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Background: Gestodene (GEST) is widely used in female contraception. It is currently being used as an oral contraceptive. However, unfortunately, oral contraceptives are often associated with several bothersome side effects and poor compliance. Therefore, a sustained delivery system for GEST to overcome these shortcomings is highly desirable. Objectives: The present study successfully developed a kind of novel dissolving microneedles (DMNs) with a potential for sustained release and a minimally invasive intradermal treatment of GEST. Methods: The dissolving microneedles containing GEST were fabricated using polyvinylpyrrolidone as the base material. The characteristics in vitro and pharmacokinetics in vivo of GEST-loaded DMNs were investigated. Results: The results showed that the microneedle could pierce the porcine skin and release the drug at an average dose of 20µg/cm2 daily for seven days. The pharmacokinetic experiment of the microneedles indicated that the plasma level of GEST in rats increased with increasing drug dosage, and the plasma drug concentration-time curves were much flatter compared with subcutaneous injection and oral administration. In addition, no cutaneous irritation was observed. Conclusions: GEST-loaded DMNs may be a promising intradermal sustained delivery system for contraceptive use.

Supplementary Materials and Methods: Description of additional methods used in this study. Supplementary References: List of references cited in Supplementary Materials and Methods. Supplementary Table 1: SCP1 expression in various tumors. Supplementary Figure 1. Screen for PMLS518 phosphatases. Supplementary Figure 2. The specificity of SCP1 antibody. Supplementary Figure 3. Representative IHC staining results for PML, pS518PML, SCP1, SCP3, and KLHL20. Supplementary Figure 4. The effects of combined inhibition of mTOR and PML degradation pathway in suppressing ccRCC. Supplementary Figure 5. The effects of combined treatment on tumor angiogenesis and mice body weight. Supplementary Figure 6. The effect of SCP1 on PML in PC-3 cells.

Three new pyrazine derivatives, named talaropyrazines A–C (1–3), were isolated from the chemical investigation of the fungus Talaromyces minioluteus. Their planar structures were established by extensive 1D and 2D NMR and HRESIMS spectroscopic data analyses. The absolute configurations of 2 and 3 were defined by the synthesis of all the four isomers, respectively. The detailed comparison of the NMR and HPLC spectra of the natural product and the synthetic ones allows us to propose a general mechanism for the formation of diastereomers of 2 and 3. Moreover, the in vitro bioassay showed the synthetic congener (2′R,3′R)-3 exhibited potential inhibitory activity in the murine splenocytes stimulated by anti-CD3/anti-CD28 mAbs, with IC50 values of 5.85 μM.

Hyperforatins L–U, ten undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) bearing a terminal double bond, together with a known compound hypericumoxide J, were isolated from the aerial parts of Hypericum perforatum L. Their structures were elucidated by spectroscopic methods, including HRESIMS, IR, UV, and NMR (1H, 13C, DEPT, HSQC, HMBC, 1H–1H COSY, and NOESY experiments). Their absolute configurations were determined by comprehensive analyses of their experimental ECD spectra in conjunction with a modified Mosher's method. Evaluation of their neuroprotective activities highlighted hyperforatin L, which displayed mild activity at a concentration of 10 μM.

Spirohypolactones A (1) and B (2) and hyperhexanones C–E (3–5), five degraded cyclohexanone-monocyclic polyprenylated acylphloroglucinol (C-MPAP) derivatives were isolated from the stems and leaves of Hypericum perforatum. The structures of these compounds were established by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of naturally occurring C-MPAPs possessing 2-oxaspiro[4.5]decane spirocyclic skeletons. Compound 5 featured an unusual dodecahydro-4H-oxocino[4,3-g]benzofuran ring system. Notably, compounds 3 and 4 induce apoptosis in the acute myeloid leukemia cell lines NB4 and HL-60 by the activation of caspase-3, and degradation of PARP. The proposed biosynthetic pathways and biological activities are also discussed.

