Inconsistent findings have been reported on the occurrence and relevance of creatine kinase (CK) isoenzymes in mammalian liver cells. Part of this confusion might be due to induction of CK expression during metabolic and energetic stress.The specific activities and isoenzyme patterns of CK and adenylate kinase (AdK) were analysed in pathological liver tissue of patients undergoing orthotopic liver transplantation.The brain-type, cytosolic BB-CK isoenzyme was detected in all liver specimens analysed. Conversely, CK activity was strongly increased and a mitochondrial CK (Mi-CK) isoenzyme was detected only in tissue samples of two primary hepatocellular carcinomas (HCCs).The findings do not support significant expression of CK in normal liver and most liver pathologies. Instead, many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. Moreover, the data suggest a possible interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC. These results, if confirmed, could provide important hints at improved therapies and cures for HCC. Creatine kinase (CK) isoenzymes catalyse the reversible transfer of the phosphate group of phosphocreatine (PCr) to ADP, to yield ATP and creatine (Cr). The CK/PCr/Cr system is present primarily in tissues with high and fluctuating energy demands such as brain, heart and skeletal muscle, and serves as a temporal and spatial "energy buffer" that helps to maintain a high intracellular phosphorylation potential in situations of increased metabolic demand .de novo. The liver is the main site of Cr production in the body . After ody see -6). Othe. Othede ody . Finallody  isoenzyme. On the other hand, besides BB-CK which was suggested to be present in endothelial and Kupffer cells, Vaubourdolle et al.  also proIn a number of studies reporting significant levels of CK activity in liver, interference by adenylate kinase (AdK) isoenzymes in the CK activity assays -21 is veBecause of these conflicting data, the goal of the present study was to analyse in detail the CK and AdK activities in pathological liver tissue of patients undergoing orthotopic liver transplantation.The present project was approved by the ethics commission of the University of Innsbruck. In total, 25 liver samples were analysed. Twenty-three samples were obtained from 18 explanted organs of liver transplant recipients, one sample was obtained at autopsy (no. 1), and the last sample was from a normal rat liver. According to pathomorphological criteria, the 25 samples can be divided into 5 groups: (1) Nine samples of cirrhotic liver tissue ; (2) six samples of neoplastic tissue ; (3) three samples of necrotizing liver tissue due to acute or subacute organ rejection ; (4) five samples of macroscopically normal liver parenchymal tissue  surrounding focal liver pathologies ; (5) two samples originating from a normal rat liver (no. 12) and from a patient with steatosis hepatis (no. 1).All steps were performed on ice or at 4°C. Approximately 5 g of liver tissue was homogenized in 45 ml buffer A . The homogenate was subjected to centrifugation for 5 min at 800 g. The pellet was discarded, and the supernatant centrifuged for 4 min at 5,100 g (centrifugation C2). The supernatant of C2 was further clarified by centrifugation for 12 min at 12,300 g, thus yielding the cytosolic fraction. The pellet of C2 was resuspended in 10 ml buffer A, followed by centrifugation for 2 min at 12,300 g (C3). After resuspension of the C3 pellet in 10 ml buffer A and centrifugation for a further 10 min at 12,300 g, the sediment was resuspended in 4 ml buffer A, thus yielding the mitochondrial fraction. One-ml aliquots of the different fractions were immediately frozen in liquid nitrogen and stored at -80°C until analysis.2, 2.1 mM ADP, 2.1 mM NADP, 21 mM N-acetylcysteine, 9 U/ml of hexokinase, and 5.8 U/ml of glucose-6-phosphate dehydrogenase (both from Sigma). Enzymatic activity was measured at 25°C as an increase in NADPH absorbance at 340 nm. For AdK, three separate measurements were made for each sample in the same assay medium. For CK measurements, 5.1 mM AMP was added to the assay medium to inhibit AdK activity. For each sample, three measurements with 10.3 mM PCr and three measurements without PCr  were made, and the CK activity was calculated as the difference of the respective means. All values in this paper represent specific activities per mg of homogenate, cytosolic or mitochondrial protein. Protein amounts were measured according to the method of Bradford -1). Therefore, in the absence of histochemical data, it cannot be concluded with certainty whether the increased levels of CK are due to increased vascularization of the tumour , or to induction of CK expression in the malignant cells.Interestingly, we observed a strong induction of both BB-CK and Mi-CK expression in two samples of primary HCC. Despite CK/AdK activity ratios Induction of CK expression has been observed previously in many types of tumours (see ) and mayA key player in the picture might be the p53 tumour suppressor gene. Mutations in p53 are quite prevalent in HCC, especially in tumours with low cellular differentiation ,43. On tin vivo models  the limited responsiveness of HCC to currently available therapeutic approaches and, thus, (ii) the poor prognosis associated with this disease. Cr analogues (cyclocreatine and β-guanidinopropionic acid) and also Cr itself were previously shown to have antitumour activity, both in cell culture and in  models . Similaepatoma; .The present findings shed light on some old enigmas and open up fascinating avenues for future research. Our findings do not support significant expression of CK in normal liver and most liver pathologies, but rather indicate that many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. On the other hand, given the need for improved understanding of the molecular pathogenesis of HCC, and for improved therapies and cures, the induction of CK expression in HCC described here calls for a more in-depth analysis of the interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC.AdK, adenylate kinase; BB-CK, brain-type cytosolic CK isoenzyme; CK, creatine kinase; Cr, creatine; HCC, hepatocellular carcinoma; Mi-CK, mitochondrial CK; MM-CK, muscle-type cytosolic CK isoenzyme; PCr, phosphocreatine.The authors declare that they have no competing interests.GM and RM covered the medical part of this study. GM, FNG and MW performed the biochemical experiments. MW drafted the manuscript.The pre-publication history for this paper can be accessed here:


The hedgehog pathway plays a critical role in the development of prostate. However, the role of the hedgehog pathway in prostate cancer is not clear. Prostate cancer is the second most prevalent cause of cancer death in American men. Therefore, identification of novel therapeutic targets for prostate cancer has significant clinical implications.Su(Fu). Furthermore, expression of sonic hedgehog protein is detected in majority of PTCH1 positive tumors (24 out of 27). High levels of hedgehog target genes are also detected in four prostate cancer cell lines . We demonstrate that inhibition of hedgehog signaling by smoothened antagonist, cyclopamine, suppresses hedgehog signaling, down-regulates cell invasiveness and induces apoptosis. In addition, cancer cells expressing Gli1 under the CMV promoter are resistant to cyclopamine-mediated apoptosis. All these data suggest a significant role of the hedgehog pathway for cellular functions of prostate cancer cells.Here we report that activation of the hedgehog pathway occurs frequently in advanced human prostate cancer. We find that high levels of hedgehog target genes, PTCH1 and hedgehog-interacting protein (HIP), are detected in over 70% of prostate tumors with Gleason scores 8–10, but in only 22% of tumors with Gleason scores 3–6. Furthermore, four available metastatic tumors all have high expression of PTCH1 and HIP. To identify the mechanism of the hedgehog signaling activation, we examine expression of Su(Fu) protein, a negative regulator of the hedgehog pathway. We find that Su(Fu) protein is undetectable in 11 of 27 PTCH1 positive tumors, two of them contain somatic loss-of-function mutations of Our data indicate that activation of the hedgehog pathway, through loss of Su(Fu) or overexpression of sonic hedgehog, may involve tumor progression and metastases of prostate cancer. Thus, targeted inhibition of hedgehog signaling may have significant implications of prostate cancer therapeutics. AftSu(Fu) is reported to affect β-catenin signaling ,28. We aIn addition to Su(Fu) inactivation, over-expression of Shh is another mechanism by which the hedgehog pathway is activated in cancer -10. We nOnce hedgehog pathway is activated, the target gene expression will be up-regulated. Thus, analysis of target gene expression using immunohistochemistry will be an effective way to detect hedgehog pathway activation in prostate cancer. Currently, PTCH1, Gli1 and HIP are good markers for the hedgehog pathway.Our findings not only provide novel basic understanding of prostate cancer, but also allow us to design new ways to treat prostate cancer. With a specific SMO antagonist, cyclopamine, it will be possible in the future to treat prostate cancers, which have over-expressed sonic hedgehog. However, as a downstream molecule, tumors with Su(Fu) inactivation may not respond to cyclopamine treatment. Therefore, additional small molecule inhibitors appear to be necessary to treat Su(Fu) inactivated prostate cancer. One possibility is to use Gli1 SiRNA since we have indicated that down-regulation of Gli1 may be an important mechanism by which inhibition of the hedgehog pathway by cyclopamine induces apoptosis Fig. . SanchezTaken together, our findings suggest that activation of the hedgehog pathway involves prostate cancer progression. There might be several mechanisms by which the hedgehog pathway is activated in advanced prostate cancers, including loss of Su(Fu) protein expression, over-expression of sonic hedgehog or other alterations. We demonstrate that activation of the hedgehog pathway is associated with DNA synthesis and cell invasiveness in prostate cancer cells. Inhibition of the hedgehog pathway, on the other hand, causes apoptosis possibly through down-regulation of Gli1. Our studies predict that targeted inhibition of the hedgehog pathway may be an effective way to prevent prostate cancer progression.