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In this paper, the 4E assessment (Energetic, Exergetic, Exergoeconomic and Exergoenvironmental) of a low-temperature ORC activated by two different alternatives is presented. The first alternative (S1) contemplates the activation of the ORC through the recovery of waste heat from a flash-binary geothermal power plant. The second alternative (S2) contemplates the activation of the ORC using direct heat from a geothermal well. For both alternatives, the energetic and exergetic models were established. At the same time, the economic and environmental impact models were developed. Finally, based on the combination of the exergy concepts and the economic and ecological indicators, the exergoeconomic and exergoenvironmental performances of the ORC were obtained. The results show higher economic, exergoeconomic and exergoenvironmental profitability for S1. Besides, for the alternative S1, the ORC cycle has an acceptable economic profitability for a net power of 358.4 kW at a temperature of 110 °C, while for S2, this profitability starts being attractive for a power 2.65 times greater than S1 and with a temperature higher than 135 °C. In conclusion, the above represents an area of opportunity and a considerable advantage for the implementation of the ORC in the recovery of waste heat from flash-binary geothermal power plants.

Background: The dismal clinical outcome of relapsed/refractory (R/R) T cell acute lymphoblastic leukemia (T-ALL) highlights the need for innovative targeted therapies. Although chimeric antigen receptor (CAR)-engineered T cells have revolutionized the treatment of B cell malignancies, their clinical implementation in T-ALL is in its infancy. CD1a represents a safe target for cortical T-ALL (coT-ALL) patients, and fratricide-resistant CD1a-directed CAR T cells have been preclinically validated as an immunotherapeutic strategy for R/R coT-ALL. Nonetheless, T-ALL relapses are commonly very aggressive and hyperleukocytic, posing a challenge to recover sufficient non-leukemic effector T cells from leukapheresis in R/R T-ALL patients., Methods: We carried out a comprehensive study using robust in vitro and in vivo assays comparing the efficacy of engineered T cells either expressing a second-generation CD1a-CAR or secreting CD1a x CD3 T cell-engaging Antibodies (CD1a-STAb)., Results: We show that CD1a-T cell engagers bind to cell surface expressed CD1a and CD3 and induce specific T cell activation. Recruitment of bystander T cells endows CD1a-STAbs with an enhanced in vitro cytotoxicity than CD1a-CAR T cells at lower effector:target ratios. CD1a-STAb T cells are as effective as CD1a-CAR T cells in cutting-edge in vivo T-ALL patient-derived xenograft models., Conclusions: Our data suggest that CD1a-STAb T cells could be an alternative to CD1a-CAR T cells in coT-ALL patients with aggressive and hyperleukocytic relapses with limited numbers of non-leukemic effector T cells., Competing Interests: Competing interests: LA-V is cofounder of Leadartis, a spin-off focused on unrelated interest. PM is cofounder of OneChain Immunotherapeutics, a spin-off company from the Josep Carreras Leukemia Research Institute. VMC is current employee of One Chain Immunotherapeutics., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CD19-directed chimeric antigen receptor (CAR) T cells have yielded impressive response rates in refractory/relapse B cell acute lymphoblastic leukemia (B-ALL); however, most patients ultimately relapse due to poor CAR T cell persistence or resistance of either CD19 + or CD19 - B-ALL clones. CD22 is a pan-B marker whose expression is maintained in both CD19 + and CD19 - relapses. CD22-CAR T cells have been clinically used in B-ALL patients, although relapse also occurs. T cells engineered with a tandem CAR (Tan-CAR) containing in a single construct both CD19 and CD22 scFvs may be advantageous in achieving higher remission rates and/or preventing antigen loss. We have generated and functionally validated using cutting-edge assays a 4-1BB-based CD22/CD19 Tan-CAR using in-house-developed novel CD19 and CD22 scFvs. Tan-CAR-expressing T cells showed similar in vitro expansion to CD19-CAR T cells with no increase in tonic signaling. CRISPR-Cas9-edited B-ALL cells confirmed the bispecificity of the Tan-CAR. Tan-CAR was as efficient as CD19-CAR in vitro and in vivo using B-ALL cell lines, patient samples, and patient-derived xenografts (PDXs). Strikingly, the robust antileukemic activity of the Tan-CAR was slightly more effective in controlling the disease in long-term follow-up PDX models. This Tan-CAR construct warrants a clinical appraisal to test whether simultaneous targeting of CD19 and CD22 enhances leukemia eradication and reduces/delays relapse rates and antigen loss., Competing Interests: Declaration of interests S.R.Z., T.V.-H., F.G.-A., D.S.-M., and P.M. are inventors of a CD22scFv pending European patent (file no. 20382175.6). P.M. is the co-founder of OneChain Immunotherapeutics (OCI). V.M.D. is employed by OCI., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Endothelin-converting enzyme-1 (ECE1) activates the endothelin-1 peptide, which upregulates pathways that are related to diverse hallmarks of cancer. ECE1 is expressed as four isoforms differing in their N-terminal domains. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE1c, enhancing its stability and promoting invasiveness of colorectal cancer cells. However, the specific residues in ECE1c that are phosphorylated by CK2 and how this phosphorylation promotes invasiveness was unknown. Here we demonstrate that Ser-18 and Ser-20 are the bona fide residues phosphorylated by CK2 in ECE1c. Thus, biphospho-mimetic ECE1c DD and biphospho-resistant ECE1c AA mutants were constructed and stably expressed in different colorectal cancer cells through lentiviral transduction. Biphospho-mimetic ECE1c DD displayed the highest stability in cells, even in the presence of the specific CK2 inhibitor silmitasertib. Concordantly, ECE1c DD -expressing cells showed enhanced hallmarks of cancer, such as proliferation, migration, invasiveness, and self-renewal capacities. Conversely, cells expressing the less-stable biphospho-resistant ECE1c AA showed a reduction in these features, but also displayed an important sensitization to 5-fluorouracil, an antineoplastic agent traditionally used as therapy in colorectal cancer patients. Altogether, these findings suggest that phosphorylation of ECE1c at Ser-18 and Ser-20 by CK2 promotes aggressiveness in colorectal cancer cells. Therefore, phospho-ECE1c may constitute a novel biomarker of poor prognosis and CK2 inhibition may be envisioned as a potential therapy for colorectal cancer patients., (Copyright © 2020 Pérez-Moreno, Quezada-Meza, Chavez-Almarza, Niechi, Silva-Pavez, Trigo-Hidalgo, Aguayo, Jara, Cáceres-Verschae, Varas-Godoy, Díaz, García de Herreros, Burzio and Tapia.)