Southern rice black-streaked dwarf virus (SRBSDV) naturally infects rice and maize plants through white-backed planthopper (Sogatella furcifera) causing significant crop losses in China and Vietnam. Thus, rapid and accurate detection methods for SRBSDV are urgently needed. Recombinase polymerase amplification (RPA) is a novel technique for rapid and sensitive detection of nucleic acids. In this research, a reverse transcription (RT)-RPA-based method was developed for the detection of SRBSDV. A pair of RPA primers targeting the conserved sequences within the SP10 (major coat protein) gene on genomic RNA S10 of SRBSDV were designed. The assay was performed in a single tube at 39 °C for 20 min and demonstrated that the RPA assay is an efficient alternative for rapid detection of SRBSDV.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets and play important roles in the host response to pathogenic infection. Although infectious bursal disease virus (IBDV)-induced apoptosis in host cells has been established, the underlying molecular mechanism is not completely unraveled. Here, we show that infection of DF-1 cells by IBDV induced gga-miR-16-5p (chicken miR-16-5p) expression via demethylation of the pre-miR-16-2 (gga-miR-16-5p precursor) promoter. We found that ectopic expression of gga-miR-16-5p in DF-1 cells enhanced IBDV-induced apoptosis by directly targeting the cellular antiapoptotic protein B-cell lymphoma 2 (Bcl-2), facilitating IBDV replication in DF-1 cells. In contrast, inhibition of endogenous miR-16-5p markedly suppressed apoptosis associated with enhanced Bcl-2 expression, arresting viral replication in DF-1 cells. Furthermore, infection of DF-1 cells with IBDV reduced Bcl-2 expression, and this reduction could be abolished by inhibition of gga-miR-16-5p expression. Moreover, transfection of DF-1 cells with gga-miR-16-5p mimics enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation, and inhibition of caspase-3 decreased IBDV growth in DF-1 cells. Thus, epigenetic upregulation of gga-miR-16-5p expression by IBDV infection enhances IBDV-induced apoptosis by targeting the cellular antiapoptotic protein Bcl-2, facilitating IBDV replication in host cells. IMPORTANCE Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive disease in young chickens, causing severe economic losses to stakeholders across the globe. Although IBD virus (IBDV)-induced apoptosis in the host has been established, the underlying mechanism is not very clear. Here, we show that infection of DF-1 cells by IBDV upregulated gga-miR-16-5p expression via demethylation of the pre-miR-16-2 promoter. Overexpression of gga-miR-16-5p enhanced IBDV-induced apoptosis associated with increased cytochrome c release and caspase-9 and -3 activation. Importantly, we found that IBDV infection induced expression of gga-miR-16-5p that triggered apoptosis by targeting Bcl-2, favoring IBDV replication, while inhibition of gga-miR-16-5p in IBDV-infected cells restored Bcl-2 expression, slowing down viral growth, indicating that IBDV induces apoptosis by epigenetic upregulation of gga-miR-16-5p expression. These findings uncover a novel mechanism employed by IBDV for its own benefit, which may be used as a potential target for intervening IBDV infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) are important human pathogens responsible for urinary tract infection and meningitis. Therefore, infection of chickens by highly pathogenic E. coli with multi-drug resistance has become a major concern to food safety. In this study, we isolated a strain of E. coli (HB2016) from the oviduct of a diseased chicken with colibacillosis. Inoculation of chickens with 2 × 106 CFU of the isolate E. coli HB2016 by intraperitoneal injection successfully reproduced colibacillosis in chickens. We also found that E. coli HB2016 harbored four more virulence genes (tsh, trat, cvaC and cvaA/B) than E. coli reference strain CVCC1428. Importantly, E. coli HB2016 was resistant to cefuroxime, tobramycin, medemycin, cefazolin, cefoperazone, streptomycin and ampicillin, and carried multiple antibiotic resistance genes such as strA, strB, blaCMY-2, blaCTX-M-19, blaTEM-1B, fosA, mph(A), floR, sul2, tet(A) and tet(B). These findings suggest that the causative E. coli act as a potential zoonotic agent affecting human health.