® according to manufacturer's instruction .A total of 55 paraffin-embedded tissue blocks from patients with prostate cancer were obtained from UTMB Surgical pathology with approval from the Institutional Review Board (IRB). Pathological reports, H#E staining of each specimen were reviewed to determine the nature of the disease and the Gleason scores. Of 55 specimens, 18 were from tumors with Gleason scores 3–6, 15 with Gleason score 7 and 22 with Gleason scores 8–10. The tumor area was first identified before tissue microarray (1.5 mm in diameter for specimens) was assembled with Beecher's Tissue arrayer-IA standard avidin-biotin immunostaining technique was performed using a kit from Vector laboratories using specific antibodies to Su(Fu) (Santa Cruz Biotechnology Cat# 10933), PTCH1 (Santa Cruz Biotechnology Cat# 6149), HIP (R&D systems Cat# AF1568) and Shh (Santa Cruz Biotechnology Cat# 9024) and PSA (Vector laboratories). Positive staining was in red or brown. The specificity of antibodies was tested using the very peptide used for raising the antibodies, which abolished the specific staining. Hematoxylin was used for counterstaining (in blue). Protein was analyzed by Western analysis with appropriate antibodies [Su(Fu) antibodies were from Santa Cruz, beta-actin antibody was purchased from Sigma). The signals were visualized with the enhanced chemiluminescence detection system (Amersham).Cell lines , with triplicates for each sample and the experiment was repeated three times with the similar results. Cell were treated with 5 μM cyclopamine (or tomatidine) before (for 12 h) and during cell invasion assay (for 24 h). The rate of cell invasion was calculated by dividing cell numbers penetrated the matrigels by the number of cell in the control chambers (without matrigels).® reagent (Invitrogen), and RT-PCR was performed using Promega's RT-PCR system according to the manufacturer's protocol. Two pairs of Su(Fu) primers were used . PCR products were first purified using Qiagen's Gel Extraction Kit. Due to existence of possible Su(Fu) splicing isoforms in humans, Su(Fu) genetic mutations were screened after the PCR products were cloned into TOPO® TA cloning vectors (Invitrogen). Several independent clones (from three experiments) of each PCR product were selected for sequencing analysis in UTMB sequencing facility. All mutations were confirmed by at least six independent clones.Total RNA was isolated using Trizol® assay reagent (P/N 4319413E) for an internal control. The primers are designed to span exon-exon junctions so as not to detect genomic DNA and the primers and probe sequences were searched against the Celera database to confirm specificity. To obtain the relative quantitation of gene expression, a validation experiment was performed to test the efficiency of the target amplification and the efficiency of the reference amplification. All absolute values of the slope of log input amount vs. ΔCT were <0.1. Separate tubes (singleplex) one-step RT-PCR was performed with 20 ng RNA for both target genes and endogenous control. The reagent we used was TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169). The cycling parameters for one-step RT-PCR was: reverse transcription 48°C for 30 min, AmpliTaq activation 95°C for 10 min, denaturation 95°C for 15 sec and annealing/extension 60°C for 1 min (repeat 40 times) on ABI7000. Triplicate CT values were analyzed in Microsoft Excel using the comparative CT(ΔΔCT) method as described by the manufacturer. The amount of target (2-ΔΔCT) was obtained by normalization to an endogenous reference (18sRNA) and relative to a calibrator.Real-time PCR We used Applied Biosystems' assays-by-demand 20× assay mix of primers and TaqMan probes (FAM™ dye-labeled) for the target genes  and pre-developed 18S rRNA (VIC™-dye labled probe) TaqManin situ cell proliferation kit  [in situ cell death kit  [BrdU labeling was performed using an emicals) . Cells wemicals) ,29. Cellpatched 1; Shh – sonic hedgehog; SMO – smoothened, BCC – basal cell carcinoma.PSA – prostate specific antigen; HIP – hedgehog-interacting protein; Su(Fu) – suppressor of fused; PTCH1 – human homologue of Tao Sheng contributed to Figures Table 1 Prostate cancer specimens and protein expression. Prostate cancer specimens and expression of several hedgehog signaling proteins are summarized in this table (A). A total of 55 specimens were used in this study. The Gleason scores and protein expression of Shh, PTCH1 and Su(Fu) are shown (B).Click here for file


There is little in biology that compares in beauty and limpidity to the development of a zebrafish embryo as viewed through a light microscope. The transparent eggshell and embryo tissues expose the minutest details of cell migrations and organ assembly to the curious viewer. Within a day, distinct vertebrate features emerge: a distinct head with the outlines of two large eyes, a quickly pumping heart, a notochord, and a growing array of somites—the bone and muscle precursors—stretching from trunk into tapering tail.The transparent zebrafish embryo has allowed geneticists to discover a large number of mutants with anomalies in the development of external and internal organs. Seven mutations, collectively known as “You-class,” turn the pointed, chevron-like somites into shallow, rounded arcs (“You” stands for “U-shaped”). Ian Woods and William Talbot now show that the You mutation disrupts a new modulator of Hedgehog signaling.Hedgehog is an extracellular signaling protein that can impose various fates on target cells at close proximity or over longer distances. Much research is focused on understanding the factors that promote or limit Hedgehog's activity and range. Woods and Talbot propose that the You protein acts in the extracellular environment to promote Hedgehog signaling.Hedgehog was originally named for mutations that cause excess brush-like denticles to grow on the surface of fruitfly embryos, but it is now known to direct countless developmental decisions in invertebrates and vertebrates alike. In addition, several cancers are known to result from inappropriate Hedgehog signaling. In fish, Hedgehog's best-documented role is in muscle development. In the absence of Hedgehog signaling, cells destined to become slow muscle fibers fail to differentiate properly. A subset of these slow muscle cells—the muscle pioneers—congregate near the dorso-ventral midline of the embryo, where the dorsal and ventral halves of somites converge. When these specialized cells are absent, abnormal somite assembly leads to the U-shaped phenotype.The authors found that You mutants showed many telltale signs of reduced Hedgehog signaling. Proteins that are normally expressed at certain times during the development of slow muscle cells were not activated in You mutants, indicating that these cells did not form. Mutant embryos also displayed reduced expression of the Hedgehog receptor Patched, a universal reporter of Hedgehog signaling activity. In addition, You mutants had specific ventral spinal chord defects that are shared by known Hedgehog pathway mutants. Yet You mutants expressed Hedgehog normally. Moreover, Hedgehog targets could still be activated in You mutants in response to excess Hedgehog signaling, suggesting that the signaling cascade is left intact. The authors concluded that the You protein was a facilitator rather than a crucial transmitter in Hedgehog signaling, likely acting at a step upstream of a cell's response to Hedgehog. Normal muscle pioneers could form in chimeric embryos (embryos made of wild-type and You mutant cells) regardless of which cells—the Hedgehog-producing cells or Hedgehog-responding muscle precursors—expressed You. This made it most likely that the You protein acted outside the cells, perhaps as a cell matrix component.The authors mapped the You mutation and found that it disrupted the coding region of a gene encoding a putative secreted protein. The predicted You protein is closely related to members of the mouse SCUBE family, a group of proteins that are defined by characteristic extracellular motifs . This observation strengthens the hypothesis that the You protein has extracellular functions, and the researchers' experimental evidence supports a role for You in transport or stabilization of Hedgehog. At later stages You could also participate in other signaling pathways, as its expression does not always coincide with that of Hedgehog during zebrafish development.