Cell migration is a multifactorial/multistep process that requires the concerted action of growth and transcriptional factors, motor proteins, extracellular matrix remodeling and proteases. In this review, we focus on the role of transcription factors modulating Epithelial-to-Mesenchymal Transition (EMT-TFs), a fundamental process supporting both physiological and pathological cell migration. These EMT-TFs (Snail1/2, Twist1/2 and Zeb1/2) are labile proteins which should be stabilized to initiate EMT and provide full migratory and invasive properties. We present here a family of enzymes, the deubiquitinases (DUBs) which have a crucial role in counteracting polyubiquitination and proteasomal degradation of EMT-TFs after their induction by TGFβ, inflammatory cytokines and hypoxia. We also describe the DUBs promoting the stabilization of Smads, TGFβ receptors and other key proteins involved in transduction pathways controlling EMT., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Snail1 transcriptional factor plays a key role in the control of epithelial to mesenchymal transition and fibroblast activation. As a consequence, Snail1 expression and function is regulated at multiple levels from gene transcription to protein modifications, affecting its interaction with specific cofactors. In this review, we describe the different elements that control Snail1 expression and its activity both as transcriptional repressor or activator., Competing Interests: The authors declare no conflict of interest.

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFβ during EMT and was required for TGFβ-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFβ-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg., (©2018 American Association for Cancer Research.)

Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein (LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation., Database: The GEO accession number for the data referred to this paper is GSE35600., (© 2016 Federation of European Biochemical Societies.)

