencing factors on the infants born to HIV/AIDS infected mothers enrolled in this cohort from Jan.2004 to Nov.2007 was carried out during Aug.to Nov.2008 in seven counties of four provinces in China. A total of 498 pairs of HIV-infected mothers and their infants were enrolled and their related information was collected. Single factor and multiple factors Cox model methods were adopted for data analysis.The total observed person-years of 498 infants was 406.22, among which, 45 infants died, and the mortality density was 110.78 per 1000 child-year. A single factor Cox model showed, the pregnancy in pre-period of HIV/AIDS and HIV/AIDS period (RR = 1.971, 95%CI: 1.143 - 3.396), living status of the pregnancy (RR = 3.062, 95%CI: 1.097 - 8.550), multipara women (RR = 0.517, 95%CI: 0.278 - 0.961), natural childbirth (RR = 0.561, 95%CI: 0.345 - 0.910), premature labor (RR = 5.302, 95%CI: 2.944 - 9.547), low birth weight (RR = 4.920, 95%CI: 2.691 - 8.994), mother-child pairs taking antiretroviral drugs (RR = 0.227, 95%CI: 0.121 - 0.428) and infants infected HIV (RR = 5.870, 95%CI: 3.232 - 10.660) could affect the infants death. The death of HIV-exposed infants was influenced by various factors. The death risk of infants born to HIV infected mothers who were in the danger of pre-period of HIV/AIDS and HIV/AIDS period was greater than the infants delivered by HIV infected mothers who were in preclinical period of HIV/AIDS (RR = 6.99, 95%CI: 1.92 - 25.64). The death risks were greater in the group that the women whose CD4(+)TLC count number lower than 200 cells/microl (RR = 2.05, 95%CI: 1.01 - 4.15). The infants whose mothers had no ARV treatment had higher possibility to die than the others (RR = 6.17, 95%CI: 1.62 - 23.26). The death risk of premature delivered infants was 2.87 times of mature delivered infants (95%CI: 1.12 - 7.35). The death risk of HIV/AIDS infected infants was 9.87 times of the HIV/AIDS uninfected infants (95%CI: 3.81 - 25.62).Some measurements including improving HIV-infected pregnant women's immunity, reducing mother to child transmission of HIV and premature birth, low birth weight are beneficial to reducing infant mortality.

Sixteen genera of soil moniliaceous hyphomycetes were isolated from 40 soil samples collected from the southern and northern slopes of Taibai Mountain, and the indices Shannon-Wiener diversity index, evenness, niche breadth, and niche overlap were used to analyze the community structure and niche of soil moniliaceous hyphomycetes in different forest types on the southern and northern slopes of Taibai Mountain. The results showed that the distribution of soil moniliaceous hyphomycetes on the study area had an obvious environmental gradient pattern. Altitude and forest type were the main determinants of the fungal community distribution. The species richness and diversity index of soil moniliaceous hyphomycetes decreased significantly with increasing altitude, but the community evenness represented an increasing trend. Among the genera of soil moniliaceous hyphomycetes, Penicillium, Aspergillus, Verticillium, Paecilomyces and Acremonium had larger niche width and greater niche overlap with other genera, suggeting that these genera had wide distribution on the southern and northern slopes of Taibai Mountain. As for Trichothecium, Gliocladium and Metarhizium, their niche width was narrower, and their niche overlap with other genera was smaller, indicating that the distribution of these genera was more affected by altitude and vegetation, with the occurrence only in special forest types.

To investigate the clinical characteristics and the pattern of precore and core promoter mutations of hepatitis B virus (HBV) subgenotypes Ba, C1 and C2.A cohort of 151 patients with chronic HBV infection in Guangdong province of China was enrolled in this study. HBV subgenotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Precore and core promoter mutations were analysed using nucleotide sequencing.Of the 151 patients, 80, 51 and 20 were infected with subgenotypes Ba, C1 and C2 respectively. No significant differences were found in HBeAg positivity and liver functional indexes among these three subgenotypes when age and sex were matched. Virologically, HBV/Ba showed the highest frequency of A1896 mutation but the lowest frequency of T1762/A1764 mutation. HBV/C1 was associated with the highest tendency to develop T1762/A1764 mutation, but the lowest prevalence of A1896 mutation. HBV/C2 was associated with an intermediate tendency to develop A1896 and T1762/A1764 mutations.Different mutation patterns in precore and core promoter regions are responsible for HBeAg-negative HBV infections among different subgenotypes.