These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed  Genetic studies in zebrafish have identified a new protein involved in Hedgehog signaling - a key pathway required for development in vertebrates and invertebrates Drosophila larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development . Misregiewed in ,3,4, mediewed in ,6,7, paniewed in , and holiewed in ,10.Drosophila, diffusion of lipid-modified Hedgehog proteins is dependent on the action of tout velu, a gene involved in the synthesis of heparan sulfate proteoglycans [growth-arrest specific gene product Gas1 [After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In oglycans ,12. The oglycans . In vertuct Gas1 , and Heduct Gas1 . Moreoveuct Gas1 ,17. Geneuct Gas1 ,19.prox1. In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of prox1 expression [Hedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation ,26,27,28pression . As devepression ,27,28. Spression ,28,29,30pression ,28.Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations ,31,32,33you-class” mutants because of their U-shaped somites. Five of the seven you-class mutations have been cloned, and four of these genes, syu/shh, yot/gli2, smu/smoh, and con/disp1, encode members of the Hedgehog signaling pathway [ubo/prdm1, which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ubo and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in ubo mutants [ubo: Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target ptc1, whereas ptc1 expression is normal in ubo mutants, indicating that they can receive Hedgehog signals [Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish ,39,40,41 pathway ,36,37,39w muscle ,42. Care mutants ,43. Exam signals ,33,36,39you-class gene, you, has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ptc1 and adaxial myod, is reduced in you mutants [you gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the you gene has remained unknown. We report the positional cloning of the you gene and show that it encodes a novel extracellular EGF-CUB  protein required for Hedgehog signaling. Functional studies provide evidence that you is essential for the transport or stability of Hedgehog signals in the extracellular environment.Previous phenotypic characterization of mutants for the eponymous  defects ,43. More mutants . These ryou mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the you class . These hedgehog genes—ehh, shh, and twhh—are expressed at the midline in early embryonic stages. To determine whether you is required for hedgehog gene transcription, we analyzed the expression of hedgehog genes in wild-type and you mutant embryos. Expression of all three hedgehog genes appeared normal in you embryos at bud stage  region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270  library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked you  and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes SCUBE2, LMO1, STK33, and ST5 exhibited conserved synteny with orthologous genes in both mouse and zebrafish . Pairwibrafish , the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB . In all ty97you mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon  injection experiments to phenocopy defects seen in you, and mRNA injection experiments to rescue the you phenotype in mutants (myod (n = 112) in the adaxial cells (n = 70) in the muscle pioneers (myod (n = 65) or Engrailed (n = 48) expression  of embryos from you/+ intercrosses showed expression of myod in adaxial cells at 12 somites (myod expression from these intercrosses showed that 137 (24%) were homozygous mutant for you. In contrast, 23.6% (n = 127) of embryos from you/+ intercrosses injected with a mutant form of you mRNA lacked expression of myod in adaxial cells  from a you/+ intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers (ty97you), whereas 24% (n = 33) of embryos injected with control mRNA lacked Engrailed expression (you mutation). In addition, injection of you MOs resulted in loss of nkx2.2 expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of you mRNA was sufficient to rescue nkx2.2 expression in you mutants (data not shown).When injected with 50 pg of synthetic wild-type  somites E. Genotyal cells F; 20 of you transcripts appear to be maternally deposited in zebrafish embryos  you was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes , in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis  of synthetic you mRNA that was sufficient to rescue the phenotypic defects observed in you mutants  was injected into embryos from a you/+ intercross, ectopic expression of myod . Because downstream targets of the Hedgehog pathway were rescued or upregulated in shh-injected you mutants, components of the Hedgehog pathway downstream of shh are most likely functional in you embryos. These results are consistent with you acting upstream of or parallel with shh in the Hedgehog pathway.To explore the possibility that the  mutants . When con = 394; B, Engrai n = 25; F, or nkx n = 17; J. This r n = 53; C and 6D, n = 78; K and 6L patched activity with MOs  or a combination of MOs targeting both ptc1 and ptc2 (n = 13 mutants). you mutant embryos injected with a ptc1 mismatch control MO did not exhibit rescued myod expression (n = 8 mutants). In similar experiments, injection of patched MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of you mutant embryos .Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down with MOs . Expressyou function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts . Similarly, cells from embryos in which you function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown).To determine whether a cell must be wild-type for pe hosts . Cells dyou function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into you mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in you mutants .you is essential for Hedgehog signaling, our analysis indicates that you mutant cells are able to produce and respond to Hedgehog signals. hedgehog gene expression in the embryonic midline is normal in you mutants, indicating that You acts downstream of hedgehog gene transcription  in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial myod, Engrailed in muscle pioneers, or nkx2.2 in the ventral spinal cord . For initial localization of you, bulked segregant analysis  was perescribed .http://www.rzpd.de). BAC end sequences were obtained from the Sanger Institute database  following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer . Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15.The CHORI211 BAC library was screened by PCR to identify positive BAC clones (you clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the you protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant you clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for you were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic you mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit .A full-length you/+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [myod [ptc1 [nkx2.2 [ehh [shh [Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from escribed . Other ped [myod , ptc1 [6od [ptc1 , nkx2.2  [nkx2.2 , ehh [20ehh [shh , and monehh [shh . Genotypyou translational initiation site (5′-GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAgAGTCgAAACAcCTgCCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting ptc1 and ptc2 were obtained from [Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described . RNA wasned from .you/+ intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran . Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from you/+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into you mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in you/+ intercrosses.Cellular transplantations were done according to standard methods . Embryosyou cDNA sequence has been deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) under accession number AY741664.The http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716), con/disp1 (LocusID 378448), Cubilin (LocusID 8029), ehh (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725), myod (LocusID 30513), nkx2.2 (LocusID 30697), Patched (LocusID 35851), prox1 (LocusID 30679), ptc1 (LocusID 30181), ptc2 (LocusID 30189), sea urchin Fibropellin (LocusID 373313), smu/smoh (LocusID 30225), syu/ssh (LocusID 30269), Tolloid (LocusID 42945), tout velu (LocusID 36614), twhh (LocusID 30444), ubo/prdm1 (LocusID 323473), yot/gli2 (LocusID 30154), human LMO1 (LocusID 4004), human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human ST5 (LocusID 6764), human STK33 (LocusID 65975), mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).The LocusLink (


FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci). Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling.FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1) maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI.The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing. Thus, mRNAs may contain different 5'UTRs and encode different protein isoforms. Furthermore, FU is able to enhance the activity of GLI2 but not of GLI1, implicating FU in some aspects of Hedgehog signaling.The  Drosophila, Hh signaling to the transcription factor Cubitus interruptus (Ci) is mediated by a protein complex consisting of Ci and three other cytosolic proteins. These are the costal 2 (cos2), suppressor of fused (su(fu)) and fused (fu), where fu is a kinase domain containing protein with positive regulatory activities in Hh induction of Ci mediated transcriptional activation. Hh binds to its receptor patched (ptc), a 12 membrane spanning protein, leading to the activation of another membrane protein smoothened (smo) . In Droed (smo) . Smo is iewed in ]. It is iewed in -7. In a iewed in . FU is aPTCH1, the human counterpart of ptc, underlie the Nevoid Basal Cell Carcinoma Syndrome (NBCCS) [SMO and SUFU mutations as well as overexpression of GLI1 or GLI2 can lead to BCC or medulloblastoma [Interestingly, it was discovered that mutations in  (NBCCS) ,10. Pati (NBCCS) ,12. Alsoblastoma -16. ThusFU gene, as well as for construction and subcloning of FU expression vectors. Using the available public databases the FU gene was found to be present in a sequenced BAC clone from chromosome 2. FU is located in the same