[Impacts of radiofrequency ablation on quality of life of atrial fibrillation patients with low CHA2DS2-VASc score].

OBJECTIVE: CHADS2 and CHA2DS2-VASc scores are two widely used systems for evaluating the risk of stroke in patients with atrial fibrillation (AF). The Quality of life (QoL) after a single radiofrequency ablation (RFA) in AF patients with low CHADS2 score has been established. This study was designed to further explore the association between the QoL and AF patients with low CHA2DS2-VASc score after RFA. METHODS: We selected 367 AF patients with low CHA2DS2-VASc score (CHA2DS2-VASc<2) from the Chinese Atrial Fibrillation Registry (CAFR) between 2011 and 2013. They all completed both baseline and 6-month Atrial Fibrillation Effect on Quality-of-Life (AFEQT) questionnaires. Propensity score matching was used to construct a final cohort of 105 patients. They were divided into two groups: RFA group (n=44) and non-RFA group (n=61). RESULTS: Baseline clinic characteristics, and each domain and global score of AFEQT were comparable between the two groups. Except for domain of treatment satisfaction in non-RFA group (mean score change from baseline to 6-month: 3.55, P= 0.13) , the other domains in this group (mean score change from baseline to 6-month: symptom, 14.66; daily activities, 12.56; treatment concern, 17.69; global score, 14.71, all P<0.05) and all the domains in RFA group improved greatly within-group comparison (mean score change from baseline to 6-month: symptom, 20.42; daily activities, 17.1; treatment concern, 20.36; global score, 18.75; treatment satisfaction, 9.66, all P<0.05). No significant differences were observed in all domains of baseline to 6-month changes between two groups (all P>0.05). CONCLUSIONS: No significant changes could be viewed in QoL between RFA and non-RFA AF patients with low CHA2DS2-VASc score, whose baseline AFEQT were balanced, and QoL from baseline to 6-month improves significantly.

Characteristics of different phenotypes of polycystic ovary syndrome based on the Rotterdam criteria in a large-scale Chinese population.

OBJECTIVE: To analyse the phenotypic spectrum of polycystic ovary syndrome (PCOS) and determine the association between metabolic, hormonal and new ultrasonographic criteria. DESIGN: Clinical cross-sectional study. SETTING: University teaching hospital. POPULATION: A total of 804 Chinese women, among whom 719 cases were diagnosed as PCOS based on the 2003 Rotterdam criteria. Eighty-five women with regular menstrual cycles and without hyperandrogenism were recruited as controls. METHODS: PCOS patients were divided into four subgroups: (i) oligo- and/or anovulation (O), hyperandrogenism (H), and polycystic ovary morphology (P); (ii) O + H; (iii) H + P; and (iv) O + P. MAIN OUTCOME MEASUREMENTS: Clinical history, ultrasonographic (ovarian follicle number and volume), hormonal and metabolic parameters. RESULTS: The composition of the two new phenotypes created by the European Society for Human Reproduction and Embryology/The American Society for Reproductive Medicine (ESHRE/ASRM) 2003 was 65.6% (O + P and H + P). BMI and F-G scores were highest in the O + H + P group and lowest in O + P and controls. Serum testosterone concentrations and insulin resistance were highest in cases with O + H + P and O + H, intermediate in cases with H + P, and lowest in cases with O + P and controls. The prevalence of metabolic syndrome in the five groups was 28.5% (O + H + P), 25.5% (O + H), 8.3% (H + P), 7.2% (O + P) and 3.5% (controls), respectively. CONCLUSIONS: Nonclassic phenotypes for PCOS (O + P, H + P and O + H + P) were more frequent than the classic phenotype (O + H). The nonhyperandrogenic PCOS phenotype (O + P), one of the new phenotypes created by the Rotterdam criteria, may represent a form of PCOS associated with milder metabolic profile compared with the other phenotypes.

The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7.

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53(R273H)) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53(R273H)-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53(R273H) binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53(R273H)-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

p53 abnormalities in primary prostate cancer: single-strand conformation polymorphism analysis of complementary DNA in comparison with genomic DNA. The Cooperative Prostate Network.