Caesaldecins A and B (1a/1b), a pair of epimeric cassane-type diterpenoids containing an ether linkage between C-6 and C-20 and an α,β-butenolide ring in the northern hemisphere, were isolated from the leaves of the medicinal plant Caesalpinia decapetala (Roth) Alston, along with a new labdane-type diterpenoid, termed as 8(17),11(Z),13(E)-trien-15,18-dioic acid (2). Structures of 1 and 2 were established by extensive spectroscopic analyses, including HRESIMS, 1D and 2D NMR, and electronic circular dichroism (ECD) calculations. The in vitro antibacterial and inhibitory activities against a panel of bacteria and cancer cell lines were evaluated, respectively. Compound 2 displayed moderate antibacterial activity against the methicillin-resistant Staphylococcus aureus subsp. aureus (MRSA) with an MIC50 value of 5.99 μg mL−1.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). IBDV VP3 is a multifunctional protein playing a key role in virus assembly and pathogenesis. To investigate the role of VP3 in pathogenesis, we transfected DF-1 cells with pRK5-FLAG-vp3 and found that VP3 enhanced type I interferon expression and suppressed IBDV replication. Furthermore we found that VP3 interacted with chicken Ribosomal Protein L18 (chRPL18) in host cells and knockdown of chRPL18 by RNAi significantly promoted Type I interferon expression and inhibited IBDV replication. Moreover, our data show that chicken double-stranded RNA-activated protein kinase (chPKR) interacted with both VP3 and chRPL18. Thus chRPL18 in association with VP3 and chPKR affects viral replication.

// Limin Zhao 1,* , Xiaoqin Tian 2,* , Xueyan Duan 1 , Yongxiu Ye 1 , Min Sun 3 and Junfang Huang 1 1 Department of General Medicine, Shenzhen Longhua New District Central Hospital, Shenzhen, Guangdong, P.R. China 2 Department of Endocrinology, Shandong Liaocheng City Central Hospital, Liaocheng, Shandong, P.R. China 3 Department of Urolgoy, The Xinjiang Uygur Autonomous Region Peoples Hospital, Uygur, Xinjiang, P.R. China * These authors have contributed equally to this work Correspondence to: Junfang Huang, email: // Keywords : body mass index, bladder cancer, risk, dose-response, meta-analysis Received : December 09, 2016 Accepted : February 07, 2017 Published : March 30, 2017 Abstract Prospective epidemiologic studies on the association between body mass index (BMI) and bladder cancer yielded inconsistent findings. This study sought to quantitatively summarize the evidence by performing a dose-response meta-analysis on prospective cohort studies. Eligible studies were retrieved via PubMed and Embase databases, and by manual review of the references. Linear and nonlinear trend analyses were conducted to explore the relationships between BMI and bladder cancer risk. Meta-analyses on the categories of overweight and obesity were also conducted. The summary relative risk (SRR) was estimated. Heterogeneity across the studies was explored through subgroup analyses based on gender, age, year of publication, sample size, assessment of BMI, geographic location, physical activity and family history of cancer. A total of 14 prospective cohort studies involving 12,642 cases were included. Result of the dose-response analysis showed a nonlinear positive relationship between BMI and bladder cancer (SRR = 1.03, 95% CI: 1.01-1.06, P -nonlinearity =0.031), suggesting that per 5 kg/m 2 increment on BMI corresponded to a 3.1 % increase of bladder cancer risk, especially BMI exceed 30kg/m 2 .Furthermore, significant positive association was also observed between obesity category and bladder cancer risk (SRR: 1.10, 95%CI: 1.03-1.17). In summary, this dose-response meta-analysis suggests a nonlinear positive association between BMI and bladder cancer risk. Further studies are required to confirm these findings and elucidate the pathogenic mechanisms.

Three previously undescribed compounds, including a meroterpenoid, guignardone T (1), and two ophiobolin-type sesterterpenoids, maydispenoids A and B (2 and 3), along with four known compounds (4–7), were isolated from the phytopathogenic fungus Bipolaris maydis collected from Anoectochilus roxburghii (Wall.) Lindl leaves. The structures of all undescribed compounds were elucidated by spectroscopic analysis, electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction. Structurally, maydispenoids A was characterized by a fascinating decahydro-3-oxacycloocta[cd]pentalene fragment. It is notable that the compounds 2 and 3 exhibited potential inhibitory activity in anti-CD3/anti-CD28 monoclonal antibodies (mAbs) stimulated murine splenocytes proliferation, with IC50 values of 5.28 and 9.38 μM, respectively, and also suppress the murine splenocytes proliferation activated by lipopolysaccharide (LPS), with IC50 values of 7.25 and 16.82 μM, respectively. This is the first report of ophiobolin-type sesterterpenoids as immunosuppressor, and may provide new chemical templates for the development of new immunosuppressive drugs for autoimmune disease treatment.