To study the relationship between hepatitis B virus (HBV) genotypes and liver fibrosis.Polymerase chain reaction-restriction fragment length polymorphism method was employed to determine HBV genotypes in 298 HBV-related liver disease patients, including 73 with hepatocellular carcinoma, 53 with liver cirrhosis, 91 with chronic hepatitis B and 82 asymptomatic HBV carriers to analyze the relationship of HBV genotypes with serum fibrous indices and serum albumin and globulin (A/G) ratio.B and C genotypes were dominant in patients with HBV-related hepatic diseases in Guangdong province. The levels of serum fibrous indices were significantly higher in patients with genotype C than in those with genotype B, and the former patients also had lower A/G ratio.Patients with HBV genotype C may sustain more serious liver injury.

To investigate the distribution of hepatitis B virus (HBV) genotype B subgenotype in China.A cohort of 511 patients with chronic HBV genotype B infection, collected from 7 centers across China, were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or nucleotide sequencing.For the 511 patients, only subgenotype Ba was identified and subgenotype Bj was not found.In China, subgenotype Ba was the most prevalent HBV strains, while subgenotype Bj was very rarely found.

Hepatitis B virus (HBV) subgenotypes Ba, C1 (Cs), and C2 (Ce) are the most prevalent HBV variants in China. To investigate the virological characteristics of these subgenotypes and their clinical implications, we enrolled a cohort of 211 patients in the Guangdong Province of China, including 132 with chronic hepatitis B virus infection (CH), 32 with liver cirrhosis (LC), and 47 with hepatocellular carcinoma (HCC) according to clinical examination, liver function test, and ultrasonograph results. Overall, HBV Ba was found in 51.2% (108/211), HBV C1 in 33.6% (71/211), and HBV C2 in 15.2% (32/211) of the cases. The distribution of HBV genotype C was greater among patients in the LC and HCC groups than among patients in the CH group, while the distribution of HBV genotype B was greater among the CH patients than among the LC and HCC patients. No significant differences in clinical features were found among patients with HBV Ba, C1, and C2. Virologically, HBV C1 had the strongest association with the A1762T G1764A double mutation, while the mutation at position 1896 resulting in A (1896A) was uncommon. In contrast, HBV Ba had the highest frequency of 1896A but the lowest of A1762T G1764A, and HBV C2 had intermediate frequencies of these mutations. Mutations of 1653T and 1753V were specifically associated with HBV C2 and C1, respectively. Multivariate analyses showed that the 1653T, 1753V, and A1762T G1764A mutations and patient age significantly increased the risk of HCC development. In conclusion, HBV Ba, C1, and C2 have different mutation patterns in the enhancer II/core promoter/precore region. Therefore, genotyping and detecting the 1653T and 1753V mutations, in addition to the A1762T G1764A double mutation, might have important clinical implications as predictive risk factors for hepatocarcinogenesis.

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.

The goal of this study was to investigate the effects of medical castration (luteinizing hormone-receptor hormone [LH-RH] agonist treatment) or surgical castration on erectile function in an animal model. New Zealand White male rabbits were either kept intact (control); surgically orchiectomized; or treated for 2, 4, or 8 weeks with the LH-RH agonist leuprolide acetate (107 microg/kg/mo). At 2 weeks, plasma testosterone levels of orchiectomized and leuprolide acetate-treated animals were 12.8% and 57.4% of intact control animals, respectively. Erectile function was assessed by continuously recording systemic arterial pressure (SAP) and intracavernosal blood pressure (ICP) and determining the ICP:SAP ratios in response to electrical stimulation of the pelvic nerve at varying frequencies (2.5-32 Hz). Androgen deprivation by surgical (orchiectomy) or medical (leuprolide acetate) castration reduced ICP at all frequencies tested but did not alter SAP. Administration of the phosphodiesterase type 5 inhibitor vardenafil (10 microg/kg) did not enhance ICP in surgically orchiectomized or leuprolide acetate-treated animals. Nitric oxide synthase and arginase activities in the corpus cavernosum were not significantly altered by surgical or medical castration. Further, Masson trichrome staining of erectile tissue from androgen-ablated animals showed a reduction in smooth muscle content. These data demonstrate that androgen deprivation achieved by surgical or medical castration adversely affects penile hemodynamics and erectile function without producing significant changes in the activities of nitric oxide synthase or arginase. We conclude that androgen deprivation produces structural alterations in the corpus cavernosum leading to corporal veno-occlusive dysfunction.