BACKGROUND: The reported frequency of mutation of the p53 tumor suppressor gene (also known as TP53) in human carcinomas of the prostate has varied widely, ranging from 3% to 42%. This variability may be a consequence of tumor heterogeneity and/or the use of different methods of analysis. Since p53 mutation has been associated with clinical outcome for a number of cancer types, determination of its true frequency in primary carcinomas of the prostate is important. PURPOSE: The principal aims of this study were as follows: 1) to validate the utility of detecting p53 gene mutations by means of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of complementary DNA (cDNA) (synthesized from prostate tissue RNA and 2) to study the concordance of RNA- and DNA-based PCR-SSCP assays in detecting p53 mutations in individual tumor fragments. METHODS: RNA and genomic DNA were isolated by means of standard techniques from specimens of 19 carcinomas of the prostate, selected on the basis of p53 data obtained in a previous analysis of cDNA (indicating that 14 were mutant and five were wild-type). RNA was converted into cDNA by means of reverse transcription (RT); the cDNA was then amplified by means of nonisotopic (i.e., nonradioactive) PCR, and the PCR products were subjected to SSCP analysis in polyacrylamide gels (RT-PCR-SSCP analysis). Genomic DNA was examined by means of SSCP analysis of isotopically labeled (32PO4) PCR products (DNA-PCR-SSCP analysis). In both approaches, the protein coding region of the p53 gene was divided into multiple, smaller fragments for study. PCR products exhibiting abnormal migration in SSCP gels were subjected to direct nucleotide sequencing or to cloning and sequencing of multiple clones. RESULTS: RT-PCR-SSCP and DNA-PCR-SSCP identified p53 gene abnormalities in 15 of the 19 selected carcinomas, including one previously reported to be wild-type for p53. Overall, PCR-SSCP analysis identified 18 p53 fragments with abnormalities; three carcinomas showed two abnormalities each. Six (33%) of the 18 abnormalities were detected by both RT-PCR-SSCP and DNA-PCR-SSCP, 10 (56%) were detected by RT-PCR-SSCP alone, and two (11%) were detected by DNA-PCR-SSCP alone. The 18 abnormalities were caused by 20 changes in the sequence of the p53 gene; in one carcinoma, double mutations in two individual p53 exons were identified. CONCLUSIONS AND IMPLICATIONS: PCR-SSCP analysis of both RNA and DNA allows the detection of more mutations than the analysis of either alone. Some primary carcinomas of the prostate contain more than one altered p53 gene, consistent with the possibility of intratumoral heterogeneity of mutation of this gene. For comprehensive analysis of p53 mutations in carcinomas of the prostate, and perhaps in other tumor tissues, SSCP analysis of cDNA should be used in combination with SSCP analysis of genomic DNA.

Antibody phage libraries for the next generation of tumor targeting radioimmunotherapeutics.

Pretargeting techniques have shown promise for enhancement of the therapeutic index of radioimmunotherapy for cancer. However, methods to vary and compare antibody configurations and select optimal combinations have proved rather formidable. New options for the construction of pretargeting molecules are provided by sophisticated use of the diversity and malleability of antibody genes. Diverse arrays of single-chain antibody fragments (scFvs) can now be obtained reactive with virtually any target antigen by selection from human naive phage antibody libraries. ScFvs can also be cloned directly from hybridoma for construction of phage libraries that facilitate subsequent manipulation: e.g., affinity maturation and modification of specificity. ScFvs affinity selected from these sources to their specific antigen targets have demonstrated a wide spectrum of binding characteristics. ScFvs selected from a large human naive phage antibody library by binding Cu-1,4,8,11-tetra-azacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA) or Y-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) have shown diversity by DNA fingerprints. DNA sequence information confirmed that the anti-TETA scFv represented diverse scFv gene families. ScFvs for Y-DOTA and those for lymphoma-associated HLA DR10 (Lym-1) were selected in a similar manner from mouse antibody gene libraries derived from hybridoma. ScFv clones for each of these antigens were chosen for further study based on the results of ELISA assays involving the respective cell membrane or metal chelate antigens. A PCR primer system built to pCANTAB 5E expression vector sequence was designed to facilitate cloning of antibody heavy (V(H)) and light (V(L)) genes from selected scFvs as cassettes into diabody modules. Thus, chosen scFvs could be expressed in the same diabody format for comparative study. Selected mouse anti-DOTA scFv and Lym-1 scFv genes were linked as V(HA) anti-DOTA-link-V(LB) Lym-1; V(HB) anti-DOTA-link-V(LA) Lym-1 and ligated into the pCANTAB 5E vector. Corresponding diabodies were expressed in Escherichia coli and purified by affinity chromatography. Here we provide a perspective on the power of antibody phage libraries and the possibilities of creating simple molecular formats that can be used en route to the development of new tumor targeting and pretargeting molecules.