Fowl adenovirus serotype 4 (FAdV-4) causes hepatitis-hydropericardium syndrome (HHS), leading to severe economic losses in the poultry industry. Although the pathogenesis of FAdV-4 infection has caused much attention, the underlying molecular mechanisms remain poorly understood. Here, we identified chaperonin containing TCP-1 subunit eta (CCT7) as an interacting partner of the FAdV-4 capsid protein hexon. We found that ectopic expression of CCT7 in leghorn male hepatocellular (LMH) cells enhanced hexon expression in pRK5-flag-hexon transfected cells. On the contrary, knockdown of cellular CCT7 by RNAi markedly reduced hexon expression in FAdV-4-infected cells and suppressed viral replication. These data suggest that CCT7 is required for FAdV-4 replication and may serve as a potential target for controlling FAdV-4 infection.

Fifteen new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperforatones A-O (1-15), along with 3 structurally related analogues (16-18), were isolated from the stems and leaves of Hypericum perforatum. Their structures and absolute configurations were established by a combination of NMR spectroscopic analyses, experimental and calculated electronic circular dichroism (ECD), modified Mosher's methods, Rh2(OCOCF3)4- and [Mo2(OAc)4]-induced ECD, X-ray crystallography, and the assistance of quantum chemical predictions (QCP) of 13C NMR chemical shifts. Compound 5 was found to be the first PPAP decorated by a rare 2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol moiety and an oxepane ring. Furthermore, the isolates were screened for their acetylcholinesterase (AChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities. Compounds 5, 10, 11, and 15 showed desirable AChE inhibitory activities (IC50 6.9-9.2 μM) and simultaneously inhibited BACE1 (at a concentration of 5 μM) with inhibition rates of 50.3%, 34.3%, 47.2%, and 34.6%, respectively. Interestingly, compound 5 showed the most balanced inhibitory activities against both AChE and BACE1 of all the tested compounds, which means that 5 could serve as the first valuable dual-targeted PPAP for the treatment of Alzheimer's disease. Preliminary molecular docking studies of 5 with BACE1 and AChE were also performed.

Background Although there are several studies to investigate the association between blood lipids and microvascular complications, these studies reported conflicting results. The aim of the current study was to explore the association between blood lipid parameters and the risk of microvascular complications, especially the dose-response association between them, among community patients with type 2 diabetes mellitus (T2DM) in Shanghai, China. Methods The cross-sectional study was conducted in 6 community health service centers in Shanghai between December 2014 and December 2016.The associations between blood lipids and diabetic kidney disease (DKD) or diabetic retinopathy (DR) were assessed using multiple logistic regression. Restricted cubic spline (RCS) was employed to estimate the dose-response relation of blood lipids and the risk of microvascular complications. Results A total of 3698 participants were included in the final analysis to study the association between blood lipids and DKD, wherein 33.2% of participants had DKD and 1374 were included for the analysis of the association between blood lipids and DR, wherein 23.2% of participants had DR. DKD odds ratio was increased by 1.16(95%CI,1.08–1.25), 1.21(95%CI,1.13–1.30), 1.18(95%CI,1.10–1.26) for comparing fourth to first quartiles of triglycerides (TG), TG/high-density lipoprotein cholesterol (HDL-C), non-HDL-C/HDL-C, respectively, and decreased by 0.83(95%CI,0.78–0.89) for comparing fourth to first quartiles of HDL-C. Furthermore, the dose-response association between TG, HDL-C, TG/HDL-C, non-HDL-C/HDL-C and the risk of DKD demonstrated turning points in TG of 1.90 mmol/L, HDL-C of 1.62 mmol/L, TG/HDL-C of 2.00, non-HDL-C/HDL-C of 3.09, respectively. However, no significant association was found between blood lipid parameters and DR. Conclusions This community-based study indicated that TG, HDL-C, TG/HDL-C, non-HDL-C/HDL-C were independently associated with DKD but not DR.