F-box proteins are the key recognition subunit of multimeric E3 ubiquitin ligase complexes that participate in the proteasome degradation of specific substrates. In the last years, a discrete number of F-box proteins have been shown to regulate the epithelial-to-mesenchymal transition (EMT), a process defined by a rapid change of cell phenotype, the loss of epithelial characteristics and the acquisition of a more invasive phenotype. Specific EMT transcription factors (EMT-TFs), such as Snail, Slug, Twist and Zeb, control EMT induction both during development and in cancer. These EMT-TFs are short-lived proteins that are targeted to the proteasome system by specific F-box proteins, keeping them at low levels. F-box proteins also indirectly regulate the EMT process by controlling EMT inducers, such as Notch, c-Myc or mTOR. Here we summarize the role that these F-box proteins (Fbxw1, Fbxw7, Fbxl14, Fbxl5, Fbxo11 and Fbxo45) play in controlling EMT during development and cancer progression, a process dependent on post-translational modifications that govern their interaction with target proteins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Notch activation in aortic endothelial cells (ECs) takes place at embryonic stages during cardiac valve formation and induces endothelial-to-mesenchymal transition (EndMT). Using aortic ECs, we show here that active Notch expression promotes EndMT, resulting in downregulation of vascular endothelial cadherin (VE-cadherin) and upregulation of mesenchymal genes such as those for fibronectin and Snail1/2. In these cells, transforming growth factor β1 exacerbates Notch effects by increasing Snail1 and fibronectin activation. When Notch-downstream pathways were analyzed, we detected an increase in glycogen synthase kinase 3β (GSK-3β) phosphorylation and inactivation that facilitates Snail1 nuclear retention and protein stabilization. However, the total activity of Akt was downregulated. The discrepancy between Akt activity and GSK-3β phosphorylation is explained by a Notch-induced switch in the Akt isoforms, whereby Akt1, the predominant isoform expressed in ECs, is decreased and Akt2 transcription is upregulated. Mechanistically, Akt2 induction requires the stimulation of the β-catenin/TCF4 transcriptional complex, which activates the Akt2 promoter. Active, phosphorylated Akt2 translocates to the nucleus in Notch-expressing cells, resulting in GSK-3β inactivation in this compartment. Akt2, but not Akt1, colocalizes in the nucleus with lamin B in the nuclear envelope. In addition to promoting GSK-3β inactivation, Notch downregulates Forkhead box O1 (FoxO1), another Akt2 nuclear substrate. Moreover, Notch protects ECs from oxidative stress-induced apoptosis through an Akt2- and Snail1-dependent mechanism., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objectives: The aim of the study was to evaluate indications, results, and clinical and neurological evolution in children who have undergone liver transplantation for classical maple syrup urine disease (MSUD)., Methods: Descriptive study of liver transplantation for MSUD between 1991 and 2012. Eight patients were transplanted., Results: Indications for transplant were poor metabolic control expressed as significant psychomotor disabilities (4 had psychomotor delays, 5 had spasticity, and 5 had epilepsy) and poor quality of life (mean number of acute metabolic decompensations and mean number of total hospitalizations before transplantation 5 and 12, respectively). Four required nasogastric tube, with a maximum 4 g/day protein-restricted diet in all of them. Seven sustained significant alterations in brain magnetic resonance imaging. Mean leucine and alloisoleucine levels were 608 (standard deviation [SD] 516) and 218 μmol/L (SD 216), respectively. All of the patients received transplants with deceased-donor livers, with ages between 1.5 and 2.5 years (mean 1.78 years). Mean posttransplantation follow-up period was 12.2 years (range 5-21 years). Final patient and graft survival was 87.5% and 75%, respectively. Following transplantation, none required hospitalization in the last 3 years nor did any have new acute metabolic decompensations following a normal diet. Five followed normal schooling, 2 had motor disabilities, and 2 had convulsive crises. Brain magnetic resonance imaging was taken in 4 patients, showing neuroimage improvement in 3 of them. Mean leucine levels were <350 μmol/L from the immediate posttransplantation period (mean 225 μmol/L, SD 78), with a maximum alloisoleucine level of 20 μmol/L., Conclusions: Liver transplantation is an effective treatment for classical MSUD that arrests brain damage, although it does not reverse the process.

The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases β-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1.

The HoxB cluster expression is activated by retinoic acid and transcribed in a collinear manner. The DNA-binding Pknox1-Pbx1 complex modulates Hox protein activity. Here, NT2-D1 teratocarcinoma cells -a model of Hox gene expression- were used to show that upon retinoic acid induction, Pknox1 co-localizes with polymeric nuclear actin. We have found that globular actin aggregates, polymeric actin, the elongating RNA polymerase II and THOC match euchromatic regions corresponding to nuclear speckles. Moreover, RNA polymerase II, N-WASP, and transcription/splicing factors p54(nrb) and PSF were validated as Pknox1 interactors by tandem affinity purification. PSF pulled down with THOC and nuclear actin, both of which co-localize in nuclear speckles. Although latrunculin A slightly decreases the general level of HoxB gene expression, inhibition of nuclear actin polymerization by cytochalasin D blocks the expression of HoxB transcripts in a collinear manner. Thus, our results support the hypothesis that nuclear actin polymerization is involved in the activation of HoxB gene expression by means of nuclear speckles.

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.

Methylation of lysine 4 (K4) within histone H3 has been linked to active transcription and is removed by LSD1 and the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here, we describe the deamination catalyzed by Lysyl oxidase-like 2 protein (LOXL2) as an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, LOXL2 activity is linked with the transcriptional control of CDH1 gene by regulating H3K4me3 deamination. These results reveal another H3 modification and provide a different mechanism for H3K4 modification., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Fibronectins are cell-secreted glycoproteins that modulate cell attachment, spreading, migration, morphology, differentiation and oncogenic transformation. Fibronectin expression is activated during EMT (epithelial-mesenchymal transition) and is a hallmark of mesenchymal cells. It is shown in the present study that a transcription factor previously unrelated with EMT, TFCP2c/LSF/LBP-1c, was translocated to the nucleus and bound to the fibronectin promoter upon EMT induction by Snail1. Consequently, the interference of TFCP2c/LSF/LBP-1c's activity prevented fibronectin expression. Moreover, TFCP2c/LSF/LBP-1c was detected in nuclei of embryonic dermal mesenchymal cells adjacent to the hair bud, a cell population that expresses endogenous nuclear Snail1 and fibronectin. Therefore we indicate a new molecular role for TFCP2c/LSF/LBP-1c in fibronectin expression.

The transcription factor SNAIL1 is a master regulator of epithelial to mesenchymal transition. SNAIL1 is a very unstable protein, and its levels are regulated by the E3 ubiquitin ligase beta-TrCP1 that interacts with SNAIL1 upon its phosphorylation by GSK-3beta. Here we show that SNAIL1 polyubiquitylation and degradation may occur in conditions precluding SNAIL1 phosphorylation by GSK-3beta, suggesting that additional E3 ligases participate in the control of SNAIL1 protein stability. In particular, we demonstrate that the F-box E3 ubiquitin ligase FBXl14 interacts with SNAIL1 and promotes its ubiquitylation and proteasome degradation independently of phosphorylation by GSK-3beta. In vivo, inhibition of FBXl14 using short hairpin RNA stabilizes both ectopically expressed and endogenous SNAIL1. Moreover, the expression of FBXl14 is potently down-regulated during hypoxia, a condition that increases the levels of SNAIL1 protein but not SNAIL1 mRNA. FBXL14 mRNA is decreased in tumors with a high expression of two proteins up-regulated in hypoxia, carbonic anhydrase 9 and TWIST1. In addition, Twist1 small interfering RNA prevents hypoxia-induced Fbxl14 down-regulation and SNAIL1 stabilization in NMuMG cells. Altogether, these results demonstrate the existence of an alternative mechanism controlling SNAIL1 protein levels relevant for the induction of SNAIL1 during hypoxia.

Control of gene expression via small interfering RNA has enormous potential for the treatment of a variety of diseases, including cancer and Huntington's disease. However, before any therapies can be developed, effective techniques for controlled delivery of these molecules must be devised. In this proof-of-concept study, small interfering RNA was complexed with a polymer and loaded into a biomaterial scaffold. The scaffold was introduced primarily to control the release of the complexes, and the polymer was introduced to improve the transfection efficiency. An optimal dose and complexation ratio were selected, at which more than 50% down-regulation of the target gene Snail1 was observed in two-dimensional culture. Delayed release of the complexes was observed, and significant sustained down-regulation of Snail1 was seen in a three-dimensional scaffold system after 7 days. Thus, the use of the scaffold altered the transfection profile significantly, demonstrating the feasibility of a collagen scaffold as a controlled release system for delivery of small interfering RNA-dendrimer complexes.

The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.

Prep1 is known to interact in vivo with Pbx1 to regulate development and organogenesis. We have identified a novel Prep1-interacting protein, p160 c-Myb binding protein (p160). p160 and Pbx1 compete for Prep1 in vitro, and p160 inhibits Prep1-dependent HoxB2 expression in retinoic acid-treated NT2-D1 cells. The N-terminal physiologically truncated form of p160, p67, binds the sequence 63LFPLL67 in the HR1 domain of Prep1. Mutation of both L63 and L66 impairs the binding of Prep1 to both p160/p67 and Pbx1. The sequences required to bind Prep1 are mainly located in residues 51 to 151. Immunofluorescence colocalization and coimmunoprecipitation of endogenous p160 and Prep1 are induced by ActD, which translocates p160 from the nucleolus to the nucleoplasm. These data therefore show that p160 is a novel regulator of Prep1-Pbx1 transcriptional activity.

Prep1 homeodomain transcription factor interacts with Pbx proteins to regulate oculogenesis, angiogenesis, and hematopoiesis in mice. To isolate new Prep1 interactors competing or copurifying with Pbx, we identified proteins copurified with Prep1-TAP by tandem affinity purification (TAP). Prep1-TAP was fully functional and allowed the isolation of a Prep1 proteome from cytoplasm and nucleus, but most interactors were nuclear. The Prep1-TAP complex included Pbx1b, Pbx2, and other nonhomeodomain proteins: p160 Myb-binding protein (p160), beta-actin, NMMHCIIA.

Colon cancer progression is associated with the activation of protein kinase C (PKC), the downregulation of functional E-cadherin and an increased expression of the serine protease urokinase (u-PA) and its receptor (u-PAR). HT29-M6 intestinal epithelial cells represent an in vitro model to study colon cancer progression. These cells are induced to scatter and to invade by phorbol esters. Using proteolytic and cell signaling inhibitors, we show that HT29-M6 cells require plasminogen for the acquisition of the scattering response to PMA. Our results indicate that, prior to inducing a state of competency for plasminogen-dependent scattering, PMA triggers an ordered succession of events where upregulation of the activity of u-PA precedes proteolysis of u-PAR and active degradation of the extracellular matrix (ECM). These events poise HT29-M6 cells to a scatter-competent state that allows the subsequent localized proteolytic activation of plasminogen to plasmin, required for the execution of scattering. Finally, we show that, in addition to its enzymatic activity directed at the degradation of ECM, plasmin generates an intracellular signal resulting in the phosphorylation of ERK 1/2. For a full motogenic activity, plasmin requires this signal since the use of a MEK inhibitor (PD98059) specifically blocks the plasmin-dependent phase of cell scattering. Our observations suggest that plasmin exerts a dual role in PMA-induced scattering of HT29-M6 cells, one directed extracellularly to promote proteolysis of the ECM and one directed to generate intracellular signaling.

Background: Overexpression of tissue plasminogen activator (t-PA) in pancreatic cancer cells promotes invasion and proliferation in vitro and tumour growth and angiogenesis in vivo., Aims: To understand the mechanisms by which t-PA favours cancer progression, we analysed the surface membrane proteins responsible for binding specifically t-PA and studied the contribution of this interaction to the t-PA promoted invasion of pancreatic cancer cells., Methods: The ability of t-PA to activate plasmin and a fluorogenic plasmin substrate was used to analyse the nature of the binding of active t-PA to cell surfaces. Specific binding was determined in two pancreatic cancer cell lines (SK-PC-1 and PANC-1), and complex formation analysed by co-immunoprecipitation experiments and co-immunolocalisation in tumours. The functional role of the interaction was studied in Matrigel invasion assays., Results: t-PA bound to PANC-1 and SK-PC-1 cells in a specific and saturable manner while maintaining its activity. This binding was competitively inhibited by specific peptides interfering with the interaction of t-PA with annexin II. The t-PA/annexin II interaction on pancreatic cancer cells was also supported by co-immunoprecipitation assays using anti-t-PA antibodies and, reciprocally, with antiannexin II antibodies. In addition, confocal microscopy showed t-PA and annexin II colocalisation in tumour tissues. Finally, disruption of the t-PA/annexin II interaction by a specific hexapeptide significantly decreased the invasive capacity of SK-PC-1 cells in vitro., Conclusion: t-PA specifically binds to annexin II on the extracellular membrane of pancreatic cancer cells where it activates local plasmin production and tumour cell invasion. These findings may be clinically relevant for future therapeutic strategies based on specific drugs that counteract the activity of t-PA or its receptor annexin II, or their interaction at the surface level.