Identification of p53 mutations in archival prostate tumors. Sensitivity of an optimized single-strand conformational polymorphism (SSCP) assay.

To correlate molecular changes with clinical information in prostate tissue, it is necessary to have accurate methods for screening for mutations in clinically available specimens. We have refined the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis for detection of p53 mutations in routine pathology specimens. These improvements help overcome technical barriers that interfere with SSCP analysis of archival tissues when only small amounts of poorly preserved formalin-fixed DNA are available. Furthermore, prostate samples are heterogeneous in containing tumor, normal tissue, and hyperplastic tissue. To address the first issue, the method included an initial selection of PCR products using exonuclease I, followed by a second-step selection using nested PCR. This step ensures adequate amplification of the target sequence while minimizing artifactual products that could otherwise interfere with mutation analysis. For the second issue, in addition to morphologic selection of appropriate tissue areas, we improved the sensitivity of detection of mutations by using restriction enzyme digestion of products prior to SSCP analysis. Detection of mutations in heterogeneous tissue was evaluated by determining the minimal detectable mutant allele frequencies in exons 4, 5, 6, 7, 8-9, and 10 by using mixtures of known mutant and wild-type cell lines, which were found to be 17.6, 9.1, 12.5, 8.1 14.0, and 14.3%, respectively. To determine the utility of this method when used on heterogeneous clinical samples, we performed study of 19 archival prostate specimens (14 primary prostate cancers, three benign prostatic hyperplasia and two metastases) and detected abnormally migrating products in six of the prostate cancer specimens (four primaries and two metastases). In five of these samples, there was sufficient DNA to perform sequencing, which disclosed single-base change mutations in all five samples.

Efficient recombination of Lym-1 scFv gene using multiple doubly-restricted DNA fragments.

In order to improve radioimmunotherapy of lymphoma, a Lym-1 single-chain antigen-binding (scFv) protein molecule was produced. Because the commonly used polymerase chain reaction (PCR) method frequently causes unexpected mutations, we developed a non-PCR method for scFv gene assembly. The method involved a stepwise linkage of doubly-restricted DNA fragments and re-digestion of the resultant concatamers. Using this strategy, the Lym-1 scFv expression gene was readily constructed without mutations. The recombinant gene was cloned into an expression vector and scFv protein was expressed. The method can be used for other genes or DNA recombination.

Anti-HLA-DR/anti-DOTA diabody construction in a modular gene design platform: bispecific antibodies for pretargeted radioimmunotherapy.

Recombinant immunoglobulin libraries of single chain molecules (sc) from the variable domains of antibody light and heavy chains (Fv), have great promise for new approaches to radioimmunotherapy (RIT). However, creating and evaluating scFv from diverse sources is time consuming and differences in molecular format can influence in vitro and in vivo characteristics. Furthermore, scFv do not have optimal characteristics for targeting therapy to tumor because of their small size and univalent binding. Diabody molecules at least twice the size of scFv are better for RIT because bivalent and bispecific molecules can be constructed. A polymerase chain reaction (PCR) based primer system was created to easily convert scFv genes into a diabody gene format, once they have been placed into pCANTAB 5E, a readily available vector. The primer system for this diabody gene platform was developed and tested by constructing an anti-lymphoma/anti-chelate, bispecific diabody (anti-HLA-DR/anti-DOTA). Two mouse scFv libraries were screened for reactive clones using recombinant phage display techniques. Selected mouse anti-HLA-DR and anti-DOTA scFv genes were combined, ligated into the pCANTAB 5E vector that co-expressed these self-assembling scFv in E. coli as two mismatched nonlinked pairs (VHA-link-VLB; VHB-link-VLA). The diabody protein that was purified from periplasm had the expected molecular characteristics when analyzed by sequencing, chromatography, electrophoresis and Western blot. This modular gene design platform provides methodology for easy and rapid creation of diabody molecules from diverse scFv libraries. Diabodies from various scFv can easily be produced, thereby facilitating comparative preclinical studies en route to development of new tumor targeting molecules.

BCL2 antisense transcripts decrease intracellular Bcl2 expression and sensitize LNCaP prostate cancer cells to apoptosis-inducing agents.

Prostate cancer (CaP) is the most commonly diagnosed cancer of aging men and the second leading cause of male cancer death in the United States. At present, no effective therapy is available for treating hormone independent CaP. Since Bcl2 is believed to play a role in protecting CaP cells from apoptosis, we investigated the effects of down-regulating Bcl2 expression on CaP cells. Genetically engineered LNCaP sublines were established by stably transfecting LNCaP cells with BCL2 antisense (BCL2-AS) transcript-expressing plasmids. Western blotting analysis showed that intracellular Bcl2 protein was decreased by 50-60% in BCL2-AS-transfected LNCaP cells. Expression of the antisense transcripts resulted in 50% growth inhibition of LNCaP cells in response to androgen withdrawal and markedly sensitized these cells to Adriamycin-induced apoptosis. These results suggest that down-regulation of Bcl2 protein using BCL2-AS transcripts could be exploited for improved treatment of advanced CaP.

Apoptosis-related gene and protein expression in human lymphoma xenografts (Raji) after low dose rate radiation using 67Cu-2IT-BAT-Lym-1 radioimmunotherapy.

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to 67Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, 67Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 micrograms Lym-1, or 335-500 microCi 67Cu on less than 400 micrograms Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-beta 1 and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. 67Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after 67Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with 67Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.

Nuclear control of cortical reorganization during sexual reproduction of Stylonychia mytilus: A study with amicronucleates.

During conjugation, Stylonychia mytilus undergoes repetitive somatic development by reorganizing the cortical ciliature consecutively for three times. The control of these reorganizations by the nucleus was investigated employing amicronucleates generated by microsurgery. When amicronucleates conjugate, they underwent only the first round of cortical reorganization; the exconjugants perished before the second cortical reorganization commenced. This shows that the micronucleus is dispensable for the first cortical reorganization, but that the micronucleus, or its divisional derivatives in sexual nucleogenesis, are essential for immediate postconjugational survival. Conjugation of amicronucleates with micronucleates in most cases resulted in the arrest of macronuclear anlage development at the post-polyteny DNA-poor stage; the second cortical reorganization could not be initiated in such cells. In others where the macronuclear anlage developed beyond the post-polyteny DNA-poor stage, the cell underwent the second, and then the third round of cortical reorganization. This correlation between macronuclear anlage development and the second cortical reorganization argues for a causal relationship between the two. Very likely, the macronuclear anlage up to the post-polyteny DNA-poor stage provides the crucial morphogenetic signal(s) for the second cortical reorganization. The nature of the first and second rounds of cortical reorganization are thus fundamentally different from each other, as their nuclear controls are radically dissimilar.

[Biodistribution and radioimmunoimaging of monoclonal antibody to human lung adenocarcinoma in tumor-bearing nude mice].

Biodistribution and radioimmunoimaging of monoclonal antibody, 5F11, against human lung adenocarcinoma (LTEP-a2) in tumor-bearing nude mice were studied. The binding rate of radioiodinated 5F11 with LTEP-a2, as determined by in vitro binding assay, was 27.64 +/- 5.01%, while the control was less than 5.0%, suggesting that 5F11 reacted specifically with LTEP-a2. Seventy-two, 96 and 120 hr after injection of 125I-5F11, the percentage of dose taken per gram tumor tissue was 4.84%, 6.29% and 6.60%, higher than those of all normal organs. From 48 to 120 hr on, the ratio of tumor to normal tissue (T/NT) in the liver, spleen, kidney, stomach and intestine was more than 2; T/NT in the lung was 3.1-4.7; T/NT in the blood increased gradually and was 1.87 at 96 after injection. Tumor location index was 4.16, while that of the normal tissue was only 1.21. These results showed that human lung adenocarcinoma xenografts in nude mice were specifically located by radioiodinated 5F11. Radioimmunoimaging was performed in tumor-bearing nude mice. The tumors were clearly visualized 72 hr after injection of 131I-5F11. Radioactivity was higher in tumor region than in other regions. The optimal imaging time was 72-96 hr after injection of radioiodinated 5F11.