The tumor-suppressor protein promyelocytic leukemia (PML) is aberrantly degraded in multiple types of human cancers through mechanisms that are incompletely understood. Here, we show that the phosphatase SCP1 and its isoforms SCP2/3 dephosphorylate PML at S518, thereby blocking PML ubiquitination and degradation mediated by the prolyl isomerase Pin1 and the ubiquitin ligase KLHL20. Clinically, SCP1 and SCP3 are downregulated in clear cell renal cell carcinoma (ccRCC) and these events correlated with PMLS518 phosphorylation, PML turnover, and high-grade tumors. Restoring SCP1-mediated PML stabilization not only inhibited malignant features of ccRCC, including proliferation, migration, invasion, tumor growth, and tumor angiogenesis, but also suppressed the mTOR–HIF pathway. Furthermore, blocking PML degradation in ccRCC by SCP1 overexpression or Pin1 inhibition enhanced the tumor-suppressive effects of the mTOR inhibitor temsirolimus. Taken together, our results define a novel pathway of PML degradation in ccRCC that involves SCP downregulation, revealing contributions of this pathway to ccRCC progression and offering a mechanistic rationale for combination therapies that jointly target PML degradation and mTOR inhibition for ccRCC treatment. Cancer Res; 74(23); 6935–46. ©2014 AACR.

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily of structurally related signaling proteins that regulate a wide array of cellular functions. The key step in BMP signal transduction is the BMP receptor-mediated phosphorylation of transcription factors Smad1, 5, and 8 (collectively Smad1/5/8), which leads to the subsequent activation of BMP-induced gene transcription in the nucleus. In this study, we describe the identification and characterization of PPM1H as a novel cytoplasm-localized Smad1/5/8-specific phosphatase. PPM1H directly interacts with Smad1/5/8 through its Smad-binding domain, and dephosphorylates phospho-Smad1/5/8 (P-Smad1/5/8) in the cytoplasm. Ectopic expression of PPM1H attenuates BMP signaling, whereas loss of PPM1H activity or expression greatly enhances BMP-dependent gene regulation and mesenchymal differentiation. In conclusion, this study suggests that PPM1H acts as a gatekeeper to prevent excessive BMP signaling through dephosphorylation and subsequent nuclear exclusion of P-Smad1/5/8 proteins.

SummaryTGFβ signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFβ responses are generally mediated through transcriptional functions of Smads. A key step in TGFβ signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFβ type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFβ signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Cα as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFβ-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFβ-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFβ signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Cα, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFβ signaling.

The objective of this study is to develop the monolithic osmotic pump tablet system (MOTS) containing isosorbide-5-mononitrate (5-ISMN), and to evaluate its in vitro and in vivo properties. The influences of tablet formulation variables, size and location of the delivery orifice, membrane variables, and pH value of the dissolution medium on 5-ISMN release from MOTS have been investigated. These results demonstrated that the tablet core played an important role in MOTS, and membrane variables could affect the 5-ISMN release rate. The optimal formulation of 5-ISMN MOTS was determined by uniform design. Furthermore, the dog pharmacokinetics and relative bioavailability of the test formulation (5-ISMN MOTS) have been compared with the reference formulation (Imdur: 60 mg/tablet, a sustained release, SR, tablet system) following an oral single dose of 60 mg given to each of six Beagle dogs. The mean drug fraction absorbed by the dog was calculated by the Wagner-Nelson technique. The results showed that drug concentration in plasma could be maintained more stable and longer after the administration of 5-ISMN MOTS compared with the matrix tablets of Imdur, and a level A "in vitro-in vivo correlation" was observed between the percentage released in vitro and percentage absorbed in vivo. It is concluded that 5-ISMN MOTS is more feasible for a long-acting preparation than 5-ISMN SR tablet system as once-a-day treatment, and it is very simple in preparation, and can release 5-ISMN at the rate of approximately zero order for the combination of hydroxypropylmethyl cellulose as retarder and NaCl as osmogent.