region of chromosome 2q34-q36 to which the human limb malformation disorder Syndactyly type 1 (SD1) has recently been mapped in a large German pedigree [FU gene in an affected member from the German family [FU has been determined using an RNA array and Northern blots. FU is expressed in all 72 tested tissues. It is clear that not only a single transcript is expressed. Instead transcripts of different sizes are seen and some tissues apparently express more than one major transcript. From the genomic structure and the cDNA clones it was possible to predict several alternative splicing events and consequently the likely expression of different protein isoforms. Two of the isoforms were expressed in HEK293 cells and tested for their ability to regulate the activity of GLI1 and GLI2, showing positive effects on GLI2 but not on GLI1.Here three FU cDNA clones have been identified and used for sequence analysis, identification and structural description of the pedigree  and confpedigree . Its posn family . The tisFU gene in a 200 kb BAC clone (AC009974) from chromosome 2. The gene is localized to 2q35 at 217.56 Mb using the Ensembl [FU structure and named the gene STK36 (serine/threonine protein kinase 36). Chromosome 2q35 is the locus of several genetically based disorders. Both Syndactyly type 1 (SD1) and Brachydactyly type A1 (BDA1) have been mapped to this region [IHH (Indian Hedgehog) one of the vertebrate Hh homologues [IHH is located in the vicinity of FU on chromosome 2 (217.94 Mb) less than 400 kb away. In order to determine if alterations of FU are responsible for SD1, the FU coding region (exons 3–29) and the flanking intronic regions were sequenced using genomic DNA from an affected member of an SD1 family whose trait maps to the 2q34-q36 region [FU mutations were detected in this study, although three single nucleotide polymorphisms were identified. These included a T to C transition in intron 10, 17 bp 5' of exon 11 (IVS10-17T>C), causing gain of a BstNI site, and a G to A transition in exon 16, 17 bp 5' of the end of the exon (1748G>A), causing substitution of glutamine for arginine at amino acid 583 (R583Q) and loss of an AciI site. The altered restriction sites created by these sequence changes were tested in 44 CEPH unrelated individuals. The results showed that both changes are normal sequence variations as previously reported in the NCBI SNP database. The third change was a G to A transition in exon 27 (3008G>A), causing substitution of aspartic acid for glycine at amino acid 1003 (G1003D). By sequencing exon 27 in 8 affected and 6 unaffected members of the SD1 family [The sequence information derived from the FU cDNA clones 1HFU, 2HFU and Ngo3689 (see Methods) allowed the identification of the  Ensembl  annotatis region ,20. ReceFU exons, but they allow determination of the exon-intron organization of FU. Figure FU gene. The cDNA clones are outlined to account for the predicted structure. Only clone 1HFU and Ngo3689 contain exons from the 5'non-coding region. To the 5' side of the sequence encoded by exon 3 these clones are different, indicating that alternative 5' untranslated regions (UTR) from different exons can be used. Exons 3 to 9 encode the N-terminal kinase domain. None of the cDNA clones encodes the FU protein that has previously been described [None of the obtained cDNA clones contain sequence from all + RNA from 72 human tissues was hybridized with a labeled probe 3' of the kinase domain. It was clear from this array that all examined tissues express FU to some extent. The highest amount of FU transcripts were detected in testis and pituitary (not shown). This is in agreement with the previous results by Northern blotting, showing highest FU expression in testis [A tissue array with poly An testis . This ann testis . The Norn testis . Since on testis .To examine more specifically the expression of exons that are involved in alternative splicing events, nested PCR was performed on cDNA probes generated from whole tissue RNA by reverse transcription. Sequences containing segments including exons 8, 13 and 24 were amplified and analyzed on agarose gels .We investigated the possibility that FU expression have shown that transcripts are detected in all tissues examined. For the first time evidence is presented showing that more than one transcript can be expressed from this gene. The Northern blots clearly show that FU transcripts of different sizes indeed exist. Here the transcripts are estimated to be slightly bigger than previously reported and in some tissues more than one transcript is evident. It is clear from the available cDNAs and RT/PCR based transcript analyses that alternative splicing occurs. Additionally, it is also clear that different 5'UTRs are present in the transcripts. At least two protein isoforms, besides the previously described L-FU [Analyses of bed L-FU , may be bed L-FU . WhetherThe assessment of functionality revealed that S-FU was not able to regulate GLI1 or GLI2 when expressed in 293 cells. In contrast, both L-FU and a variant lacking a full kinase domain (2HFU) were able to enhance GLI2 induced transcription. These results are qualitatively similar to those previously reported in C3H/10T½ cells . L-FU anFU is localized on chromosome 2q35 very close to IHH. Though SD1 has been mapped to this region, we have not identified a causative role for FU in this disorder. FU consists of 29 exons of which 1 and 2 encode 5'UTRs and 3 to 9 encode a kinase domain. For the first time it is shown that transcripts of different sizes are expressed and alternative splicing takes place, probably leading to the generation of different protein isoforms. FU protein is likely to be involved in the Hedgehog signaling pathway since it can enhance the activity of GLI2. In contrast, it has no effect on GLI1 and an effect on SUFU cannot be observed in 293 cells.+. The human BAC clone AC009974 was obtained from Research Genetics . The human GLI, human SUFU, 12GLI-RE-luciferase reporter and β-galactosidase vectors have been described previously [Two almost full-length human FU clones were identified in the Incyte database. Both 1HFU and 2HFU were cloned in the vector pINCY. A third clone was available from Kazusa DNA Research Institute  and termed Ngo3689 (Gene name KIAA1278). This clone was in the vector pBluescript II SKeviously .R DNA polymerase . The cDNA for the long form of FU (L-FU) was subcloned into pCDNA3.1-HisB using the NotI and XbaI sites. 2HFU and the short FU (S-FU) cDNAs were subcloned into pCDNA3.1-HisC using the KpnI and XbaI sites.Expression constructs for different isoforms of FU was obtained by direct PCR or extension overlap PCR, using end-primers having specific restriction sites and the high fidelity VentAll PCR generated products were analyzed by DNA sequencing. The Big-Dye Terminator Cycle Sequencing kit  was used according to instructions. Sequencing was performed at CyberGene AB . Sequence alignments were done using the DIALIGN program  availablFU either as single exons with flanking intronic sequences or as products containing two exons with flanking intronic sequence and the complete intervening intron. The primer sequences can be obtained upon request. PCR was performed in a standard fashion and products were sequenced using either the Thermosequenase CyTM5.5 Dye Terminator or DYEnamic ET Dye Terminator Cycle Sequencing kits . Electrophoresis and analysis were performed on either an Automated Laser Fluorescence (ALF) DNA sequencer or MegaBACE DNA sequencer (Amersham Biosciences) after purification with Autoseq columns (Amersham Biosciences). For exon 27, the PCR product was purified using the enzymatic ExoI-SAP purification method, sequenced using the Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech) and analysed on an ABI 3100 genetic analyzer (Applied Biosystems). PCR products containing exon 11 or exons 15/16 were digested with BstNI or AciI, respectively, and the bands resolved on 3–4% agarose gels to confirm sequence changes in the patient with SD1 and to determine their frequency in a panel of 44 CEPH individuals.After informed consent was obtained, blood was taken from affected and unaffected family members and DNA extracted from peripheral blood leukocytes according to standard methods. Intronic primers were designed to amplify exons 3–29 of 32P-ATP using the High Prime DNA labeling kit  according to instructions. Hybridization of Northern blots was done with labeled DNA probes in ExpressHyp (Clontech) at 68°C according to instructions. The blots were then analyzed with a Fujix Bas 2000 phosphoimager .Commercially available Human MTN 12-lane Blot 2, Human Fetal MTN Blot II and Human Endocrine System MTN Blot Northern blots  were obtained and used with PCR generated hybridization probes. DNA probes were made by direct PCR, amplifying the sequences corresponding to exon 13 and 28. The generated fragments were then labeled with The expression of exon 8, 13 and 24 sequences in mRNA was assessed by nested PCR on RT/PCR generated cDNA samples from eight different tissues as provided in Human Multi Tissue cDNA Panel II (Clontech). Two sets of primers were made for each exon to be investigated. The outer pairs were used in a first PCR using 5 μl of the cDNA and Vent polymerase. In a second PCR 0.5 μl of the first PCR products was used together with the inner primer pairs. These pairs were also used for PCR of FU cDNA clones that served as controls. The primer pairs are listed in Table The cDNA clones were used in transfections of HEK293 cells in 24 well culture plates. Basically this was done as previously described . In shorTØ contributed to the experimental design; participated in sequencing, sequence analysis and subcloning; did the gene analysis, Northern blots, nested PCR, cell experiments; and made the manuscript draft. DBE and CES designed and carried out the patient analysis; and contributed to the manuscript. MM provided clones; contributed with subcloning; made the array analysis; and contributed to the manuscript. MMN and RCB provided the SD1 patient material; performed segregation analyses in the SD1 family; and edited the manuscript. PGZ contributed to the experimental design and subcloning; and edited the manuscript. RT contributed to the experimental design; did data base analysis; and edited the manuscript.


The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported.EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas.RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease.All three prostate cancer cell lines expressed the EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target. Prostate cancer is the most frequent cause of cancer death in men in Australia. Although many genetic changes have been detected in human prostate cancer, the role of most of these in initiation and progression of the disease is unclear. Receptor tyrosine kinases (RTKs) couple ligand binding to downstream signalling cascades and gene transcription and are key regulators of normal cellular processes such as growth, differentiation, migration and apoptosis. RTKs are also critically involved in the development and progression of human cancers and are therefore useful targets for anti-cancer therapies ,2. At leLigand binding induces receptor autophosphorylation of intracellular tyrosine, threonine and serine residues and allows interactions with a variety of different proteins that regulate cell functions such as contact inhibition, cytoskeletal organisation and cell motility ,8. SeverEphB4 was identified as one of the RTKs expressed in the xenograft tissue. More recently, Xia et al (2005) report that EphB4 is commonly expressed in prostate tumour tissues and cell lines and knockdown of EphB4 protein using siRNA and antisense approaches inhibited cell growth/viability, migration and invasion both in vitro and in vivo [EphB4 gene and its protein product in prostate cancer cell lines and tumour tissue samples.Members of both classes of Eph receptor have been found to have increased gene expression and/or protein levels in tumours from many different human tissues -20. In p in vivo . Concurr2 vented tissue culture flasks at 37°C in a 5% CO2 environment. Cells were collected at >90% confluency by trypsin digestion and centrifugation for 5 min at 1000 rpm, resuspended in phosphate buffered saline (PBS) and counted using a haemocytometer.Human prostate cancer cell lines LNCaP, DU145 and PC3 were cultured in HEPES-buffered RPMI 1640 medium , pH 7.4. The medium was supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 160 μg/ml L-glutamine and 10% heat-inactivated foetal bovine serum  in 75 cmRNA was isolated from cell lines using Tri Reagent (Invitrogen). Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Tri Reagent. After a five min incubation with gentle rocking, the cell lysate was removed to a 2 ml eppendorf tube. The RNA fraction was extracted using the manufacturer's recommendations.DC Protein Assay Kit from Biorad  following the manufacturer's protocol and using bovine serum albumin diluted from 0.1 mg/ml to 10.0 mg/ml to determine the standard curve.Growth medium was aspirated from the flask of growing cells (>90% confluent) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of Cell Lytic M Cell Lysis Reagent (Sigma) supplemented with 5 μl Protease Inhibitor Cocktail (Sigma). Protein lysate was removed to a 1.5 ml microfuge tube and the solution mixed for 30 min at 4°C on a rotary mixer. Insoluble protein was pelleted by centrifugation at 4°C for 30 min and the supernatant containing the soluble proteins was stored at -80°C until required for Western analysis. Protein concentrations were determined using the 6 primers (Invitrogen), 200 μM each deoxyribonucleoside triphosphate (dNTP)  and 200 U Superscript III reverse transcriptase (Invitrogen) in a reaction volume of 30 μl. Primers specific to either EphB4 or the housekeeping genes Porphobilinogen deaminase (PBGD) and Hypoxanthine phosphoribosyl-transferase 1 (HPRT 1) were used in a PCR reaction carried out in a BioRad iCycler MyiQ Real Time thermocycler or a Corbett Rotorgene 3000 . Cycling conditions included an initial denaturation at 94°C for 15 min, followed by 45 cycles of 94°C for 30 sec, 67°C or 68°C for 30 sec, and 72°C for 30 sec, with a final extension of 72°C for 7 min.Total RNA (2 μg) was reverse transcribed at 37°C using 3 μl pD(N) primers nvitrogenProteins (50 μg) from samples extracted from prostate tumour cell lines were separated on duplicate 8% Tris-Glycine iGels . The protein separated on one of the gels was visualised by coomassie staining and the protein on the duplicate gel was electrophoretically transferred to MFS nitrocellulose membrane . Non-specific binding was blocked by incubation for 1 h at room temperature using a Western blocking buffer containing 1% casein in maleic acid buffer (Roche) diluted using TBS with 0.1% Tween-20 (TBS-T). EphB4 antigens were detected using a 1:1000 dilution of a mouse monoclonal antibody raised to human EphB4  in blocking buffer. After a 1 h incubation at room temperature, the primary antibody was detected using a HRP-labelled anti-mouse secondary antibody (Roche) and the ECL Western Blotting System from Amersham Biosciences  following the manufacturer's recommendation. Blots were exposed to Hyperfilm™ECL™ (Amersham Biosciences) for between 5 and 30 sec. The chemiluminescence was removed from the blot by incubation in a stripping solution containing 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl pH 6.7 for 20 min at 50°C with gentle agitation. The filter was then washed with TBS-Tween 20 before blocking and re-probing with a mouse anti-α-actin monoclonal antibody  then it was stripped again and probed with a rabbit anti-calnexin antibody (Sigma) for loading comparison.Cell were grown to >50% confluence in the wells of 8 well chambered slides. Before staining, the medium was aspirated and the cells washed gently with PBS. Cells were then fixed in 4% paraformaldehyde and washed well before non-specific binding sites were blocked with 3% serum in PBS for 20 min at room temperature. Cells were then incubated overnight at 4°C with a 1:200 dilution of the EphB4-specific rabbit polyclonal antibody H-200 . After rinsing with PBS, the sections were incubated with an Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes). Immunofluorescence was visualised using a TE2000E microscope with a C1 confocal scanning head .Four consecutive 8 μm sections of formalin-fixed paraffin-embedded tissue from 15 different patients with prostate cancer were a gift from Dr Michael Brown, Medical Oncology, Royal Adelaide Hospital, Adelaide, Australia. A fifth slide, stained to visualize the histological features of the tissue, was used to identify the foci of tumour tissue within the adjacent normal prostate tissue by a trained pathologist. The paraffin was removed by incubation in Histoclear  and the section re-hydrated in ethanol before antigen retrieval by boiling in 10 mM citric acid pH 6 for 10 min. Sections were cooled, then washed in PBS before removal of endogenous peroxidase activity by incubation in 0.5% hydrogen peroxide/methanol for 30 min at room temperature. Non-specific binding sites were blocked with 3% normal goat serum in PBS for 20 min at room temperature and the Vector Laboratories  Avidin/Biotin blocking kit following the manufacturer's instructions. The sections were then incubated overnight at 4°C with a 1:200 dilution of the H-200 EphB4-specific antibody (Santa Cruz Biotechnology). After rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature followed by washing with PBS. Immunoreactivity was detected with the avidin-biotin system (Vector Laboratories) using 18.5 mM 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as a chromogen for 2 min. The sections were then counterstained using Harris haematoxylin, dehydrated, cleared using SUB-X clearing solution  and mounted using Entellan New . Sections were visualised and images recorded using a Nikon Eclipse E800 microscope with a Spot Camera 2.3.1 (Diagnostic Instruments) and Spot Advanced Version 3.5 software.EphB4 in three prostate cancer cell lines was determined using reverse transcription and real time PCR using two different real-time PCR machines. Cell lines used included the androgen-dependent line LNCaP derived from a lymph node biopsy (moderately differentiated), the androgen-independent line DU145 from a central nervous system metastasis (moderately differentiated with foci of poorly differentiated cells) and the androgen-independent line PC3 from a bone metastasis (poorly differentiated) [EphB4 gene and the house-keeping control gene PBGD were amplified in triplicate from cDNA made using three different RNA extractions for each cell line . Comparison of the combined data for each cell line showed that there was no statistical difference in the level of EphB4 expression in the three prostate cancer cell lines .The relative expression of ntiated) -24. For e Figure  and a see Figure  using a 0 Figure . For eacEphB4/PBGD and EphB4/HPRT 1 were all close to 1 indicating that there were comparable amounts of EphB4 and PBGD/HPRT 1 templates in each cell line RNA sample. There was no statistical difference between the ratios obtained using serial dilutions of a single cDNA sample for each cell line showing that the amplification is consistent regardless of the starting amount of template. These results support the use of both PBGD and HPRT 1 as normalisation controls for the EphB4 message.In these experiments the ratios of EphB4 under control of the constitutive CMV promoter was used as a positive control  that is dramatically up-regulated on many epithelial cancers, displays limited expression on normal adult tissue and would therefore appear to be a potential target for monoclonal therapies. EphB4 was identified by Robinson et al (1996) as one of several RTKs that were expressed in a xenograft model of prostate cancer [In recent years, experiments using monoclonal antibodies with limited normal-tissue reactivity have indicated that these are good candidates for development as therapeutic agents against cancer. e cancer  and untiEphB4 gene expression as real-time PCR using normalisation to two different house-keeping genes demonstrated that there was approximately 20% less EphB4 transcript expressed in the DU145 cell line than in LNCaP and PC3 (P < 0.05). However, as there was no significant difference in the activity of the gene in LNCaP and PC3, it is possible that EphB4 may also be regulated post-transcriptionally. The presence of a second band detected by western analysis with the EphB4-specific antibody may also indicate that EphB4 is regulated post-translationally and this needs to be explored in more detail.During the review of this article, Xia et al (2005) presented results showing that EphB4 is increased in 66% (41/62) of prostate tumours tested with low intensity expression in only 15% (3/20) normal prostate samples . Our wesAlthough Xia et al's study of cell lines also indicated an association between the level of EphB4 expression and aggressive growth, they did not report a direct correlation between increased EphB4 expression with higher grade of the tumour tissue samples. We examined a panel of 15 prostate cancer clinical specimens collected by transurethral resection. The prostate tumour samples we examined contained both normal prostate and tumour foci and this enabled a comparison of EphB4 immunoreactivity in normal prostate and tumour cells simultaneously from the same patient and the determination of whether the level of EphB4 also correlates with histological grade and/or stage of prostate carcinoma. Using immunohistochemical techniques, we showed that EphB4 is produced in increased amounts in human prostate cancer tissue compared with adjacent normal tissue and that this immunoreactivity was associated with the tumour epithelial cells themselves. There also appeared to be trend towards an increasing level of EphB4 protein in the tumours from the well-differentiated to the moderately and poorly differentiated cancers.EphB4 expression [A positive correlation between histological grade, stage of carcinoma and level of EphB4 protein has also been reported in breast and endometrial carcinoma -16. EphBpression . Howeverpression . One of pression .EphB4 are relevant to prostate cancer but despite substantial evidence in the literature that EphB4 has an important role in progression of many epithelial tumours, the mechanism by which these receptors contribute to tumorigenesis is still being resolved. In only a very few cases have Eph receptors been demonstrated to have transforming potential [EphA2 expression transforms MCF10A cells as judged by conversion to a fibroblastic morphology, growth in soft agar and the ability to engraft and metastasise in a nude mouse model [The results presented here suggest that elevated levels of otential -30. In pse model .EphB4 and neuT suggested that EphB4 over-expression by itself is not tumorigenic but provides convincing evidence that it favours an invasive/metastatic phenotype [EphB4 and neuT, tumour appearance was significantly accelerated relative to neuT-only animals, and in addition metastases were observed in the lung. It was not clear if this was an effect of EphB4 on metastasis or a result of accelerated tumour growth but these results clearly implicate over-expression of EphB4 in tumour growth and/or establishment of the invasive phenotype in the adult mammary tumours. While the single transgenic EphB4 animals did not develop tumours during this experiment it is possible that these studies were not taken out far enough to conclude that EphB4 over-expression is insufficient to induce transformation with a long latency.Although similar experiments over-expressing EphB4 in non-transformed cell lines have yet to be reported, a study by Munarini et al (2000) using transgenic mice expressing henotype . AlthougEphB4 expression in vitro have shown that EphB4 is involved in growth/viability, migration and invasion of prostate cancer cell lines and supports Munarini et al's study [Xia et al's (2005) recent experiments targeting EphB4 using siRNA to knockdown 's study . Further's study  and it hXenopus [A direct role for EphB4 in tumour angiogenesis has been suggested by experiments showing that A375 melanomas form smaller tumours in the presence of soluble EphB4  possiblyXenopus ,34. NoreXenopus . This maFurther investigation is needed to determine the roles of EphB4 in prostate cancer development and progression. The trend of increasing EphB4 reactivity toward higher grade disease seen in this pilot study might suggest that it is more important in the later stages of the tumour development such as metastasis and further investigation of EphB4 in the development of prostate cancer is warranted. Therapies that target EphB4 may prove to be successful in preventing the metastatic spread of the disease.The author(s) declare that they have no competing interests.YCL carried out most of the studies with the assistance of JP and ED and drafted the manuscript. JP and ED also participated in the analysis and interpretation of the data and revision of the manuscript. MR optimised the real-time PCR experiments, participated in the analysis and interpretation of the data and revision of the manuscript. MB optimised the Western analysis, participated in the analysis and interpretation of the data and revision of the manuscript. PB participated in the analysis and interpretation of the data and revision of the manuscript. SS conceived of and co-ordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Deleted-in-AZoospermia-Like (DAZL) gene has homologs required for germ cell development in many organisms. Recently, we showed that there are several common polymorphisms within the DAZL gene that are associated with age at ovarian failure/menopause and sperm count.The DAZL and examine their phenotypes in men and women. We sequenced the DAZL gene in 519 individuals; sequences spanned the entire coding region of the gene.Here we sought to identify rare mutations in DAZL. Three individuals that were heterozygous for a DAZL mutation reported having children, while two individuals that were homozygous reported no children. These mutations were found only in infertile men and women.We report the identification of four putative missense mutations in DAZL polymorphisms and deletions with fertility in humans and model organisms, we suggest that these mutations may be associated with age at menopause and/or sperm count and warrant further biochemical and genetic investigation.Given the strong data associating  DAZ (Deleted-in-AZoospermia) gene, which are associated with azoospermia (no sperm in the ejaculate) and oligozoospermia (< 20 million sperm/mL of ejaculate) [Though infertility affects 10–15% of couples, only a small number of genes have been shown to be associated with infertility in women and men ,3. In adaculate) -8.DAZL (DAZ-Like) gene, an autosomal homolog of the Y chromosome DAZ gene [DAZL expression is specific to germ cells, and mutations in DAZL homologs are limited to defects in development of the germ cell lineage [DAZL could impact measures of germ cell numbers in men and women, namely sperm count and age at menopause, respectively.As many features of germ cell development are similar in both sexes, we expect that some genes may function similarly in men and women. In humans and other mammals, male and female germ cells initially develop along the same path, but take different courses after the mitotic expansion of premeiotic germ cells . OocytesDAZ gene ,11. In a lineage . We thusDAZL gene in three study populations [DAZL [DAZL, as in other organisms, may have a function in human germ cell development [In a previous study, we directly sequenced the human ulations . The firulations . The thins [DAZL . A compans [DAZL . Our worelopment . NotablyStudy populations and DNA samples are as previously described . BrieflyDAZL was sequenced, including all exons and flanking regions. Primers, primer concentrations, and PCR conditions were as indicated [DNA extraction and genotype were performed as described . The entndicated . PCR prindicated . SequencDAZL gene, listed in Table DAZL gene; all were juxtaposed to, or within, the RNA-binding domain.Single nucleotide polymorphisms (SNPs) are defined as naturally-occurring variants with a frequency greater than one percent in the human population; in contrast, mutations occur with frequencies less than one percent. Here, we identified four putative missense mutations in the 2) group predisposes this residue to being on the surface of the protein exposed to water. Histidine is also a bulky amino acid that can be either neutrally or positively charged, depending on its environment. Overall, since both amino acids are large and generally localized to the surface of the protein, this amino acid change is most likely to affect the structure of the protein surface. In this study, we identified one woman who was heterozygous for this mutation, with onset of menopause at age 45, and no family history of early menopause. She was fertile, having had three children prior to menopause.This mutation changes a highly conserved proline to a histidine in the N-terminus of the protein, just upstream of the highly-conserved RNA Recognition Motif (RRM). Although proline is usually considered a hydrophobic amino acid whose unique cyclic structure causes it to influence protein architecture, its secondary amino . It is five amino acids upstream of the RNP2 sequence which is one of the most highly conserved parts of the DAZL protein. Isoleucine and alanine are both classified as hydrophobic amino acids; however, alanine, with its shorter side chain, is not as hydrophobic as isoleucine. The structure of the homologous RNA binding domain in another protein has been deduced, and in that structure, the amino acid corresponding to the isoleucine forms a hydrogen bond with another amino acid in the structure . The shoArg115 is near the carboxy-terminal end of the RRM in a region that interacts with several other proteins . This muDAZL gene for sequence variations that are associated with male and female fertility produced not only a number of interesting common SNPs or variants [Our screen of the variants , but alsvariants .Dazl knockout allele are fertile (though it is currently debated whether they may be subfertile) and homozygous mutant mice are infertile in both sexes [Interestingly, we observed that possessing these putative missense mutations, especially a heterozygous missense mutation, was compatible with producing offspring. Each patient that was heterozygous for a missense mutation was able to produce at least one child. In contrast, the patient that was homozygous for the Arg115Gly mutation experienced 46,XX spontaneous premature ovarian failure at the early age of 34 years and did not bear any children; similarly, the male patient that was homozygous for the Asn10Cys mutation produced no sperm. This is consistent with data from the mouse, where mice heterozygous for a th sexes .in vitro, in the future [Recently, a number of potential RNA targets for DAZL have been identified, making it possible to test the effects of mutations on RNA-binding e future -39. In ae future ,40,41.DAZL gene in both men and women. Two individuals possessed homozygous mutations. Future work on how these putative mutations affect RNA and protein partner binding could provide key insights into the requirement of DAZL in human germ cell development, especially regarding key residues required for DAZL protein function. In addition, further studies may shed a light on the role of DAZL in human infertility.Intriguing mutations were observed in the The author(s) declare that they have no competing interests.JT and MR carried out the molecular genetic studies, sequence alignment and drafted the manuscript. LMN, PJT, DWC, and MIC participated in the design of the study, especially clinical aspects. JSW performed statistical analysis. RARP conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors edited, read and approved the final manuscript.