[Localization of human lung cancer xenografts in nude mice using monoclonal antibody].

Radiolabeled monoclonal antibodies to human lung cancer were injected into nude mice bearing xenografts; and the biodistribution and tumor localization were studied. Xenografts were able to concentrate radioiodinated McAb. Radioactivities of tumor which were 5.09, 5.79 and 7.57% dose/g respectively 3-5 days after injection were higher than those of blood or organ. Tumor/blood ratios were 1.82-2.36 whereas tumor/organ ratios were more than 2. The localization indices of two McAbs were 3.4-5.6 and less than 1.5 in normal organs. The xenografts were visualized after injection of radiolabeled McAbs. These results have shown that anti-human lung cancer McAbs can specifically localize on xenografts in nude mice and it is possible to use them clinically for imaging of lung cancer.

miR-30 as a tumor suppressor connects EGF/Src signal to ERG and EMT.

Src tyrosine kinase (Src) is implicated in the development of bone metastasis and castration resistance of prostate cancer. Src inhibitors are currently being tested in clinical trials for such diseases. Understanding the molecular and cellular actions of Src inhibitors holds the key to future improvement of this line of therapy. Here we describe the microRNA expression profiles modulated by two Src inhibitors and demonstrate that the miR-30 family members are the most prominently induced species. Consistent with its tumor suppressor role, miR-30 is downmodulated by oncogenic signals such as epidermal growth factor (EGF) and hepatocyte growth factor, and is generally underexpressed in prostate cancer specimens. A number of epithelial-to-mesenchymal transition (EMT)-associated genes are predicted targets of miR-30. Among these genes the Ets-related gene (ERG) is the most frequently overexpressed oncogene in prostate cancer activated by genomic fusion events between promoter upstream sequences of the TMPRSS2 and coding sequences of ERG. We showed by ERG 3' untranslated region reporter and mutagenesis assays that ERG is a direct target of miR-30. Overexpression of miR-30 in prostate cancer cells suppresses EMT phenotypes and inhibits cell migration and invasion. It also inhibits the in vitro and in vivo growth of VCaP cells, which depends on TMPRSS2-ERG for proliferation. TMPRSS2-ERG is generally regulated by androgen at the transcriptional level. Our finding reveals a new post-transcriptional mechanism of TMPRSS2-ERG regulation by Src and growth signals via miR-30 providing a rationale for targeting ERG-positive castration-resistant tumors with Src inhibitors.

Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model.

Macro-autophagy is associated with drug resistance in various cancers and can function as an adaptive response to maintain cell survival under metabolic stresses, including androgen deprivation. Androgen deprivation or treatment with androgen receptor (AR) signaling inhibitor (ARSI), Enzalutamide (MDV-3100, ENZA) or bicalutamide induced autophagy in androgen-dependent and in castration-resistant CaP (castration-resistant prostate cancer (CRPC)) cell lines. The autophagic cascade triggered by AR blockage, correlated with the increased light chain 3-II/I ratio and ATG-5 expression. Autophagy was observed in a subpopulation of C4-2B cells that developed insensitivity to ENZA after sustained exposure in culture. Using flow cytometry and clonogenic assays, we showed that inhibiting autophagy with clomipramine (CMI), chloroquine or metformin increased apoptosis and significantly impaired cell viability. This autophagic process was mediated by AMP-dependent protein kinase (AMPK) activation and the suppression of mammalian target of rapamycin (mTOR) through Raptor phosphorylation (Serine 792). Furthermore, small interfering RNA targeting AMPK significantly inhibited autophagy and promoted cell death in CaP cells acutely or chronically exposed to ENZA or androgen deprivation, suggesting that autophagy is an important survival mechanism in CRPC. Lastly, in vivo studies with mice orthotopically implanted with ENZA-resistant cells demonstrated that the combination of ENZA and autophagy modulators, CMI or metformin significantly reduced tumor growth when compared with control groups (P<0.005). In conclusion, autophagy is as an important mechanism of resistance to ARSI in CRPC. Antiandrogen-induced autophagy is mediated through the activation of AMPK pathway and the suppression of mTOR pathway. Blocking autophagy pharmacologically or genetically significantly impairs prostate cancer cell survival in vitro and in vivo, implying the therapeutics potential of autophagy inhibitors in the antiandrogen-resistance setting.

Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells.

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

Inappropriate activation of androgen receptor by relaxin via beta-catenin pathway.

We have previously demonstrated that human H2-relaxin can mediate androgen-independent growth of LNCaP through a mechanism that involves the activation of the androgen receptor (AR) signaling pathway. The goal of the current study is to elucidate the mechanism(s) by which H2-relaxin causes activation of the AR pathway. Our data indicate that there is cross-talk between AR and components of the Wnt signaling pathway. Addition of H2-relaxin to LNCaP cells resulted in increased phosphorylation of protein kinase B (Akt) and inhibitory phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) with subsequent cytoplasmic accumulation of beta-catenin. Immunoprecipitation and immunocytochemical studies demonstrated that the stabilized beta-catenin formed a complex with AR, which was then translocated into the nucleus. Chromatin immunoprecipitation analysis determined that the AR/beta-catenin complex binds to the proximal region of the prostate-specific antigen promoter. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, using LY294002, prevented both H2-relaxin-mediated phosphorylation of Akt and GSK-3beta and translocation of beta-catenin/AR into the nucleus. Knockdown of beta-catenin levels using a beta-catenin-specific small interfering RNA inhibited H2-relaxin-induced AR activity. The combined data demonstrate that PI3K/Akt and components of the Wnt pathway can facilitate H2-relaxin-mediated activation of the AR pathway.

Morphology and development of mirror-image doublets of Stylonychia mytilus.

This paper describes the cortical anatomy and development of mirror-image doublets of Stylonychia mytilus, analyzed using the protargol technique. The reversed, or "left-handed" (LH) component of these doublets is a mirror image of the normal or "right handed" (RH) component with regard to the arrangement of cortical structures. The mirror-image patterning is imperfect, however, as the individual ciliary structures of the LH component all are of normal internal asymmetry, and the orientation of membranelles is inverted. Certain structures that would be expected to form near the line of symmetry are absent. During cell division and cortical reorganization, ciliary primordia arise and become arranged in a mirror-image pattern that is more perfect than that exhibited by the mature structures. Deviations from a mirror-image pattern appear at late stages when organelle sets differentiate within ciliary primordia: for example, the membranelle set differentiates within the oral primordium of the LH component in a sequence that is an inversion rather than a mirror image of the corresponding sequence of the RH component. This mixed control of oral development by different cortical "informational systems" accounts for some of the characteristic abnormalities of the mature oral structures of the LH component.

Effects of sequential deletions of residues from the N- or C-terminus on the functions of epsilon subunit of the chloroplast ATP synthase.

Ten truncated mutants of chloroplast ATP synthase epsilon subunit from spinach (Spinacia oleracea), which had sequentially lost 1-5 amino acid residues from the N-terminus and 6-10 residues from the C-terminus, were generated by PCR. These mutants were overexpressed in Escherichia coli, reconstituted with soluble and membrane-bound CF(1), and the ATPase activity and proton conductance of thylakoid membrane were examined. Deletions of as few as 3 amino acid residues from the N-terminus or 6 residues from the C-terminus of epsilon subunit significantly affected their ATPase-inhibitory activity in solution. Deletion of 5 residues from the N-terminus abolished its abilities to inhibit ATPase activity and to restore proton impermeability. Considering the consequence of interaction of epsilon and gamma subunit in the enzyme functions, the special interactions between the epsilon variants and the gamma subunit were detected in the yeast two-hybrid system and in vitro binding assay. In addition, the structures of these mutants were modeled through the SWISS-MODEL Protein Modeling Server. These results suggested that in chloroplast ATP synthase, both the N-terminus and C-terminus of the epsilon subunit show importance in regulation of the ATPase activity. Furthermore, the N-terminus of the epsilon subunit is more important for its interaction with gamma and some CF(o) subunits, and crucial for the blocking of proton leakage. Compared with the epsilon subunit from E. coli [Jounouchi, M., Takeyama, M., Noumi, T., Moriyama, Y., Maeda, M., and Futai, M. (1992) Arch. Biochem. Biophys. 292, 87-94; Kuki, M., Noumi, T., Maeda, M., Amemura, A., and Futai, M. (1988) J. Biol. Chem. 263, 4335-4340], the chloroplast epsilon subunit is more sensitive to N-terminal or C-terminal truncations.