The existing methods for the extraction and determination of isosorbide-5-mononitrate (5-ISMN) in biological samples are too complicated, poorly reproducible, and time-consuming. Therefore a procedure is described for the extraction of 5-ISMN from plasma with ethyl acetate and saturated potassium carbonate solution. The procedure of extraction is simple, and has a high recovery. At the same time, an improved, simple, rapid, and specific analytical method for the determination of 5-ISMN in dog plasma by capillary gas chromatography with electron-capture detection was developed. O-Dinitrobenzene was used as an internal standard. This method was successfully applied to a pharmacokinetic study on 5-ISMN.

To accurately forecast container throughput is crucial to the success of any port operation policy. In this article, Attribute Theory is used for forecast port container throughput. The method of container throughput forecast based on Attribute Theory is provided. Then the application process of the method is presented in detail combining container throughput forecast of Shanghai Port as an example. The result shows that this method is reasonable and effective. It offers a more practical and reliable way to forecast container throughput in related research.

Enterprise's Core Competence Evaluating Model is set based on the Method of Arrtibute Theory. A data base of 300 enterprises' core competence level is imitated and sorted by computer programming. The sorted result can reflect different decision makers' mentally preference and be adjusted by different decision makers' mentally preference which is called weight. The use of the method of attribute theory has added a new and effective approach to the evaluation on enterprise's core competence.

Somatostatin receptor subtype 5 (SSTR5) mediates the inhibitory effect of somatostatin and its analogs on insulin expression/secretion and islet cell proliferation. We provide biochemical and genetic evidence that SSTR5 exerted its physiological actions via down-regulating pancreatic and duodenal homeobox-1 (PDX-1), a β-cell-specific homeodomain-containing transcription factor. Cotransfection of SSTR5 with PDX-1 resulted in dose-dependent inhibition of PDX-1 expression in human embryonic kidney 293 cells. SSTR5 agonist RPL-1980 inhibited PDX-1 expression and abolished glucagon-like peptide 1-stimulated PDX-1 expression in mouse insulinoma β-TC-6 cells. SSTR5 knockdown by short hairpin RNA led to increased PDX-1 expression that was accompanied by enhanced insulin secretion stimulated by high glucose in β-TC6 cells and alternated expressions of cell cycle proteins that favor cell proliferation in mouse insulinoma MIN6 cells. Quantitative RT-PCR analysis showed that cotransfected SSTR5 inhibited PDX-1 mRNA expression, whereas knockdown of SSTR5 increased PDX-1 mRNA expression. In addition, we found that cotransfected wild-type SSTR5 increased PDX-1 ubiquitination in human embryonic kidney 293 cells, whereas SSTR5 P335L, a hypofunctional single nucleotide polymorphism of SSTR5, inhibited PDX-1 ubiquitination. SSTR5 knockout resulted in increased expression of PDX-1, insulin, and proliferating cell nuclear antigen in the islets of sstr−/− mice. Immunohistochemistry analysis showed that SSTR5 P335L was associated with elevated expression of PDX-1 in human pancreatic neuroendocrine tumor. Taken together, our studies demonstrated that SSTR5 is a negative regulator for PDX-1 expression and that SSTR5 may mediate the inhibitory effects of somatostatin and its analogs on insulin expression/secretion and cell proliferation via down-regulating PDX-1 at both transcriptional and posttranslational levels.

In eukaryotes, regulation of signaling mediators/effectors in the nucleus is one of the principal mechanisms that govern duration and strength of signaling. Smads are a family of structurally related intracellular proteins that serve as signaling effectors for transforming growth factor beta (TGF-beta) and TGF-beta-related proteins. Accumulating evidence demonstrates that Smads possess intrinsic nucleocytoplasmic shuttling capacity, which enables them to transmit TGF-beta signals from cell membrane to nucleus. We recently identified two important steps in the termination of nuclear Smad signaling. The first step is initiated by a serine/threonine phosphatase PPM1A that dephosphorylates Smad2/3 in the nucleus, thereby shutting down signaling capacity of phosphorylated Smad2/3. The second step involves nuclear export of dephosphorylated Smad2/3 with the aid of nuclear protein RanBP3 to terminate Smad signaling. This chapter introduces methods for examining nuclear export of Smad2/3 in TGF-beta signaling.

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation.