Artículos de revistas: "D1-class receptor"

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Watanabe, Katsushige y Minoru Kimura. "Dopamine Receptor–Mediated Mechanisms Involved in the Expression of Learned Activity of Primate Striatal Neurons". Journal of Neurophysiology 79, n.º 5 (1 de mayo de 1998): 2568–80. http://dx.doi.org/10.1152/jn.1998.79.5.2568.

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Watanabe, Katsushige and Minoru Kimura. Dopamine receptor–mediated mechanisms involved in the expression of learned activity of primate striatal neurons. J. Neurophysiol. 79: 2568–2580, 1998. To understand the mechanisms by which basal ganglia neurons express acquired activities during and after behavioral learning, selective dopamine (DA) receptor antagonists were applied while recording the activity of striatal neurons in monkeys performing behavioral tasks. In experiment 1, a monkey was trained to associate a click sound with a drop of reward water. DA receptor antagonists were administered by micropressure using a stainless steel injection cannula (300 μm ID) through which a Teflon-coated tungsten wire for recording neuronal activity had been threaded. Responses to sound by tonically active neurons (TANs), a class of neurons in the primate striatum, were recorded through a tungsten wire electrode during the application of either D1- or D2-class DA receptor antagonists (total volume <1 μl, at a rate of 1 μl/5–10 min). Application of the D2-class antagonist, (−)-sulpiride (20 μg/μl, 58 mM, pH 6.8), abolished the responses of four of five TANs examined. In another five TANs, neither the D2-class antagonist nor the D1-class antagonists, SCH23390 (10 μg/μl, 31 mM, pH 5.7) or cis-flupenthixol (30 μg/μl, 59 mM, pH 6.6) significantly suppressed responses. In experiment 2, four- or five-barreled glass microelectrodes were inserted into the striatum. The central barrel was used for extracellular recording of activity of TANs. Each DA receptor antagonist was iontophoretically applied through one of the surrounding barrels. SCH23390 (10 mM, pH 4.5) and (−)-sulpiride (10 mM, pH 4.5) were used. The effects of iontophoresis of both D1- and D2-class antagonists were examined in 40 TANs. Of 40 TANs from which recordings were made, responses were suppressed exclusively by the D2-class antagonist in 19 TANs, exclusively by the D1-class antagonist in 3 TANs, and by both D1- and D2-class antagonists in 7 TANs. When 0.9% NaCl, saline, was applied by pressure (<1 μl) or by iontophoresis (<30 nA) as a control, neither the background discharge rates nor the responses of TANs were significantly influenced. Background discharge rate of TANs was also not affected by D1- or D2-class antagonists applied by either micropressure injection or iontophoresis. It was concluded that the nigrostriatal DA system enables TANs to express learned activity primarily through D2-class and partly through D1-class receptor–mediated mechanisms in the striatum.

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Ibañez-Sandoval, Osvaldo, Adán Hernández, Benjamin Florán, Elvira Galarraga, Dagoberto Tapia, Rene Valdiosera, David Erlij, Jorge Aceves y José Bargas. "Control of the Subthalamic Innervation of Substantia Nigra Pars Reticulata by D1 and D2 Dopamine Receptors". Journal of Neurophysiology 95, n.º 3 (marzo de 2006): 1800–1811. http://dx.doi.org/10.1152/jn.01074.2005.

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The effects of activating dopaminergic D1 and D2 class receptors of the subthalamic projections that innervate the pars reticulata of the subtantia nigra (SNr) were explored in slices of the rat brain using the whole cell patch-clamp technique. Excitatory postsynaptic currents (EPSCs) that could be blocked by 6-cyano-7-nitroquinoxalene-2,3-dione and d-(−)-2-amino-5-phosphonopentanoic acid were evoked onto reticulata GABAergic projection neurons by local field stimulation inside the subthalamic nucleus in the presence of bicuculline. Bath application of ( RS)-2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine hydrochloride (SKF-38393), a dopaminergic D1-class receptor agonist, increased evoked EPSCs by ∼30% whereas the D2-class receptor agonist, trans-(−)-4aR-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo(3,4-g)quinoline (quinpirole), reduced EPSCs by ∼25%. These apparently opposing actions were blocked by the specific D1- and D2-class receptor antagonists: R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepinehydrochloride (SCH 23390) and S-(−)-5-anino-sulfonyl- N-[(1-ethyl-2-pyrrolidinyl)-methyl]-2-methoxybenzamide (sulpiride), respectively. Both effects were accompanied by changes in the paired-pulse ratio, indicative of a presynaptic site of action. The presynaptic location of dopamine receptors at the subthalamonigral projections was confirmed by mean-variance analysis. The effects of both SKF-38393 and quinpirole could be observed on terminals contacting the same postsynaptic neuron. Sulpiride and SCH 23390 enhanced and reduced the evoked EPSC, respectively, suggesting a constitutive receptor activation probably arising from endogenous dopamine. These data suggest that dopamine presynaptically modulates the subthalamic projection that targets GABAergic neurons of the SNr. Implications of this modulation for basal ganglia function are discussed.

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Wang, Chenguang, Nagarajan Pattabiraman, Jian Nian Zhou, Maofu Fu, Toshiyuki Sakamaki, Chris Albanese, Zhiping Li et al. "Cyclin D1 Repression of Peroxisome Proliferator-Activated Receptor γ Expression and Transactivation". Molecular and Cellular Biology 23, n.º 17 (1 de septiembre de 2003): 6159–73. http://dx.doi.org/10.1128/mcb.23.17.6159-6173.2003.

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ABSTRACT The cyclin D1 gene is overexpressed in human breast cancers and is required for oncogene-induced tumorigenesis. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor selectively activated by ligands of the thiazolidinedione class. PPARγ induces hepatic steatosis, and liganded PPARγ promotes adipocyte differentiation. Herein, cyclin D1 inhibited ligand-induced PPARγ function, transactivation, expression, and promoter activity. PPARγ transactivation induced by the ligand BRL49653 was inhibited by cyclin D1 through a pRB- and cdk-independent mechanism, requiring a region predicted to form an helix-loop-helix (HLH) structure. The cyclin D1 HLH region was also required for repression of the PPARγ ligand-binding domain linked to a heterologous DNA binding domain. Adipocyte differentiation by PPARγ-specific ligands (BRL49653, troglitazone) was enhanced in cyclin D1−/− fibroblasts and reversed by retroviral expression of cyclin D1. Homozygous deletion of the cyclin D1 gene, enhanced expression by PPARγ ligands of PPARγ and PPARγ-responsive genes, and cyclin D1−/− mice exhibit hepatic steatosis. Finally, reduction of cyclin D1 abundance in vivo using ponasterone-inducible cyclin D1 antisense transgenic mice, increased expression of PPARγ in vivo. The inhibition of PPARγ function by cyclin D1 is a new mechanism of signal transduction cross talk between PPARγ ligands and mitogenic signals that induce cyclin D1.

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Yamamoto, Kei, Olivier Mirabeau, Charlotte Bureau, Maryline Blin, Sophie Michon-Coudouel, Michaël Demarque y Philippe Vernier. "Evolution of Dopamine Receptor Genes of the D1 Class in Vertebrates". Molecular Biology and Evolution 30, n.º 4 (28 de noviembre de 2012): 833–43. http://dx.doi.org/10.1093/molbev/mss268.

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Sasikumar, T. K., D. A. Burnett, H. Zhang, A. Smith-Torhan, A. Fawzi y J. E. Lachowicz. "Hydrazides of clozapine: A new class of D1 dopamine receptor subtype selective antagonists". Bioorganic & Medicinal Chemistry Letters 16, n.º 17 (septiembre de 2006): 4543–47. http://dx.doi.org/10.1016/j.bmcl.2006.06.022.

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Vargo, J. M., B. B. Bromberg, P. J. Best, J. V. Corwin y J. F. Marshall. "D1-class dopamine receptor involvement in the behavioral recovery from prefrontal cortical injury". Behavioural Brain Research 72, n.º 1-2 (diciembre de 1995): 39–48. http://dx.doi.org/10.1016/0166-4328(95)00028-3.

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Shayegan, Darius K. y Stephen M. Stahl. "Atypical Antipsychotics: Matching Receptor Profile to Individual Patient's Clinical Profile". CNS Spectrums 9, S11 (octubre de 2004): 6–14. http://dx.doi.org/10.1017/s1092852900025086.

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AbstractUnderstanding common pharmacologic and clinical “class” actions associated with atypical antipsychotics certainly reveals how these agents are alike, but what about unique differences from one agent to another? Atypical antipsychotics are also a heterogeneous group of agents that have complex pharmacologic entities, acting upon multiple dopamine receptors (D2, D1 (D3, and D4) and multiple serotonin receptors (5-HT2A, 5-HT2C, 5-HT1A, and 5-HT1D, among others). Atypical antipsychotics also interact with noradrenergic (α1- and α2-adrenergic receptor blockade), histaminergic (H1-receptor blockade), and cholinergic (muscarinic M1 blockade) neurotransmitter systems as well as with monoamine (D, 5-HT, and norepinephrine reuptake blockade) transporters. However, no two atypical antipsychotics possess the same portfolio of actions upon all of these additional neurotransmitter systems.

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Sakurai, Atsuko, Julie Gavard, Yuliya Annas-Linhares, John R. Basile, Panomwat Amornphimoltham, Todd R. Palmby, Hiroshi Yagi et al. "Semaphorin 3E Initiates Antiangiogenic Signaling through Plexin D1 by Regulating Arf6 and R-Ras". Molecular and Cellular Biology 30, n.º 12 (12 de abril de 2010): 3086–98. http://dx.doi.org/10.1128/mcb.01652-09.

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ABSTRACT Recent studies revealed that a class III semaphorin, semaphorin 3E (Sema3E), acts through a single-pass transmembrane receptor, plexin D1, to provide a repulsive cue for plexin D1-expressing endothelial cells, thus providing a highly conserved and developmentally regulated signaling system guiding the growth of blood vessels. We show here that Sema3E acts as a potent inhibitor of adult and tumor-induced angiogenesis. Activation of plexin D1 by Sema3E causes the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix (ECM) and causing the retraction of filopodia in endothelial tip cells. Sema3E acts on plexin D1 to initiate a two-pronged mechanism involving R-Ras inactivation and Arf6 stimulation, which affect the status of activation of integrins and their intracellular trafficking, respectively. Ultimately, our present study provides a molecular framework for antiangiogenesis signaling, thus impinging on a myriad of pathological conditions that are characterized by aberrant increase in neovessel formation, including cancer.

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Prommer, Eric. "Olanzapine". American Journal of Hospice and Palliative Medicine® 30, n.º 1 (10 de abril de 2012): 75–82. http://dx.doi.org/10.1177/1049909112441241.

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Olanzapine is an atypical antipsychotic agent of the thienobenzodiazepine class. Olanzapine blocks multiple neurotransmitter receptors, including dopaminergic (D1, D2, D3, and D4), serotonergic (5-hydroxytryptamine 2A [5-HT2A], 5-HT2C, 5-HT3, and 5-HT6), adrenergic (α1), histaminic (H1), and muscarinic (M1, M2, M3, and M4) receptors. Olanzapine has a high affinity for the 5HT2A receptor, which is up to 5 times greater than the dopamine receptor, resulting in less propensity to the development of extrapyramidal side effects. The affinity of olanzapine for multiple receptors has lead to the identification of olanzapine as an important agent in the treatment of delirium, nausea, and vomiting. Olanzapine has been demonstrated to have opioid-sparing properties. Olanzapine is principally metabolized by glucuronidation, with a smaller metabolic contribution from the cytochrome oxidase system. Adverse effects of olanzapine include somnolence, postural hypotension, constipation, dizziness, restlessness, and weight gain. The purpose of this article is to outline the pharmacodynamics, pharmacology, and evidence for the use of olanzapine in palliative care.

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Martini, Michael L., Jing Liu, Caroline Ray, Xufen Yu, Xi-Ping Huang, Aarti Urs, Nikhil Urs et al. "Defining Structure–Functional Selectivity Relationships (SFSR) for a Class of Non-Catechol Dopamine D1 Receptor Agonists". Journal of Medicinal Chemistry 62, n.º 7 (15 de marzo de 2019): 3753–72. http://dx.doi.org/10.1021/acs.jmedchem.9b00351.

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Tseng, K. Y., L. A. Riquelme y M. G. Murer. "Impact of d1-class dopamine receptor on striatal processing of cortical input in experimental parkinsonism in vivo". Neuroscience 123, n.º 2 (enero de 2004): 293–98. http://dx.doi.org/10.1016/j.neuroscience.2003.10.005.

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Iype, Elizabeth Mathew, Rajan Balakrishna, Lakshmi Subhadradevi, Jissa Vinoda Thulaseedharan, Bharath Veerabadhran y Jayasree Kattoran. "Expression of proliferation markers in laryngeal squamous cell carcinoma: an association with clinico-pathological factors and treatment outcomes". International Journal of Otorhinolaryngology and Head and Neck Surgery 6, n.º 5 (21 de abril de 2020): 858. http://dx.doi.org/10.18203/issn.2454-5929.ijohns20201675.

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Background: The study was done with the objective to study the expression of epidermal growth factor receptor (EGFR), cyclin D1 and Ki-67 in laryngeal squamous cell carcinoma and to assess the correlation of all three proliferation markers with various clinic-pathological parameters, the treatment outcomes as well as survival.Methods: We prospectively evaluated the surgical specimens of 72 laryngeal squamous cell carcinoma (LSCC) patients, treated with primary surgery and post-operative adjuvant therapy. Tumor tissue samples were analysed for the expression of EGFR, cyclin D1 and Ki-67 markers and analysis were done by immune-histochemistry and western blot test. Results: EGFR showed significant expression in 67.6% and was insignificant in 31.9% patients in our analysis of 72 tumor samples. Cyclin D1 showed intense expression in 43%, and was insignificant in 57% patients. Ki-67 was intensely expressed in 43% patients. There was no correlation between expression of these markers with age, T-stage and N-stage. However, all the three markers showed significantly intense expression in tumours with extra capsular disease as well as perineural invasion (PNI) both of which are features of invasiveness of the tumor.Conclusions: Estimation of biomarkers such as EGFR, cyclin Dl, and Ki-67 could be beneficial in predicting tumor aggressiveness, prognosis and survival in LSCC patients. Thus, all the three proliferation markers can be categorized as markers of invasiveness. Combination of proliferation markers-EGFR, cyclin D1 and Ki-67 is useful pre-operatively in planning surgical strategies so as to decide a more radical approach for the resection of the primary as well as neck dissection.

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Błaszczak-Świątkiewicz, Katarzyna. "New Selective Progesterone Receptor Modulators and Their Impact on the RANK/RANKL Complex Activity". Molecules 25, n.º 6 (13 de marzo de 2020): 1321. http://dx.doi.org/10.3390/molecules25061321.

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Breast cancer depends on women’s age. Its chemotherapy and hormone therapy lead to the loss of bone density and disruption of the skeleton. The proteins RANK and RANKL play a pivotal role in the formation of osteoclasts. It is also well established that the same proteins (RANK and RANKL) are the main molecules that play an important role in mammary stem cell biology. Mammary stem cells guarantee differentiation of the epithelial mammary cells, the growth of which is regulated by the progesterone-induced RANKL signaling pathway. The crosstalk between progesterone receptor, stimulated by progesterone and its analogues results in RANKL to RANK binding and activation of cell proliferation and subsequently unlimited expansion of the breast cancer cells. Therefore downstream regulation of this signaling pathway is desirable. To meet this need, a new class of selective estrogen receptor modulators (SPRMs) with anti- and mesoprogestin function were tested as potential anti-RANK agents. To establish the new feature of SPRMs, the impact of tested SPRMs on RANK-RANKL proteins interaction was tested. Furthermore, the cells proliferation upon RANKL stimulation, as well as NFkB and cyclin D1 expression, induced by tested SPRMs were analyzed. Conducted experiments proved NFkB expression inhibition as well as cyclin D1 expression limitation under asoprisnil and ulipristal treatment. The established paracrine anti-proliferative activity of antiprogestins together with competitive interaction with RANK make this class of compounds attractive for further study in order to deliver more evidence of their anti-RANK activity and potential application in the breast cancer therapy together with its accompanied osteoporosis.

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Ali, Mohamed, Sara Mahmoud Shahin, Nagwa Ali Sabri, Ayman Al-Hendy y Qiwei Yang. "Activation of β-Catenin Signaling and its Crosstalk With Estrogen and Histone Deacetylases in Human Uterine Fibroids". Journal of Clinical Endocrinology & Metabolism 105, n.º 4 (25 de diciembre de 2019): e1517-e1535. http://dx.doi.org/10.1210/clinem/dgz227.

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Abstract Context Uterine fibroids (UF) are the most common benign tumor of the myometrium (MM) in women of reproductive age. However, the mechanism underlying the pathogenesis of UF is largely unknown. Objective To explore the link between nuclear β-catenin and UF phenotype and β-catenin crosstalk with estrogen and histone deacetylases (HDACs). Design Protein/RNA levels of β-catenin (CTNNB1 gene), its responsive markers cyclin D1 and c-Myc, androgen receptor (AR), p27, and class-I HDACs were measured in matched UF/MM tissues or cell populations. The effects of chemical inhibition/activation and genetic knockdown of CTNNB1 on UF phenotype were measured. The anti-UF effect of 2 HDAC inhibitors was evaluated. Main Outcome Measure β-catenin nuclear translocation in response to β-catenin inhibition/activation, estrogen, and HDAC inhibitors in UF cells. Results UF tissues/cells showed significantly higher expression of nuclear β-catenin, cyclin D1, c-Myc, and HDACs 1, 2, 3, and 8 than MM. Estradiol induced β-catenin nuclear translocation and consequently its responsive genes in both MM and UF cells, while an estrogen receptor antagonist reversed this induction effect. Treatment with β-catenin or HDAC inhibitors led to dose-dependent growth inhibition, while Wnt3a treatment increased proliferation compared with control. Chemical inhibition of β-catenin decreased cyclin D1 and c-Myc expression levels, while β-catenin activation increased expression of the same markers. Genetic knockdown of CTNNB1 resulted in a marked decrease in β-catenin, cyclin D1, c-Myc, and AR expression. Treatment of UF cells with HDAC inhibitors decreased nuclear β-catenin, cyclin D1, and c-Myc expression. Moreover, HDAC inhibitors induced apoptosis of UF cells and cell cycle arrest. Conclusion β-catenin nuclear translocation contributes to UF phenotype, and β-catenin signaling is modulated by estradiol and HDAC activity.

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Amenta, Francesco, Livio Chiandussi, Maurizio Mancini, Alberto Ricci, Marina Schena y Franco Veglio. "Pharmacological characterization and autoradiographic localization of dopamine receptors in the human adrenal cortex". European Journal of Endocrinology 131, n.º 1 (julio de 1994): 91–96. http://dx.doi.org/10.1530/eje.0.1310091.

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Amenta F, Chiandussi L, Mancini M, Ricci A, Schena M, Veglio F. Pharmacological characterization and autoradiographic localization of dopamine receptors in the human adrenal cortex. Eur J Endocrinol 1994;131:91–6. ISSN 0804–4643 The pharmacological characteristics and the anatomical localization of dopamine D1-like and D2-like receptors were studied in sections of the human adrenal cortex using radioligand binding and autoradiographic techniques. [3H]SCH23390 was used as a ligand of D1-like receptors, whereas [3H]spiroperidol was used to label D2-like receptors. No specific [3H]SCH 23390 binding was detectable in sections of the human adrenal cortex. On the other hand, [3H]spiroperidol was bound to sections of the adrenal gland in a manner consistent with the labelling of dopamine D2-like receptor sites. The binding was time, temperature and concentration dependent, belonging in the range of concentrations of the radioligand used for a single class of high-affinity sites. The dissociation constant (Kd) averaged 2.7 nmol/l, whereas the maximum density of binding sites (Bmax) was 160 nmol/mg tissue. Experiments on the pharmacological specificity of [3H]spiroperidol binding to sections of the human adrenal cortex revealed that clozapine was the most powerful displacer of [3H]spiroperidol from sections of the human adrenal cortex. This suggests the presence in the human adrenal cortex of dopamine receptors of the D4 subtype. Light microscope autoradiography showed the highest density of specific [3H]spiroperidol binding sites in the zona glomerulosa and to a lesser extent in the zona reticularis. Only sparse [3H]spiroperidol binding sites were localized in the zona fasciculata. The possible functional consequences of this localization of dopamine D2-like receptor sites in the human adrenal cortex are discussed. Francesco Amenta, Istituto di Farmacologia, Via Scalzino 5, 62032 Camerino, Italy

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Fyfe, Tim J., Peter J. Scammells, J. Robert Lane y Ben Capuano. "Enantioenriched Positive Allosteric Modulators Display Distinct Pharmacology at the Dopamine D1 Receptor". Molecules 26, n.º 13 (22 de junio de 2021): 3799. http://dx.doi.org/10.3390/molecules26133799.

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(1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively); (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with superior allosteric properties.

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Boyle, Kathleen A. y Teresa Compton. "Receptor-Binding Properties of a Soluble Form of Human Cytomegalovirus Glycoprotein B". Journal of Virology 72, n.º 3 (1 de marzo de 1998): 1826–33. http://dx.doi.org/10.1128/jvi.72.3.1826-1833.1998.

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ABSTRACT The human cytomegalovirus (HCMV) glycoprotein B (gB) (also known as gpUL55) homolog is an important mediator of virus entry and cell-to-cell dissemination of infection. To examine the potential ligand-binding properties of gB, a soluble form of gB (gB-S) was radiolabeled, purified, and tested in cell-binding experiments. Binding of gB-S to human fibroblast cells was found to occur in a dose-dependent, saturable, and specific manner. Scatchard analysis demonstrated a biphasic plot with the following estimated dissociation constants (Kd ): K d1, 4.96 × 10−6 M; K d2, 3.07 × 10−7 M. Cell surface heparan sulfate proteoglycans (HSPGs) were determined to serve as one class of receptors able to facilitate gB-S binding. Both HSPG-deficient Chinese hamster ovary (CHO) cells and fibroblast cells with enzymatically removed HSPGs had 40% reductions in gB-S binding, whereas removal of chondroitin sulfate had no effect. However, a significant proportion of gB-S was able to associate with the cell surface in the absence of HSPGs via an undefined nonheparin component. Binding affinity analysis of gB-S binding to wild-type CHO-K1 cells demonstrated biphasic binding kinetics (K d1, 9.85 × 10−6M; Kd2 , 4.03 × 10−8 M), whereas gB-S binding to HSPG-deficient CHO-677 cells exhibited single-component binding kinetics (Kd , 7.46 × 10−6 M). Together, these data suggest that gB-S associates with two classes of cellular receptors. The interaction of gB with its receptors is physiologically relevant, as evidenced by an inhibitory effect on HCMV entry when cells were pretreated with purified gB-S. This inhibition was determined to be manifested at the level of virus attachment. We conclude that gB is a ligand for HCMV that mediates an interaction with a cellular receptor(s) during HCMV infection.

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Opazo, Juan C., Kattina Zavala, Soledad Miranda-Rottmann y Roberto Araya. "Evolution of dopamine receptors: phylogenetic evidence suggests a later origin of the DRD2l and DRD4rs dopamine receptor gene lineages". PeerJ 6 (13 de abril de 2018): e4593. http://dx.doi.org/10.7717/peerj.4593.

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Dopamine receptors are integral membrane proteins whose endogenous ligand is dopamine. They play a fundamental role in the central nervous system and dysfunction of dopaminergic neurotransmission is responsible for the generation of a variety of neuropsychiatric disorders. From an evolutionary standpoint, phylogenetic relationships among the DRD1 class of dopamine receptors are still a matter of debate as in the literature different tree topologies have been proposed. In contrast, phylogenetic relationships among the DRD2 group of receptors are well understood. Understanding the time of origin of the different dopamine receptors is also an issue that needs further study, especially for the genes that have restricted phyletic distributions (e.g., DRD2l and DRD4rs). Thus, the goal of this study was to investigate the evolution of dopamine receptors, with emphasis on shedding light on the phylogenetic relationships among the D1 class of dopamine receptors and the time of origin of the DRD2l and DRD4rs gene lineages. Our results recovered the monophyly of the two groups of dopamine receptors. Within the DRD1 group the monophyly of each paralog was recovered with strong support, and phylogenetic relationships among them were well resolved. Within the DRD1 class of dopamine receptors we recovered the sister group relationship between the DRD1C and DRD1E, and this clade was recovered sister to a cyclostome sequence. The DRD1 clade was recovered sister to the aforementioned clade, and the group containing DRD5 receptors was sister to all other DRD1 paralogs. In agreement with the literature, among the DRD2 class of receptors, DRD2 was recovered sister to DRD3, whereas DRD4 was sister to the DRD2/DRD3 clade. According to our phylogenetic tree, the DRD2l and DRD4rs gene lineages would have originated in the ancestor of gnathostomes between 615 and 473 mya. Conservation of sequences required for dopaminergic neurotransmission and small changes in regulatory regions suggest a functional refinement of the dopaminergic pathways along evolution.

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Singh-Jasuja, Harpreet, René E. M. Toes, Pieter Spee, Christian Münz, Norbert Hilf, Stephen P. Schoenberger, Paola Ricciardi-Castagnoli et al. "Cross-Presentation of Glycoprotein 96–Associated Antigens on Major Histocompatibility Complex Class I Molecules Requires Receptor-Mediated Endocytosis". Journal of Experimental Medicine 191, n.º 11 (6 de junio de 1999): 1965–74. http://dx.doi.org/10.1084/jem.191.11.1965.

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Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I–restricted re-presentation of gp96-associated peptides and CTL activation; non–receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

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Omeljaniuk, Robert J. "Specific [3H]spiperone binding sites in the pituitary of rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus)". Canadian Journal of Physiology and Pharmacology 73, n.º 5 (1 de mayo de 1995): 585–93. http://dx.doi.org/10.1139/y95-074.

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Dopamine, a catecholamine neurohormone, modulates pituitary hormone release in teleost fishes and other vertebrates. The existence and binding parameters of a pituitary dopamine–neuroleptic receptor from trout were examined and compared with those from goldfish. Pituitary homogenate was incubated with [3H]spiperone (D2 antagonist) under several experimental paradigms; incubations were terminated by filtration and bound 3H radioactivity was assessed by liquid scintillation spectroscopy. Specific binding of [3H]spiperone was tissue dependent. Equilibrium displacement analyses using domperidone (D2 antagonist) indicated a single class of binding site (LIGAND) with Kd = 2.49 ± 0.89 μM and a capacity of 3.10 ± 0.45 nmol/mg protein; the goldfish Kd and capacity were both significantly (p < 0.05) larger: Kd = 4.63 ± 0.30 μM and capacity = 20.66 ± 2.03 nmol/mg protein. The Kd and capacity for the trout pars distalis (2.45 ± 0.33 μM and 3.27 ± 0.24 nmol/mg protein, respectively) did not differ significantly (p < 0.05) from that of the neurointermediate lobe (2.50 ± 0.08 μM and 3.58 ± 0.56 nmol/mg protein, respectively). Dopamine D2 receptor ligands differentially displaced [3H]spiperone from the trout pituitary, while D1 ligands, a D4 ligand, and a 5-hydroxytryptamine (5HT2) receptor antagonist had only small nonspecific effects. Comparison of the trout and goldfish pituitary dopamine–neuroleptic receptor indicates conservation of receptor affinity (Kd); however, differences in receptor numbers and in the distribution of receptors between the pars distalis and neurointermediate lobe in the two species may be due in part to species or developmental differences, and may reflect differences in the role(s) and degrees of influence of dopamine in these fishes.Key words: pituitary, dopamine, receptor, rainbow trout, goldfish.

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Sinnett-Smith, James, Yang Ni, Jia Wang, Ming Ming, Steven H. Young y Enrique Rozengurt. "Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling". American Journal of Physiology-Cell Physiology 306, n.º 10 (15 de mayo de 2014): C961—C971. http://dx.doi.org/10.1152/ajpcell.00048.2014.

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We examined whether class IIa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1 (PKD1) in IEC-18 intestinal epithelial cells. Our results show that class IIa HDAC4, HDAC5, and HDAC7 are prominently expressed in these cells. Stimulation with ANG II, a potent mitogen for IEC-18 cells, induced a striking increase in phosphorylation of HDAC4 at Ser246 and Ser632, HDAC5 at Ser259 and Ser498, and HDAC7 at Ser155. Treatment with the PKD family inhibitors kb NB 142-70 and CRT0066101 or small interfering RNA-mediated knockdown of PKD1 prevented ANG II-induced phosphorylation of HDAC4, HDAC5, and HDAC7. A variety of PKD1 activators in IEC-18 cells, including vasopressin, lysophosphatidic acid, and phorbol esters, also induced HDAC4, HDAC5, and HDAC7 phosphorylation. Using endogenously and ectopically expressed HDAC5, we show that PKD1-mediated phosphorylation of HDAC5 induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5 with Ser259 and Ser498 mutated to Ala was localized to the nucleus in unstimulated and stimulated cells. Treatment of IEC-18 cells with specific inhibitors of class IIa HDACs, including MC1568 and TMP269, prevented cell cycle progression, DNA synthesis, and proliferation induced in response to G protein-coupled receptor/PKD1 activation. The PKD1-class IIa HDAC axis also functions in intestinal epithelial cells in vivo, since an increase in phosphorylation of HDAC4/5 and HDAC7 was demonstrated in lysates of crypt cells from PKD1 transgenic mice compared with matched nontransgenic littermates. Collectively, our results reveal a PKD1-class IIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling.

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22

Avila, DM, SA Fuqua, FW George y MJ McPhaul. "Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment". Journal of Endocrinology 159, n.º 3 (1 de diciembre de 1998): 403–11. http://dx.doi.org/10.1677/joe.0.1590403.

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In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.

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23

Seiler, Max P., Alexander Hagenbach, Hans Juerg Wuethrich y Rudolf Markstein. "trans-Hexahydroindolo[4,3-ab]phenanthridines ("benzergolines"), the first structural class of potent and selective dopamine D1 receptor agonists lacking a catechol group". Journal of Medicinal Chemistry 34, n.º 1 (enero de 1991): 303–7. http://dx.doi.org/10.1021/jm00105a047.

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24

Tang, Jinhua, Yanli Yan, Ting C. Zhao, George Bayliss, Haidong Yan y Shougang Zhuang. "Class I histone deacetylase activity is required for proliferation of renal epithelial cells". American Journal of Physiology-Renal Physiology 305, n.º 3 (1 de agosto de 2013): F244—F254. http://dx.doi.org/10.1152/ajprenal.00126.2013.

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The process of renal regeneration after acute kidney injury is thought to recapitulate renal development, and proliferation of renal proximal tubular cells (RPTCs) is a critical step in the regenerative response. Recent studies indicate that class I histone deacetylases (HDACs) are required for embryonic kidney gene expression, growth, and differentiation. The role and underlying mechanisms of class I HDAC activation in RPTC proliferation, however, remain unclear. In this study, we used cultured RPTCs to examine this issue since four class I HDAC isoforms (1, 2, 3, and 8) are abundantly expressed in this cell type. Blocking class I HDAC activity with a highly selective inhibitor, MS-275, induced global histone H3 hyperacetylation, reduced RPTC proliferation, and diminished expression of cyclin D1 and proliferating cell nuclear antigen. Silencing HDAC1, 3, or 8 with small interfering RNA resulted in similar biological effects. Activation of epidermal growth factor receptor (EGFR) and signal transducers and activators of transcription 3 (STAT3) was required for RPTC proliferation, and STAT3 functioned downstream of EGFR. Treatment with MS-275 or knockdown of HDAC1, 3, or 8 suppressed EGFR expression and phosphorylation, and silencing HDAC1 and 3 also reduced STAT3 phosphorylation. However, HDAC2 downregulation did not affect RPTC proliferation and phosphorylation of EGFR and STAT3. Collectively, these data reveal a critical role of class I HDACs in mediating proliferation of renal epithelial cells through activation of the EGFR/STAT3 signaling pathway.

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Orr, Sarah K., Romain A. Colas, Jesmond Dalli, Nan Chiang y Charles N. Serhan. "Proresolving actions of a new resolvin D1 analog mimetic qualifies as an immunoresolvent". American Journal of Physiology-Lung Cellular and Molecular Physiology 308, n.º 9 (1 de mayo de 2015): L904—L911. http://dx.doi.org/10.1152/ajplung.00370.2014.

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Resolution of inflammation is an active process driven by several new families of endogenous lipid mediators collectively coined specialized proresolving mediators (SPM). Here, we report a synthetic analog of resolvin D1 (RvD1) and aspirin-triggered RvD1, benzo-diacetylenic-17 R-RvD1-methyl ester (BDA-RvD1), which was prepared using fewer steps than required for total organic synthesis of natural SPM. BDA-RvD1 was resistant to further metabolism by human recombinant 15-prostaglandin dehydrogenase, a major inactivation pathway for RvD1. In ischemia-reperfusion-initiated second organ injury, BDA-RvD1 intravenously (1 μg) reduced neutrophil infiltration into the lungs by 58 ± 9% and was significantly more potent than native RvD1. BDA-RvD1 at 100 ng/mouse also shortened the resolution interval, Ri, of Escherichia coli peritonitis with a similar potency as RvD1, by ∼57%, from Ri 10.5 h to 4.5 h. With isolated human phagocytes, BDA-RvD1 at picomolar concentrations (10−12 M) stimulated phagocytosis of zymosan A particles. BDA-RvD1 activated human recombinant G protein-coupled receptor 32/DRV1, an RvD1 receptor, in a dose-dependent manner. These results indicate that, both in vivo in mice and with isolated human cells, BDA-RvD1 shares defining proresolving actions of RvD1, including inhibiting leukocyte infiltration and stimulating phagocytosis. Moreover, they provide evidence for a new analog mimetic and example of an immunoresolvent, namely an agent that stimulates active resolution of inflammation, for a potential new therapeutic class.

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SEILER, M. P., A. HAGENBACH, H. J. WUETHRICH y R. MARKSTEIN. "ChemInform Abstract: trans-Hexahydroindolo(4,3-ab)phenanthridines (“Benzergolines”), the First Structural Class of Potent and Selective Dopamine D1 Receptor Agonists Lacking a Catechol Group." ChemInform 22, n.º 24 (23 de agosto de 2010): no. http://dx.doi.org/10.1002/chin.199124230.

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27

Mitrofanova, L. B., A. A. Perminova, N. S. Goncharova y E. N. Mikhailov. "Histological and immunohistochemical study of nerve fibers and ganglia in the periarterial adipose tissue of the pulmonary artery bifurcation in patients with and without pulmonary hypertension". "Arterial’naya Gipertenziya" ("Arterial Hypertension") 25, n.º 5 (31 de enero de 2020): 498–509. http://dx.doi.org/10.18705/1607-419x-2019-25-5-498-509.

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Background. One of the possible methods of reducing pressure in the pulmonary artery (PA) in pulmonary hypertension (PH) is radiofrequency damage of the periarterial nerve fibers. At the same time, the impact of the autonomic nervous system has not been fully determined yet. It is also known that dopamine induces LA vasorelaxation, however, the subtypes of dopamine receptors involved in this mechanism have not yet been identified.Objective. To assess the morphology of nerve fibers and ganglia in the periarterial adipose tissue of the PA bifurcation in patients with and without PH. Design and methods. Tissue samples of the PA bifurcation zone with surrounding fatty tissue were taken from 8 patients with PH and 9 patients without PH (autopsy material): 7 women aged 59 ± 22 years and 10 men 62 ± 15 years. An immunohistochemical reaction was performed with antibodies to S100 protein, tyrosine hydroxylase (TH), M1 muscarinic receptor, D1, D2, D5 dopamine receptors. The number of nerve fibers and ganglia per area of the preparation, their diameter and depth relative to the PA intima were estimated.Results. There were no statistically significant differences in the structure and size of nerve fibers and ganglia in patients with and without PH. In general, the average number of nerve fibers per area of the preparation (4 cm2) was 29,1 ± 15,5; ganglia — 1,1 ± 1,3; the average fiber diameter was 130,6 ± 35,1 pm, ganglia — 532,0 ± 790,7 pm; the average fiber-intima distance was 2141,4 ± 663,3 pm, the ganglion-intima — 1799,0 ± 500,5 pm. The density of fibers around the PA bifurcation was significantly higher (p = 0,04) in patients with chronic heart failure (CHF) II NYHA functional class (FC) (20 ± 10 fibers / 4 cm2) compared to patients with CHF III-IV FC (11 ± 4 fibers / 4 cm2). The expression of M1 and TH was determined on nerve fibers and ganglia, in the endothelium and smooth muscle cells of the PA, in the epithelium of the bronchi. At the same time, unlike M1, the expression of TH was not observed in all nerve cells, and its level ranged from 1 to 4 points. No D2 receptor expression was detected, D1 expression was mild, and D5 was 4 points in all cases in the endothelium and smooth muscle cells of the LA.Conclusions. Morphometric analysis of nerve fibers and ganglia revealed no differences between patients with and without PH. There was a significant decrease in the number of nerve fibers with the progression of heart failure. TH expression on nerve fibers and ganglia was less pronounced and was not observed in all cells as compared with the M1 receptor. Expression of dopamine receptors was detected only in the endothelium and smooth muscle cells of the PA. Further morphological study involving larger sample will allow the substantiation for the validity of the interventional innervation of the PA.

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Yang, Quan-en, Andrew G. Stephen, Joseph W. Adelsberger, Paula E. Roberts, Weimin Zhu, Michael J. Currens, Yaxiong Feng et al. "Discovery of Small-Molecule Human Immunodeficiency Virus Type 1 Entry Inhibitors That Target the gp120-Binding Domain of CD4". Journal of Virology 79, n.º 10 (15 de mayo de 2005): 6122–33. http://dx.doi.org/10.1128/jvi.79.10.6122-6133.2005.

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ABSTRACT The interaction between human immunodeficiency virus type 1 (HIV-1) gp120 and the CD4 receptor is highly specific and involves relatively small contact surfaces on both proteins according to crystal structure analysis. This molecularly conserved interaction presents an excellent opportunity for antiviral targeting. Here we report a group of pentavalent antimony-containing small molecule compounds, NSC 13778 (molecular weight, 319) and its analogs, which exert a potent anti-HIV activity. These compounds block the entry of X4-, R5-, and X4/R5-tropic HIV-1 strains into CD4+ cells but show little or no activity in CD4-negative cells or against vesicular stomatitis virus-G pseudotyped virions. The compounds compete with gp120 for binding to CD4: either immobilized on a solid phase (soluble CD4) or on the T-cell surface (native CD4 receptor) as determined by a competitive gp120 capture enzyme-linked immunosorbent assay or flow cytometry. NSC 13778 binds to an N-terminal two-domain CD4 protein, D1/D2 CD4, immobilized on a surface plasmon resonance sensor chip, and dose dependently reduces the emission intensity of intrinsic tryptophan fluorescence of D1/D2 CD4, which contains two of the three tryptophan residues in the gp120-binding domain. Furthermore, T cells incubated with the compounds alone show decreased reactivity to anti-CD4 monoclonal antibodies known to recognize the gp120-binding site. In contrast to gp120-binders that inhibit gp120-CD4 interaction by binding to gp120, these compounds appear to disrupt gp120-CD4 contact by targeting the specific gp120-binding domain of CD4. NSC 13778 may represent a prototype of a new class of HIV-1 entry inhibitors that can break into the gp120-CD4 interface and mask the gp120-binding site on the CD4 molecules, effectively repelling incoming virions.

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Mackay, Simon P. y Patrick J. O’Malley. "Molecular Modelling of the Interaction of Cyanoacrylate Inhibitors with Photosystem II Part 1. The Effect of Hydrophobicity of Inhibitor Binding". Zeitschrift für Naturforschung C 48, n.º 9-10 (1 de octubre de 1993): 773–81. http://dx.doi.org/10.1515/znc-1993-9-1015.