Background & Aims: Overexpression of tissue-type plasminogen activator (t-PA) in exocrine pancreatic tumors might be a determinant of the aggressive biological behavior of these tumors., Methods: Endogenous t-PA production was suppressed by antisense oligonucleotides or transcripts in CAPAN-1 and RWP-1 cell lines. Reciprocally, the t-PA non-expressing BxPC-3 and PANC-1 cells were stably transfected to overexpress t-PA. Recombinant t-PA and chemical inhibitors were also used on these cells. Clones were assayed for invasion and growth in vitro and in vivo., Results: In vitro, specific inhibition of t-PA expression or activity significantly inhibited the proliferation of t-PA-producing RWP-1, CAPAN-1, and SK-PC-1 cells. Antisense constructs were used to generate RWP-1 clones stably suppressed for t-PA expression (AS clones). These clones had a significantly reduced invasion and proliferation on plastic and in soft agar. The addition of recombinant t-PA rescued the growth of the AS clones to parental levels and was mitogenic for other independent pancreas cell lines. This effect did not require plasmin activity. In athymic mice, RWP-1 AS clones produced tumors fivefold smaller than control clones. AS tumors contained a significantly reduced number of Ki67-positive nuclei, fewer mitotic cells, and a remarkably reduced angiogenic network. Finally, the generation of tetracycline-repressed t-PA transfectants in PANC-1 cells confirmed the activity of t-PA in invasion and proliferation in vitro and in vivo., Conclusions: t-PA, in addition to its known role in invasion, plays other critical roles in pancreas tumor progression, stimulating cancer cell proliferation and tumor-associated angiogenesis.

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon-intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua-UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua-UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua-UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua-UEV protein confers new biological properties to this regulator of variant polyubiquitination.

Because hepatocyte growth factor (HGF) is a potent mitogen for normal human exocrine pancreas cells (NPCs) in vitro, we have analyzed the expression of HGF and its receptor, Met, in NPC and pancreas cancer cells and studied its effects in vitro. Using immunohistochemistry, Northern blotting, and reverse transcription-polymerase chain reaction, we examined the expression of HGF and Met in normal pancreas and pancreas cancer. Scatter assays, wound-healing assays, and migration through transwell filters were used to study HGF-stimulated motility of IMIM-PC-2 cancer cells. In tumors, HGF is mainly detected in stromal cells, whereas Met is overexpressed in cancer cells with an unpolarized distribution. In vitro, HGF stimulates motogenesis but not proliferation in cancer cells. Cell motility is accompanied by a rapid decrease in the cytoskeleton-bound E-cadherin, an acceleration of cellular adhesion to the substrate, an up-regulation of urokinase plasminogen activator (u-PA) RNA and protein, and a change in the solubility and proteolysis of the u-PA receptor. Cell motility is significantly reduced by inhibitors of u-PA proteolytic activity such as antibodies neutralizing u-PA activity, plasminogen activator inhibitor 1, and amiloride. These results show that a paracrine loop of HGF activation may participate in the development or progression of pancreas cancer. In vitro, the HGF-stimulated motogenesis of pancreas cancer cells involves the activation of the u-PA/u-PA receptor proteolytic system, suggesting its role in the invasive stages of tumor progression.

Plasminogen activators (PAs) play an important role in tumor cell invasion. We have analysed the expression of tissue-type PA (t-PA), urokinase-type PA (u-PA), and their respective receptors, annexin II and u-PAR, in normal and neoplastic cultures of pancreatic cells, as well as in pancreatic tissues, and have examined their role in tumor invasiveness in vitro. Using Northern blotting, Western blotting, and ELISA, t-PA is detected in cultured pancreas cancer cells displaying a well differentiated phenotype but it is undetectable in less differentiated cells and in normal pancreatic cultures. In contrast, u-PA transcripts, protein, and enzymatic activity are detected both in cancer cells and in normal cultures. Higher levels of u-PAR and annexin II are present in cancer cells than in normal cultures and, in SK-PC-1 cells, both receptors are localized in the basolateral membrane. In vitro invasion assays indicate that both t-PA and u-PA contribute to the invasiveness of SK-PC-1 cells through reconstituted extracellular matrix. To determine the relevance of these studies to pancreas cancer, immunohistochemical assays have been used to examine the expression of t-PA, u-PA, and their receptors in normal and neoplastic tissues. t-PA is absent from normal pancreas and from tumor associated pancreatitis, whereas it is detected in the majority of pancreas cancer tissues (16/17). Annexin II is also overexpressed in some tumors (5/13). u-PAR is overexpressed in most tumor samples examined (14/15), while u-PA is weakly detected in a low number of cases (3/14); both u-PAR and u-PA are overexpressed in areas of tumor associated pancreatitis. Indirect evidences indicate that K-ras and p53 mutated proteins can regulate the expression of PAs. In pancreatic cancer we have found an association between codon 12 K-ras mutations and t-PA expression (P=0.04). These results support the contention that, in the exocrine pancreas, activation of t-PA is more specifically associated to neoplastic transformation and to the invasive phenotype, whereas the induction of u-PA/u-PAR system might be more relevant to inflammatory or non-neoplastic events.