Schistosoma, depends upon the release of parasite eggs from the human host. However, approximately 50% of eggs produced by schistosomes fail to reach the external environment, but instead become trapped in host tissues where pathological changes caused by the immune responses to secreted egg antigens precipitate disease. Despite the central importance of egg production in transmission and disease, relatively little is understood of the molecular processes underlying the development of this key life stage in schistosomes. Here, we describe a novel parasite-encoded TGF-β superfamily member, Schistosoma mansoni Inhibin/Activin (SmInAct), which is key to this process. In situ hybridization localizes SmInAct expression to the reproductive tissues of the adult female, and real-time RT-PCR analyses indicate that SmInAct is abundantly expressed in ovipositing females and the eggs they produce. Based on real-time RT-PCR analyses, SmInAct transcription continues, albeit at a reduced level, both in adult worms isolated from single-sex infections, where reproduction is absent, and in parasites from IL-7R−/− mice, in which viable egg production is severely compromised. Nevertheless, Western analyses demonstrate that SmInAct protein is undetectable in parasites from single-sex infections and from infections of IL-7R−/− mice, suggesting that SmInAct expression is tightly linked to the reproductive potential of the worms. A crucial role for SmInAct in successful embryogenesis is indicated by the finding that RNA interference–mediated knockdown of SmInAct expression in eggs aborts their development. Our results demonstrate that TGF-β signaling plays a major role in the embryogenesis of a metazoan parasite, and have implications for the development of new strategies for the treatment and prevention of an important and neglected human disease.Over 200 million people have, and another 600 million are at risk of contracting, schistosomiasis, one of the major neglected tropical diseases. Transmission of this infection, which is caused by helminth parasites of the genus  Schistosomes are parasitic worms that infect hundreds of millions of people in developing countries. They cause disease by virtue of the fact that the eggs that they produce, which are intended for release from the host in order to allow transmission of infection, can become trapped in target organs such as the liver, where they induce damaging inflammation. Egg production by female schistosomes is critically dependent on the presence of male parasites, without which females never fully develop, and (counterintuitively) on the contribution of signals from the host's immune system. Very little is understood about the molecular basis of these interactions. Here, we describe a newly discovered schistosome gene, which is expressed in the reproductive tract of the female parasite and in parasite eggs. The protein encoded by this gene is made only when females are paired with males in an immunologically competent setting. Using recently developed tools that allow gene function to be inhibited in schistosomes, we show that the product of this gene plays a crucial role in egg development. Examining how the expression of this gene is controlled has the potential to provide insight into the molecular nature of the interactions between male and female parasites and their hosts. Moreover, the pivotal role of this gene in the egg makes it a potential target for blocking transmission and disease development. Amongst the Bilateria, transforming growth factor–β (TGF-β) signaling is recognized as playing an essential role in embryogenesis in deuterostomes and in arthropod protostomes, but its role in lophotrochozoan protostomes is unclear . SchistoSchistosoma mansoni receptor kinase-1 [SmRK1], S. mansoni transforming growth factor–β type I receptor [SmTβ RI]), one type II receptor [S. mansoni, with nearly all components localized to either the surface of the worm or reproductive tissues of the female [S. mansoni has been examined with the identification of 163,000 expressed sequence tags (ESTs) [Components of TGF-β signaling have been molecularly characterized in metazoans throughout the animal kingdom. Activation of this pathway begins at the cell surface when a dimeric ligand binds a complex consisting of types I and II receptor serine/threonine kinases . Upon lireceptor ,7  , a ligans (ESTs) ,13,14, aS. mansoni live within the mesenteric vasculature, where each female produces approximately 300 eggs each day. Transmission of schistosomiasis depends upon the release of parasite eggs from the human host. Development of an immature egg into a mature egg containing a miracidium, the stage of the parasite that invades the intermediate fresh water snail host, occurs outside of the female worm, and takes approximately 5 d. Many of the eggs produced by schistosomes fail to reach the external environment, but instead become trapped in host tissues, where pathological changes caused by the immune responses to secreted egg antigens cause disease [Sexually mature  disease . Despite disease ,16–18, eS. mansoni TGF-β homolog, S. mansoni Inhibin/Activin (SmInAct). Although we found SmInAct to be expressed in adult male and female parasites, and in eggs, the localization of SmInAct expression to the reproductive organs of female parasites focused our attention on the role of this gene in egg production. A role for SmInAct in reproduction was supported by analyses of female parasites recovered from infertile infections, in which we found that SmInAct protein was undetectable. Confirmation of the importance of this TGF-β superfamily member in the reproductive process was obtained from RNA interference (RNAi) studies, in which targeted knockdown of SmInAct in female worms or directly in the eggs that they produce resulted in a marked cessation of embryogenesis.In this study, we describe the cloning and characterization of a SmInAct was identified through a tblastn search of the Wellcome Trust's Sanger Institute's S. mansoni genome sequence using the C-terminal region of the Drosophila melanogaster dActivin sequence. We were unable to identify SmInAct in EST databases regardless of whether we searched using the coding or 3′–untranslated region (UTR) sequences. The 5′ and 3′ ends of SmInAct were amplified via rapid amplification of cDNA ends (RACE) using primers designed from within putative coding sequence and adult S. mansoni cDNA as template. The 1.3-kb, full-length SmInAct transcript contains 10 base pairs (bp) of 5′UTR, 808 bp of 3′UTR, and a poly-A tail. The deduced amino acid sequence of SmInAct is 161 residues long and contains many of the molecular hallmarks for a TGF-β, including a putative basic proteolytic cleavage site located at position 32 as RQRR where the bioactive, C-terminal domain (126 amino acids) is enzymatically separated from the N-terminal pro-domain. Nine invariant cysteine moieties, and invariant proline and glycine residues  and the parasitic nematodes Brugia malayi (Bm-TGH-2) and Strongyloides stercoralis (Ss-TGH-1).residues A essenti TGF-β 1 A. Phylog TGF-β 1 B, and fuSmInAct at the transcript level, real-time reverse transcriptase–polymerase chain reaction (RT-PCR) was performed on cDNA from eggs, adult male parasites, and adult female parasites from mixed-sex infections. As seen in SmInAct is expressed in all stages tested at relatively similar levels. Western analyses using polyclonal antibodies against recombinant SmInAct were used to determine the protein expression profile of SmInAct. The anti-SmInAct serum recognized a 28-kDa protein in egg antigen extracts and a doublet (32 kDa and 28 kDa) in adult male and female extracts  mice carrying mixed-sex infections, which produce a significant number of dead eggs [SmInAct mRNA levels were significantly decreased, but not absent, in females from these infections  or an irrelevant control dsRNA (luciferase) for 1 wk in vitro, followed by RNA extraction and real-time RT-PCR analyses. SmInAct dsRNA–treated worms showed a consistent and significant decrease in SmInAct expression of >40% when compared to SmInAct expression in worms soaked in the irrelevant control dsRNA . To specifically address the role of SmInAct in egg development, we treated eggs directly with SmInAct dsRNA. Approximately 20% of eggs laid by adult parasites during the first 2 d of in vitro culture will develop over the ensuing 5 d to contain miracidia [ex vivo were collected and soaked in dsRNA (1 μg/ml) corresponding to SmInAct or an irrelevant dsRNA for 5 d, and their development was scored. Relative to eggs soaked in an irrelevant dsRNA, where ∼20% of the eggs developed through stage 6, eggs treated with SmInAct dsRNA aborted development at stage 2 , a cathepsin B detectable in eggs –like homolog in D. melanogaster, acts as a morphogen by determining cell fate along the dorsal–ventral axis in a gradient-dependent manner [D. melanogaster, a type I receptor, baboon, stimulates cellular proliferation and is essential for normal embryonic development [C. elegans are important for patterning or growth of the embryo [tig-2 and Y46E12BL.1), and, intriguingly, serial analysis of gene expression (SAGE) tags for both homologs have been found in the C. elegans embryo [C. elegans TGF-β homologs that are resistant to RNAi affects, tig-2 and Y46E12BL.1 have no phenotype in genome-wide RNAi screens [Understanding of the developmental processes regulated by TGF-β in invertebrates is based largely on data from the model organisms t manner . Also inelopment . Presumae embryo –33; howes embryo . Like th screens ,36; therS. mansoni, a member of the Platyhelminthes, the earliest branch of the Bilateria [S. mansoni, there appears to be at least three type I receptors and two type II receptors present in the genome based on a preliminary blast search for homologs. It will be important to delineate which of the S. mansoni type I and type II TGF-β receptors are involved in SmInAct signaling and to identify the Smads important for transmitting the signal induced by this growth factor. Furthermore, identifying the genes regulated by SmInAct signaling will provide information regarding the precise function that this growth factor serves in egg maturation, as well as the functions the pathway may serve in other life stages of the parasite, including the adult male.The identification of SmInAct, a TGF-β superfamily member, as a key component of egg development in ilateria , undersc−/− mice, despite the fact that these parasites contained SmInAct transcripts . This strongly indicates that SmInAct is both transcriptionally and post-transcriptionally regulated by worms of the opposite sex as well as by signals from the host. It is well established that parasites recovered from hosts lacking CD4+ T cells are developmentally stunted and produce significantly fewer fertile eggs than those recovered from mixed-sex infections of immunocompetent hosts. Translation of SmInAct mRNA is the first identified molecular process downstream of the effect of the host immune system on schistosome development [S. mansoni. We are unaware of a link between the site of expression of SmInAct in the male schistosome and reproductive events, and further work is required to elucidate the function of SmInAct in male worms.SmInAct protein was not detectable in infertile females recovered from single-sex infections or from IL-7Relopment –24, and + T cells may be considered stressed due to the lack of signals received from the opposite sex and immunocompetent host, thereby restricting the translation of non-essential transcripts. SmInAct protein expression may be considered expendable considering the role it plays in embryogenesis rather than in crucial cellular functions linked to the survival of the adult worm. A more thorough investigation of the S. mansoni homologs of translation factors involved in the stress response and of the regulation of other transcripts and protein expression will be required to evaluate this possibility.In other settings, the uncoupling of transcription and translation is linked to the activation of the integrated stress response –41. ThisSmInAct has two exact repeats of “UUUCTAUUUA” that contain the consensus “AUUUA” ARE (underlined). Furthermore, the 3′UTR of SmInAct is U-rich (43% uridines). It will be interesting to determine whether these repeats, or other regions of the long 3′UTR, play a role in the post-transcriptional regulation of SmInAct expression.Post-transcription regulation of eukaryotic transcripts is controlled in part by the 3′UTR . This reDrosophila BMP homologs DPP and 60A are able to induce bone development when injected into the skin of rats [Drosophila DPP mutants [It is of interest when considering the relationship of schistosomes with their mammalian hosts to note that in other systems, TGF-β superfamily members have been shown to function across phylum boundaries ,46. For  of rats , and mam mutants , and SmI mutants , a solutS. mansoni was used in all experiments. Adult schistosomes were recovered by hepatic-portal perfusion from C57BL/6 female mice or B6 IL-7R−/−  that had each been percutaneously exposed to ∼60 cercariae 8 wk earlier. Adult parasites and eggs laid were maintained in vitro in M199 , 10% fetal calf serum, 1% Antibiotic/Antimycotic (Gibco), and 1% HEPES in a 37 °C/5% CO2 atmosphere as previously described [The Puerto Rican/NMRI strain of escribed ,28.Drosophila activin homolog (dActivin) (amino acids 565–669) was used to search the Wellcome Trust's Sanger Institute's S. mansoni genome assembly using the tblastn algorithm. A contig (0020320) with significant similarity to dActivin was identified. Full-length cDNA corresponding to SmInAct was isolated using total RNA (1 μg) from adult parasites and the SuperScript III GeneRacer 5′ and 3′ RACE kit  as per manufacturer's instructions. Gene-specific primers were designed for isolation of the 5′-end (5′-GGTTCAAAACTTTTCGGGTGTA-3′) and 3′-end (5′-AATCTTGTTGTCATCCAACTCAA-3′) of SmInAct and used in RT-PCR with GeneRacer 5′ and 3′ primers according to manufacturer's suggestions. Resulting amplicons were cloned into the TOPO cloning vector (Invitrogen) and sequenced. To verify the full-length sequence of SmInAct, primers designed from the 5′ and 3′ ends of the transcript were used in RT-PCR, and the resulting fragment was cloned and sequenced.The C-terminal, translated region of the http://www.ncbi.nlm.nih.gov). An unrooted phylogram was drawn using amino acids within the conserved C-terminal domain of SmInAct, and known TGF-β superfamily members and distances were drawn using the Dayhoff Pam matrix and neighbor-joining algorithm in the PHYLIP software package developed by J. Felsenstein, University of Washington, Seattle, Washington, United States (http://evolution.genetics.washington.edu/phylip.html). Percentages at branch points are based on 1,000 bootstrap runs.Sequence similarities between the deduced amino acid sequence of SmInAct and other members of the TGF-β superfamily were determined through multiple sequence alignments using the ClustalW algorithm, as well as the Align 2 sequences (bl2seq) program at the National Center for Biotechnology Information (http://www.qiagen.com), and contaminating genomic DNA was removed by DNase treatment using the Turbo DNA-free endonuclease . First-strand cDNA was synthesized using 500 ng of RNA, SuperScript II reverse transcriptase (Invitrogen), and oligo dT as a primer. RT-minus controls were performed to confirm the absence of genomic DNA (unpublished data).Total RNA was extracted from parasites using Qiagen's RNeasy Mini kit (http://www.appliedbiosystems.com). Total reaction volume was 10 μl with 300 nM of each primer, 5 μl of SYBR green PCR Master Mix, and 0.5 μl of cDNA as template (or water as a negative control). SmInAct primers were: forward 5′-AATCTTGTTGTCATCCAACTCAA-3′ and reverse 5′-AACTACAAGCACATCCTAAAACAA-3′. α-Tubulin primers were: forward 5′-CCAGCAAATCAGATGGTGAA-3′ and reverse 5′-TTGACATCCTTGGGGACAAC-3′. PCR efficiency (E) was determined for both primer sets by plotting cycle thresholds from a 10-fold serial dilution of cDNA and inputting the slope in the equation E = 10(−1/slope). For expression analyses, quantification of SmInAct transcript relative to α-tubulin was calculated using the equation: ratio = (ESmα-tubulin)CT/(SmInActE)CT where ESmα-tubulin is the PCR efficiency of the reference gene, SmInActE is the PCR efficiency of target gene, and CT is the cycle threshold. For analysis of RNAi-induced knockdown, quantification of SmInAct transcript relative to paramyosin (paramyosin primers were: forward 5′-CGTGAAGGTCGTCGTATGGT-3′ and reverse 5′-GACGTTCAAATTTACGTGCTTG-3′) was calculated using the 2−ΔΔCt method. Dissociation curves were generated for each real-time RT-PCR to verify the amplification of only one product.SmInAct transcript levels in egg and adult stages were quantified relative to α-tubulin using Applied Biosystems' 7500 real-time PCR system and SYBR green PCR Master Mix  and sequenced to verify the absence of any mutations. Expression of recombinant SmInAct was induced in Escherichia coli BL21(DE3) by addition of 1 mM IPTG when cultures reached an OD600 of 0.5 at 37 °C, followed by 3 hours of shaking at room temperature. Recombinant SmInAct was expressed in bacteria as insoluble inclusion bodies. Exhaustive attempts to refold the protein using gluathione and reduced glutathione proved unsuccessful. We therefore purified the protein via nickel column chromatography under denaturing conditions (6 M urea) as per the manufacturer's protocol (Novagen). Antiserum was generated by Cocalico Biologicals  through subcutaneous inoculation of a rabbit with 100 μg of purified protein in complete Freund's adjuvant, followed by three boosts of 50 μg in incomplete Freund's adjuvant on days 14, 21, and 49, followed by exsanguinations on day 64.Eco RI (forward) and Xho I (reverse) tagged primers were designed to amplify the C-terminal bioactive region of SmInAct (forward 5′-GGAATTCTCATTAACTAAAGGAGATGA-3 and reverse 5′-CCGCTCGAGTTAACTACAAGCACATCCTA-3′). The amplified product was cloned into the expression vector pET28a+  were separated by SDS-PAGE, electroblotted, and probed with anti-SmInAct antiserum , pre-immune serum , or a monoclonal antibody (4B1) against paramyosin. Affinity purified HRP-conjugated goat anti-rabbit IgG  was used to detect bound rabbit antibodies, while an affinity purified HRP-conjugated horse anti-mouse IgG  was used to detect the anti-paramyosin monoclonal antibody. The secondary antibodies were detected using ECL reagents as per manufacturer's instructions .For detection of SmInAct protein, 10 μg of protein extracted from eggs, adult males, and adult females via Dounce homogenizing in lysis buffer  supplemented with a protease inhibitor cocktail  as per manufacturer's instructions with T7-tagged amplicons as template . The hybridized DIG-probes were detected using an alkaline-phosphatase conjugated anti-DIG antibody (Roche), and visualized using NBT (337.5 μg/ml) and BCIP (175 μg/ml) in 0.1M Tris-HCl, 0.1M NaCl, 0.05 MgCl2. Worm sections were photographed using a Leica DMIRB microscope and DC500 camera .Localization of SmInAct in 5-μm sections of adult escribed . DIG-labSmInAct-dsRNA template encompassing the active ligand domain . Luciferase and SmCB1 dsRNAs (negative controls) were generated as described [dsRNA was synthesized using the T7 Megascript kit (Ambion) as per manufacturer's instructions. T7-tagged primers were used to generate a 381-bp escribed . For dsRt-test was used for statistical analyses of dsRNA-induced knockdown of SmInAct expression, change in expression of SmInAct in single-sex and IL-7R−/− mice, and egg developmental phenotypes (control versus SmInAct dsRNA). Chi-square analyses were used to test the statistical significance of the egg developmental phenotype. The Yates correction was applied because we specified only two categories: undeveloped and developed Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) under accession number DQ863513. Other GenBank accession numbers of genes and sequences used in this study include: B. malayi TGH-1 (AAB71839); B. malayi TGH-2 (AAD19903); C. elegans DAF-7 (NP_497265); C. elegans DBL-1 (NP_504709); Danio rerio Activinβ A isoform 2 (AAX68505); D. melanogaster Activin (NP_651942); D. melanogaster dActivin (AF454392); D. melanogaster decapentaplegic (NP_477311); Homo sapiens Activinβ E (NP_113667); H. sapiens BMP-2 (NP_001191); H. sapiens BMP-3 (NP_001192); H. sapiens BMP-4 (NP_031580); H. sapiens BMP-5 (NP_066551); H. sapiens BMP-6 (NP_001709); H. sapiens BMP-7 (NP_001710); H. sapiens BMP-8 (NP_861525); H. sapiens GDF-5 (NP_000548); H. sapiens GDF-6 (NP_001001557); H. sapiens GDF-7 (NP_878248); H. sapiens GDF-10 (NP_004953); H. sapiens Inhibinβ A precursor (NP_002183); H. sapiens Inhibinβ B (NP_002184); H. sapiens Inhibinβ C (NP_005529); H. sapiens TGF-β 1 (NP_000651); H. sapiens TGF-β 2 (NP_003229); H. sapiens TGF-β 3 (NP_003230); Mus musculus BMP-2 (NP_031579); M. musculus BMP-3 (NP_775580); M. musculus BMP-4 (NP_031580); M. musculus GDF-10 (NP_665684); M. musculus Inhibinβ A (NP_032406); M. musculus Inhibinβ B (NP_032407); M. musculus TGF-β 1 (NP_035707); M. musculus TGF-β 2 (NP_033393); M. musculus TGF-β 3 (NP_033394); S. mansoni α-tubulin (M80214); S. mansoni paramyosin (M35499); and Strongyloides stercoralis TGH-1 (AAV84743).Sequence data reported in this manuscript are available from GenBank (