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Abstract Cyanoacrylate Inhibitors, Photosystem II, Hydrophobicity The secondary quinone binding site of photosystem II is also the binding site for many dif ferent herbicides. The 2-cyanoacrylate inhibitors are a potent class of electron transfer inhibitors which bind at this site and are extremely sensitive to minor structural variation. In order to understand their mode of binding, we have studied the interaction between the inhibitors and receptor in the D1 protein binding region (residues Leu 210 to Val 280) in terms of non bonded intermolecular forces. The intermolecular energy was calculated by van der Waals and electrostatic interactions after energy minimization of the combined structures to reduce inter and intramolecular strain. We have identified specific amino acid residues within the binding protein which are instrumental in binding the herbicide and have shown that the spatial arrangement of the herbicide functional groups within the binding site rather than their lipophilicity is the determining factor in binding efficiency.

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30

Lewis, C. A. y D. S. Faber. "Properties of spontaneous inhibitory synaptic currents in cultured rat spinal cord and medullary neurons". Journal of Neurophysiology 76, n.º 1 (1 de julio de 1996): 448–60. http://dx.doi.org/10.1152/jn.1996.76.1.448.

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1. To identify the type(s) and properties of inhibitory postsynaptic receptor(s) involved in synaptic transmission in cultured rat embryonic spinal cord and medullary neurons, we have used whole cell patch-clamp techniques to record miniature inhibitory postsynaptic currents (mIPSCs) in the presence of tetrodotoxin, DL-2-amino-5-phosphonovaleric acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. 2. The mIPSCs recorded from both spinal cord and medullary neurons had skewed amplitude distributions. 3. The glycinergic antagonist strychnine and the GABAergic antagonist bicuculline each decreased both the frequency and mean peak amplitudes of mIPSCs. We conclude that both glycine and gamma-aminobutyric acid (GABA) are neurotransmitters at inhibitory synapses in our cultured cells. 4. Most (approximately 96-97%) mIPSCs decay with single-exponential time constants, and decay time distributions were consistently best fitted by the sum of four Gaussians with decay constants as follows: D1 = 5.8 +/- 0.1 (SE) ms (n = 63), D2 = 12.2 +/- 0.2 ms (n = 61), D3 = 23.2 +/- 0.4 ms (n = 54), and D4 = 44.7 +/- 1.0 ms (n = 57). We conclude that the four classes of decay times represent kinetically different inhibitory postsynaptic receptor populations. 5. Strychnine and bicuculline usually had one of two different effects on the mIPSC decay time constant distributions; either selective decreases in the frequency of mIPSCs with decay times in certain classes (i.e., the D1 class was reduced by bicuculline, the D2 class by strychnine, and the D3 and D4 classes by both antagonists) or a nonselective depression in the frequency of mIPSCs with decay times in all four classes. The particular effect observed in a given neuron was correlated with the presence or absence of ATP and guanosine 5'-triphosphate (GTP) in the patch pipette. Namely, in 71% of the antagonist applications where the pipette contained ATP and GTP, the result was a nonselective decrease in mIPSCs in all decay time constant classes. Conversely, in 54% of the antagonist applications in their absence, the result was a selective decrease in the frequency of mIPSCs in specific decay time constant classes. 6. In some experiments, mIPSCs reappeared in antagonist solution after an essentially complete block. Recovery from block in the continued presence of antagonist was never observed in the absence of ATP and GTP (8 neurons), and, at the same time, 5 of 9 neurons patched with ATP and GTP in the pipette did show recovery (56%).

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Thompson, Peter, Jinhua Lu y Gerardo G. Kaplan. "The Cys-Rich Region of Hepatitis A Virus Cellular Receptor 1 Is Required for Binding of Hepatitis A Virus and Protective Monoclonal Antibody 190/4". Journal of Virology 72, n.º 5 (1 de mayo de 1998): 3751–61. http://dx.doi.org/10.1128/jvi.72.5.3751-3761.1998.

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ABSTRACT The hepatitis A virus cellular receptor 1 (HAVcr-1) cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function which serves as an African green monkey kidney (AGMK) cell receptor for HAV. The extracellular domain of havcr-1 has an N-terminal Cys-rich region that displays homology with sequences of members of the immunoglobulin superfamily, followed by a Thr/Ser/Pro (TSP)-rich region characteristic of mucin-like O-glycosylated proteins. The havcr-1 glycoprotein contains four putative N-glycosylation sites, two in the Cys-rich region and two in the TSP-rich region. To characterize havcr-1 and define region(s) involved in HAV receptor function, we expressed the TSP-rich region in Escherichia coli fused to glutathione S-transferase and generated antibodies (Ab) in rabbits (anti-GST2 Ab). Western blot analysis with anti-GST2 Ab detected 62- and 65-kDa bands in AGMK cells and 59-, 62-, and 65-kDa bands in dog cells transfected with the HAVcr-1 cDNA (cr5 cells) but not in dog cells transfected with the vector alone (DR2 cells). Treatment of AGMK and cr5 cell extracts with peptide-N-glycosidase F resulted in the collapse of the havcr-1-specific bands into a single band of 56 kDa, which indicated that different N-glycosylated forms of havcr-1 were expressed in these cells. Treatment of AGMK and cr5 cells with tunicamycin reduced binding of protective monoclonal Ab (MAb) 190/4, which suggested that N-glycans are required for binding of MAb 190/4 to havcr-1. To test this hypothesis, havcr-1 mutants lacking the N-glycosylation motif at the first site (mut1), second site (mut2), and both (mut3) sites were constructed and transfected into dog cells. Binding of MAb 190/4 and HAV to mut1 and mut3 cells was highly reduced, while binding to mut2 cells was not affected and binding to dog cells expressing an havcr-1 construct containing a deletion of the Cys-rich region (d1− cells) was undetectable. HAV-infected cr5 and mut2 cells but not mut1, mut3, d1−, and DR2 cells developed the characteristic cytoplasmic granular fluorescence of HAV-infected cells. These results indicate that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for binding of protective MAb 190/4 and HAV receptor function.

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Weinberger, P., P. Kountourakis, C. Sasaki, B. Haffty, R. Camp, D. Rimm y A. Psyrri. "Oropharyngeal squamous cell cancers (OSCC) bearing transcriptionally active human papillomavirus (HPV) display distinct protein expression profile". Journal of Clinical Oncology 24, n.º 18_suppl (20 de junio de 2006): 10028. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.10028.

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10028 Background: It is known that among HPV DNA+ OSCCs only the transcriptionally active ones are biologically and clinically relevant. pRb downregulation by HPVE7 results in p16 upregulation. We previously showed (JCO, Feb 2006) that p16 expression defines transcriptionally active HPV+ tumors. Thus, OSCC tumors can be Class I: HPV−/p16−, Class II: HPV+/p16−, or Class III: HPV+/p16+. We hypothesized that tumors classified by these criteria would show different protein expression profiles. Methods: Following institutional review board approval, paraffin-embedded specimens from 77 patients were collected from Yale-New Haven Hospital archives. Patients with OSCC treated with radiotherapy (RT) or surgery and RT were eligible. We used real-time polymerase chain reaction for HPV 16 and immunohistochemistry for p16 protein expression to classify 77 patients with OSCC. Tumors were classified into a 3-class model based on p16 expression and HPV DNA presence: Class I (HPV−, p16−) Class II (HPV+, p16−) and Class III (HPV+, p16+). A tissue microarray including these cases was constructed and examined for 13 target proteins with known involvement in tumor suppression or progression. We used a method of in-situ automated quantitative protein expression analysis (AQUA). Protein signal (AQUA score) was scored on a scale of 0–255.Protein expression between groups was analyzed by analysis of variance (ANOVA). Results: Thirty tumors were class I (39%), 29 were class II (38%), and 18 were class III (23%). There were significant differences for protein expression of Met (p=0.005), β-catenin (p=0.009), pRb (p=0.001), p53(p=0.026), epidermal growth factor receptor (EGFR) (p=0.009) and Vascular Endothelial Growth Factor (VEGF) (p=0.028). There was no difference for ki-67, COX2, p27, p21 or Cyclin D1 expression, while ERK2 and p14 trended towards significance (p=0.09 and 0.054, respectively). Conclusions: We demonstrated that tumors classified by HPV DNA presence and p16 expression status have different molecular phenotypes. Of particular note, class III tumors demonstrated increased expression of Met, β-catenin, EGFR and VEGF. These findings may prove useful for patient selection for molecular-targeted therapies. [Table: see text]

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Singh, Amareshwar T. K., Mistuni Ghosh, C. Shad Thaxton, Trudy M. Forte, Robert O. Ryan y Leo I. Gordon. "The Bioactive Polyphenol Curcumin (diferuloylmethane) In Human Apolipoprotein A-1 Nanodisks Enhances Apoptosis and G1 Cell Cycle Arrest In Mantle Cell Lymphoma Compared with Free Curcumin". Blood 116, n.º 21 (19 de noviembre de 2010): 3934. http://dx.doi.org/10.1182/blood.v116.21.3934.3934.

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Abstract Abstract 3934 Background: Mantle cell lymphoma (MCL) is a pre–germinal center neoplasm characterized by cyclin D1 overexpression resulting from translocation of the cyclin D1 gene on 11q13 to the promoter of the immunoglobulin heavy chain locus on 14q32. Since MCL is incurable with standard lymphoma therapies, new treatment approaches are needed that target specific biologic pathways. The bioactive polyphenol curcumin (Curc), derived from the rhizome of Curcuma longa Linn, has been shown to have pleiotropic activities related to its complex chemistry and its influence on multiple signaling pathways including NF-kB, Akt, Nrf2 and pathways involved in metastasis and angiogenesis. Curc has been shown to cause growth arrest and apoptosis of BKS-2 immature B-cell lymphoma by downregulating growth and survival promoting genes (Clin Immunol 1999; 93:152). However, because of poor aqueous solubility Curc has had limited clinical utility, so investigators have explored nanoparticle drug delivery approaches (J Nanobiotech 2007, 5:3, MCT 2010; 9:2255). We reasoned that effective and targeted drug delivery by nanoparticles required appropriate receptors to facilitate binding. We therefore screened lymphoma cell lines for receptors that recognize apolipoprotein (apo) A-1. We hypothesized that a novel discoidal nanoparticle (ND) consisting of apoA-1, phospholipid and Curc (Curc ND) would bind to such receptors to facilitate drug delivery. Methods: We compared biologic activity of free Curc vs. Curc-ND in MCL cell lines expressing receptors for apoA-1. Cell lines were grown and maintained in culture, treated, and apoptosis and cell cycle progression was measured by flow cytometry. Relevant signaling intermediates and presence of apoA-1 receptors were measured by immunoblotting using specific antibodies. Results: Granta and Jeko cells (both MCL cell lines) expressed apoA-1 receptors including class B scavenger receptor (SR-B1) and the ATP-binding cassette transporter of the sub-family G1 (ABCG1). To compare the pro-apoptotic effect of free Curc and Curc-ND, Granta cells were incubated with free Curc, Curc-ND, empty ND, and medium alone (untreated). Compared to medium alone, empty ND had no effect while free Curc (20 μM) induced apoptosis. Curc-ND produced a dose-dependent increase in apoptosis, with ∼70% apoptosis at 20 μM. To investigate the mechanism of Curc-ND induced apoptosis, apoptosis-related proteins were studied in cultured Granta cells. A time-dependent decrease in caspase-9 levels was observed following incubation with Curc-ND or free Curc. The decrease in caspase-9 seen with Curc-ND, however, occurs much earlier (between 2–4 h of incubation) than for free-Curc. Caspase-3 was undetectable after 16 h with either treatment. Loss of this band implies activation of caspase-3, which was confirmed by PARP cleavage, wherein a decrease in the 116 kD band was accompanied by an increase in the 85 kD cleavage product. Unlike free Curc, Curc-ND induced PARP cleavage even at 16 h of incubation, suggesting sustained drug release. Curc-ND downregulated cyclin D1, decreased Akt phosphorylation and enhanced cleavage of caspases-9 and -3, and PARP. In addition, Curc-ND induced G1 cell cycle arrest to a greater extent than free Curc in Granta and Jeko cells (Granta: Control 34% G1, Curc 37% G1, Curc-ND 46% G1; Jeko: Control 39% G1, Curc 49% G1, Curc-ND 54% G1). Conclusion: We have shown that the MCL cell lines Granta and Jeko express apoA-1 receptors, making them likely targets for discoidal nanoscale delivery vehicles stabilized with Apo-A1. These nanodisks, when carrying the polyphenol Curc, can result in increased caspase -dependent apoptosis, cell cycle arrest, downregulation of cyclin-D1 and decreased p-Akt. These data suggest that the pleiotropic polyphenol Curc has cell killing/arrest activity in MCL and that Curc-ND may be a potential therapeutic with drug targeting ability. Disclosures: Forte: Lypro Biosciences: Employment.

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Klasa, Richard J., Alan F. List y Bruce D. Cheson. "Rational Approaches to Design of Therapeutics Targeting Molecular Markers". Hematology 2001, n.º 1 (1 de enero de 2001): 443–62. http://dx.doi.org/10.1182/asheducation-2001.1.443.

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Abstract This paper introduces novel therapeutic strategies focusing on a molecular marker relevant to a particular hematologic malignancy. Four different approaches targeting specific molecules in unique pathways will be presented. The common theme will be rational target selection in a strategy that has reached the early phase of human clinical trial in one malignancy, but with a much broader potential applicability to the technology. In Section I Dr. Richard Klasa presents preclinical data on the use of antisense oligonucleotides directed at the bcl-2 gene message to specifically downregulate Bcl-2 protein expression in non-Hodgkin's lymphomas and render the cells more susceptible to the induction of apoptosis. In Section II Dr. Alan List reviews the targeting of vascular endothelial growth factor (VEGF) and its receptor in anti-angiogenesis strategies for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In Section III Dr. Bruce Cheson describes recent progress in inhibiting cell cycle progression by selectively disrupting cyclin D1 with structurally unique compounds such as flavopiridol in mantle cell lymphoma as well as describing a new class of agents that affect proteasome degradation pathways.

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Schroeder, Frederick A., Krista L. Penta, Anouch Matevossian, Sara R. Jones, Christine Konradi, Andrew R. Tapper y Schahram Akbarian. "Drug-Induced Activation of Dopamine D1 Receptor Signaling and Inhibition of Class I/II Histone Deacetylase Induce Chromatin Remodeling in Reward Circuitry and Modulate Cocaine-Related Behaviors". Neuropsychopharmacology 33, n.º 12 (20 de febrero de 2008): 2981–92. http://dx.doi.org/10.1038/npp.2008.15.

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Aiello, Aurora, Giuseppe Pandini, Francesco Frasca, Enrico Conte, Antonella Murabito, Antonella Sacco, Marco Genua, Riccardo Vigneri y Antonino Belfiore. "Peroxisomal Proliferator-Activated Receptor-γ Agonists Induce Partial Reversion of Epithelial-Mesenchymal Transition in Anaplastic Thyroid Cancer Cells". Endocrinology 147, n.º 9 (1 de septiembre de 2006): 4463–75. http://dx.doi.org/10.1210/en.2005-1610.

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Anaplastic thyroid cancer (ATC) is an extremely aggressive tumor characterized by marked epithelial mesenchymal transition, which leads, almost invariably, to death. Peroxisomal proliferator-activated receptor (PPAR)-γ agonists have recently emerged as potential antineoplastic drugs. To establish whether ATC could be a target of PPARγ agonists, we first examined PPARγ protein expression in a panel of six ATC cell lines and then studied the biologic effects of two PPARγ agonists, ciglitazone and rosiglitazone, that belong to the class of thiazolidonediones. PPARγ protein was present and functional in all ATC cell lines. Both ciglitazone and rosiglitazone showed complex biological effects in ATC cells, including inhibition of anchorage-dependent and -independent growth and migration, and increased apoptosis rate. Rosiglitazone-induced growth inhibition was associated with cell cycle arrest and changes in cell cycle regulators, such as an increase of cyclin-dependent kinases inhibitors p21cip1 and p27kip1, a decrease of cyclin D1, and inactivation of Rb protein. Rosiglitazone-induced apoptosis was associated with a decrease of Bcl-XL expression and caspase-3 and -7 activation. Moreover, rosiglitazone antagonized IGF-I biological effects by up-regulating phosphatase and tensin homolog deleted from chromosome 10 with subsequent inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway. Finally, rosiglitazone increased the expression of thyroid-specific differentiation markers. In conclusions, these data suggest that PPARγ agonists induce a partial reversion of the epithelial mesenchymal transition in ATC cells by multiple mechanisms. PPARγ agonists may, therefore, have a role in the multimodal therapy currently used to slow down ATC growth and dissemination.