Of 1,065 persons sampled during a house-to-house survey of an area of the Department of Curuzú Cuatiá, Province of Corrientes, Argentina, 21 were seropositive to Brucella canis antibodies by the gel-diffusion test, using a saline-extracted B. ovis surface R antigen. Two positive reactors were similarly identified during a survey of rural schools in the Province of Neuquén, which included 887 persons. The findings are discussed in terms of current knowledge of this recently recognized zoonotic infection.

The ultrastructure of the hydatid cyst germinal membrane was studied. It was divided into 3 regions, the tegument, the tegumental cell region, and the innermost area bordering the cyst cavity. The morphology of tegumental, muscular, flame, duct, and glycogen-containing cells and cells containing lysosomal-like bodies is described. The significance of these findings is discussed in terms of the possible function of these structures and present knowledge on the penetration of macromolecules into hydatid cysts.

Fluid was collected from cysts of Taenia hydatigena in 60 adult sheep and fluid from each animal pooled separately. By double diffusion antigen 5 was demonstrated in all pools but one. The criteria are described for selection and standardization of these preparations for use as antigens for the immunodiagnosis of human hydatid disease. Sera from 50 persons harbouring hydatid cysts and from 50 patients with other disease conditions were examined by the arc-5 double-diffusion test, using two antigens prepared from Echinococcus granulosus and T. hydatigena cyst fluids, respectively. The results showed that a higher diagnostic sensitivity was obtained with the hydatid antigen. The significance of the findings is discussed in terms of their application to human immunodiagnosis in areas where hydatidosis, but not cysticercosis, is rare in livestock.

In a study of 662 sera from a hydatidosis endemic area, the indirect hemagglutination test based on a minimal nonspecificity criterion of positivity and the latex agglutination (LA) test were found to be suitable screening techniques for the detection of sera positive to the arc 5, diagnostic of hydatid infection. The lower nonspecificity of the LA test, its greater simplicity and its excellent correlation with the immunoelectrophoresis test suggest that it is the choice screening technique for use in field surveys or seroepidemiologic studies of hydatid disease. The advantages and limitations of this serologic approach for the detection of human hydatid cyst carriers in field studies are discussed.

The comparative sensitivity and specificity of the immunoelectrophoresis (IEP), latex agglutination (LA) and indirect hemagglutination (IHA) test for hydatidosis were evaluated using a single hydatid cyst fluid pool as antigen and the same hydatid and nonhydatid sera and were found to vary with the type of IHA test, the criterion for IHA test positively and the group of sera selected for study. The sensitivity of the LA and IEP tests was comparable, both tests correlated well and neither gave a positive result in the nonhydatid sera studied. The sensitivity of the IEP test was higher when IHA test positivity was based on diagnostically significant titers but not when all IHA test reactors were considered as positive to this test. The merits and limitations of the LA and IHA tests in diagnostic and seroepidemiologic studies of human hydatidosis are discussed. On the basis of the present findings, the use of the LA test and possibly of the tannic acid IHA test, to screen for IEP test positive sera is proposed as the most reliable immunodiagnostic approach to estimate the prevalence of human hydatidosis for seroepidemiologic purposes and also for detecting hydatidosis cases in the field.

Human sera from one case of polycystic hydatidosis due to Echnincoccus vogeli and from a case of cysticercosis and multiple myeloma were positive to the immunoelectrophoresis (IEP) test for hydatidosis based on the E. granulosus arc 5 positivity criterion. Arc 5 can therefore be elicited in IEP tests of human sera not only by E. granulosus and E. multilocularis but also by E. vogeli antigens. Whether the cross reaction observed in the second serum was due to the multiple myeloma or to the cysticerci remains to be determined. Although arc 5 antigens are known to be present in Taenia hydatigena cyst fluid this is the first report of an arc 5 due to antigens other than Echinococcus in IEP of human sera.