The addition of 2 at.% Ta or 1 at.% Ti into a (Nd,Dy)-(Fe,Co)-B alloy is very effective in improving room temperature coercivity and thermal stability. The Ta containing alloy shows 30% higher coercivity value than that of a Ta-free alloy at temperatures higher than 100 degrees C. Both Ta and Ti alloying greatly reduce the high temperature reversible losses at temperatures of up to 225 degrees C. They are thus potential in high temperature applications. >

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 μM cGMP. The IC 50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (K i = 4.5 nM) than sildenafil (K i = 14.7 nM) in the same cellular extracts. In intact cells, 10 μM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 ± 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 μM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27–60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.

In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger cyclic nucleotides in response to vasodilators and determined the activity and kinetics of phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3′,5′-cyclic GMP phosphodiesterase). Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E and β-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE 1 , or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE 1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue. The cells retained phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity ( K i = 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone.

We investigated the biochemical and physiological mechanisms of action of phentolamine mesylate (Vasomax) in regulating erectile tissue smooth muscle contractility in human and rabbit corpus cavernosum.The binding activity of phentolamine was investigated in a cell-free system by displacement of specific and selective radiolabelled ligands to alpha 1 and 2 adrenergic receptors. The physiologic activity of phentolamine-mediated relaxation of adrenergic and non-adrenergic pre-contracted erectile tissue strips of human and rabbit corpus cavernosum were studied in organ bath chambers.In corpus cavernosum membranes, phentolamine displaced binding of the selective alpha 1 receptor antagonists [125I]HEAT and [3H]prazosin and the alpha 2 receptor antagonists [3H]rauwolscine and [3H]RX 821002 with relatively high affinity. Phentolamine caused concentration dependent relaxation in erectile tissue strips pre-contracted with adrenergic agonists phenylephrine, norepinephrine, oxymetazoline and UK 14,304, as well as with non-adrenergic contractile agents endothelin and KCl. Biochemical and physiologic studies reveal that the concentration of phentolamine required to displace half maximal binding or to produce half-maximal relaxation was similar to that found in human plasma 30 min after ingestion of 40 mg of Vasomax. Reversible inhibition of nitric oxide synthase by L-nitroarginine or mechanical disruption of endothelium diminished non-adrenergic phentolamine-mediated erectile tissue relaxation.Phentolamine mesylate induced relaxation of corpus cavernosum erectile tissue by direct antagonism of alpha 1 and 2 adrenergic receptors and by indirect functional antagonism via a non-adrenergic, endothelium-mediated mechanism suggesting nitric oxide synthase activation.

We have identified and characterized a 55 kDa nuclear protein (referred to as nmt55) from human breast tumors and MCF-7, human adenocarcinoma breast cell line, using site-directed monoclonal antibodies. Measurements of estrogen receptors (ER) and progesterone receptors (PR), by ligand binding assays, in cytosols of 63 human breast tumors permitted classifications of these tumors into four phenotypes (ER+/PR+, ER+/ PR-, ER-/PR-, ER-/PR+). Nuclear protein (nmt55) expression in these tumors, as determined from Western blot analyses, showed a statistically significant association (p = 0.001) with tumor hormonal phenotype. Review of the pathologic characteristics of tumors analyzed suggested that lack of nmt55 expression was significantly associated with mean tumor size (p < 0.03), mean ER (p = 0.001) and mean PR (p < 0.002), but was not associated with tumor stage, grade, or type. To further study this protein, we cloned and sequenced a 2.5 kb cDNA using a monoclonal antibody to nmt55. The complete predicted open reading frame encodes a protein with 471 amino acids and a calculated molecular mass of 54,169 Da. The deduced amino acid sequence exhibited unique regions rich in glutamine, histidine, arginine, and glutamic acid. Northern blot analysis of RNA from MCF-7 cells and ER+/PR+ human breast tumors showed a 2.6 kb mRNA. Southern blot analysis suggested the presence of a single copy of this gene. Chromosomal mapping, using fluorescent in situ hybridization (FISH), located nmt55 gene to the X chromosome, region q13. The extensive homology between nmt55 and RNA binding proteins suggested that nmt55 may be involved in hnRNA splicing. The strong association observed between expression of nmt55, tumor hormonal phenotype, mean tumor size, mean ER, and mean PR content suggests that loss of nmt55 expression may be related to events involved in hormone insensitivity, tumor differentiation, and unregulated tumor cell growth and metastases.