The sequential development of the hydatid immunodiagnostic activities of the control program of the Province of Neuquén, Argentina is described. Test results were used to obtain immunological confirmation of clinical cases and to detect asymptomatic cyst carriers amongst residents of rural endemic areas. The information was also valuable for improving the accuracy of prevalence estimates of human hydatidosis and the quality of surveillance data in different areas of the Province, characterized by varying degrees of environmental contamination by Echinococcus granulosus. In population groups examined by radiologic and immunologic methods, the latter detected more cases. When only immunodiagnostic surveys were carried out, mostly liver but also pulmonary hydatidosis cases were detected. This experience illustrates the advantages which may be obtained in endemic areas through the local application of hydatid immunodiagnosis based on arc 5 positivity.

The comparative sensitivity and specificity of four technical variants of the indirect hemagglutination test (IHA) for hydatidosis with tannic acid, glutaraldehyde, benzidine and formol treated cells, the same pool of hydatid cyst fluid and sera from hydatid and non-hydatid persons was studied. The number of reactors in each group of sera and the degree of reactivity of each serum sample varied with the type of IHA test. The sensitivity and specificity of each technique was related to the criterion on which IHA test positivity was based and to the group of sera examined. Of the four techniques, that employing tannic acid was considered as the choice IHA test for hydatid immunodiagnosis. The findings are discussed in terms of their implications to the use of a standard serum of known IHA test titer for reference purposes and to the significance of IHA test results for diagnostic and seroepidemiological studies.

The antibody response to rabies virus was studied in twenty volunteers immunized with different schemes of suckling mouse brain and human diploid cell culture rabies vaccines. Throughout the study period, titers in serum neutralization and indirect fluorescent antibody tests, as well as the class of immunoglobulins with antirabies activity, varied in different individuals with the treatment scheme and the antigenic potency of the vaccine. The results suggest that measurement of the IgG class of antirabies antibodies, and possibly IgA as well, may be a more adequate criterion to assess the immunogenicity of rabies vaccines than the determination of SN titers alone.

Host IgG was detected in 15 out of 18 peritoneal hydatid cysts obtained from experimentally infected gerbils and in 18 out of 61 mouse hydatid cysts which had been implanted into the peritoneal cavity of gerbils for periods ranging from one day to two years. A wide variation in host IgG concentration was observed among the different cysts in which these immunoglobulins were found. These findings are discussed in terms of the possible manner by which macromolecules may enter into the hydatid cysts.

This report deals with an evaluation of a double diffusion (DD5) test which employs a control antiserum against Echinococcus granulosus arc 5 antigens to recognize arc 5-positive sera by a reaction of identity. The DD5 test was more sensitive than the immuno-electrophoresis (IEP5) test based on the same positivity criterion and was equally specific for the immunodiagnosis of human hydatidosis. Its greater simplicity suggest its application as a substitute for the IEP5 test in diagnosis, particularly areas. The application of both DD5 and latex agglutination tests is recommended for hydatid immunodiagnosis on the basis of the present findings.

A Mangosta street rabies virus strain antigen not shared by the challenge virus standard (CVS) was identified by counterimmunoelectrophoresis. This strain antigen persisted following nine mouse passages of the virus, during which the CVS strain antigen was not acquired. These observations suggest that the antigen composition of the two virus strains is different. Antigenic differences during the course of adaptation of Mangosta virus were also revealed by crossed serum neutralization tests. The findings are discussed in terms of the occurrence of variations in the number and localizaion of antigen sites in the virus and of the possible existence of antigenic competition by non-infective viral antigens in serum neutralization tests for rabies.

A diagnosis of hydatid disease was established by the arc 5 double diffusion (DD5) test in two persons who had no symptoms, of whom one had had prior surgery for this parasitic infection. The location of some cysts which were removed surgically from these patients could not be determined in preoperative radiological and scintillographic studies. Postoperative serological monitoring by DD5 showed that one of these patients, though without symptoms, was harbouring an additional cyst. Abdominal x-rays showed no abnormalities, a partially calcified 2.7 cm cyst was detected in the right lobe of the liver by computerized axial tomography.