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37

Bäsecke, Jörg, Anneke Lehmann, Ulla Bergholz, James McCubrey, Lorenz Trümper, Carol Stocking y Stefan Horn. "The Glycogensynthase Kinase 3b (GSK-3β) Is Involved in Leukemic Transformation". Blood 112, n.º 11 (16 de noviembre de 2008): 4489. http://dx.doi.org/10.1182/blood.v112.11.4489.4489.

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Abstract Purpose: Signal transduction pathways, such as the PI3K/AKT cascade, are frequently activated in acute myeloid leukemia and stimulate the proliferation and survival of leukemic cells. Mutations in upstream genes such as Class III receptor tyrosine kinases are frequent but not exclusive causes of that activation. An important downstream target of PI3K/AKT is the key regulator GSK-3β. It controls anti-apoptotic genes such as NF-kappaB and Cyclin D1, is involved in wnt-pathway activation and drug resistance of leukemic cells. Deregulated signaling by GSK-3β occurs through inhibition by AKT-mediated phosphorylation. We have observed that constitutive phosphorylation of GSK-3β occurred in hematopoietic cells with pro-leukemogenic PI3K-mutations. We wanted to evaluate the relevance of GSK-3β inactivation in the transformation process of hematopoietic cells. Methods and Results: We used an in vitro factor-independent growth assay, GSK-3b inhibitors (Lithium, BIO) and established a second hit model using retroviral gene transfer of the weak oncogene Bcl XL. Signaling cascades were analysed by western blot. We demonstrate that inactivation of GSK-3b alone was not sufficient to induce factor-independent growth in IL-3 dependent early hematopoietic cells (Ba/F3). Induction of apoptosis upon growth factor withdrawal was reduced, but not prevented, in the presence of GSK-3b inhibitors, leading to a delayed Caspase 3 activation, PARP cleavage and DNA fragmentation. Overexpression of Bcl-XL also did not result in a prevention of apoptosis. GSK-3b inhibition in synergy with Bcl-XL overexpression resulted in the establishement of several growth factor-independent cell lines, which were characterized by the activation of multiple signaling cascades including AKT, MAPK, STAT5, but not STAT3. Also Cyclin D1 was overexpressed in contrast to other cyclines (D2, D3) which are no substrates of GSK-3b. Conclusions: Our data show that GSK-3β is part of the apoptotic response to growth factor withdrawal and suggest that GSK-3β is causaly involved in the transformation process of hematopoietic cells and seems to have an synergistic role in addition to other pro-leukemic mutations.

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38

Wang, Qinghua, Jing Tang, Shujun Jiang, Zan Huang, Anying Song, Siyuan Hou, Xiang Gao y Hai-Bin Ruan. "Inhibition of PPARγ, adipogenesis and insulin sensitivity by MAGED1". Journal of Endocrinology 239, n.º 2 (noviembre de 2018): 167–80. http://dx.doi.org/10.1530/joe-18-0349.

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Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipogenesis and a target of the thiazolidinedione (TZD) class of antidiabetic drugs; therefore, identifying novel regulators of PPARγ action in adipocytes is essential for the future development of therapeutics for diabetes. MAGE family member D1 (MAGED1), by acting as an adaptor for ubiquitin-dependent degradation pathways and a co-factor for transcription, plays an important role in neural development, cell differentiation and circadian rhythm. Here, we showed that MAGED1 expression was downregulated during adipogenesis and loss of MAGED1 promoted preadipocyte proliferation and differentiation in vitro. MAGED1 bound to PPARγ and suppressed the stability and transcriptional activity of PPARγ. Compared to WT littermates, MAGED1-deficient mice showed increased levels of PPARγ protein and its target genes, more CD29+CD34+Sca-1+ adipocyte precursors and hyperplasia of white adipose tissues (WATs). Moreover, MAGED1-deficient mice developed late-onset obesity as a result of decreased energy expenditure and physical activity. However, these mice were metabolically healthy as shown by improved glucose clearance and insulin sensitivity, normal levels of serum lipids and enhanced secretion of adipokines such as leptin and adiponectin. Taken together, our data identify MAGED1 as a novel negative regulator of PPARγ activity, adipogenesis and insulin sensitivity in mice. MAGED1 might therefore serve as a novel pharmaceutical target to treat obesity-associated insulin resistance.

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39

Zhou, Yixin, Linhua Jin, Stefania Pittaluga, Mark Raffeld, Takashi Miida y Yoko Tabe. "PI3K a Selective Inhibitor Induces Growth Inhibition In Mantle Cell Lymphoma". Blood 116, n.º 21 (19 de noviembre de 2010): 1812. http://dx.doi.org/10.1182/blood.v116.21.1812.1812.

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Abstract Abstract 1812 Deregulation of the phosphatidylinositol 3-kinase (PI3K)-mediated signaling plays an important role in the development of cell proliferation of mantle cell lymphoma (MCL). The PI3K pathway activation in MCL has been shown to result from constitutive B cell receptor (BCR) activation which is directly mediated by the Class IA PI3K p110 isoforms (a, β, and d). However, their relative contribution in MCL is not fully understood. In this study, the activity and molecular mechanisms of isoform-selective PI3K inhibitors which target different isoforms of the p110-kDa subunit has been investigated. We utilized the isoform-selective PI3K inhibitors; PI3-Ka inhibitor IV (p110a), TGX115 (p110b), IC87114 (p110d) and the non-specific PI3K inhibitor LY294002 (all inhibitors were purchased commercially). The p110a and p110d but not p110b isoform protein expression was detected in all tested MCL cell lines (Granta 519, JVM-2, Z138, Jeko-1, MINO). PI3-Ka inhibitor IV as well as non-specific PI3K inhibitor LY294002 induced cell growth inhibition with dose-dependent manner (IC50 at 48 hrs; PI3-Ka inhibitor IV: 17.5 μM for Granta 519, 14.3 μM for Jeko-1, 16.5 μM for Z138, LY294002: 14.8 μM for Granta 519, 19.4 μM for Jeko-1, 15.0 μM for Z138, MTT test). However, neither IC87114 nor TGX115 showed significant cell growth inhibition up to 40mM. Low dose of PI3-Ka inhibitor IV (5 μM) or LY294002 (5 μM) induced G0/G1 cell cycle arrest (increase of G0/G1 phase: PI3-Ka inhibitor IV 17.9 % for Granta 519, 28.2 % for Jeko-1, LY294002 19.3 % for Granta 519, 14.5 % for Jeko-1), and the higher dose (10 μM) increased apoptosis(specific apoptosis: PI3-Ka inhibitor IV 10.8 % for Granta 519, 15.3 % for Jeko-1, LY294002 13.6 % for Granta 519, 19.6 % for Jeko-1). No induction of cell cycle arrest/apoptosis by IC87114 or TGX115 treatment was observed. We then tried to assess the inhibition of PI3K/Akt signaling activation by p110a and p110d inhibitors. PI3-Ka inhibitor IV (10 μM) completely diminished phosphorylated (p-) Akt in all cell lines analyzed. Further investigation with 1–10 μM PI3-Ka inhibitor IV or IC87114 in Granta 519 and Jeko-1 cells declared that 1 μM PI3-Ka inhibitor IV almost diminished p-Akt and p-S6rp in both cells. The phosphorylation level of other PI3K/Akt signaling downstream substrates, GSK3-b and 4E-BP1, were down-regulated in dose dependent manner. Recently, GSK3-b kinase has been shown to negatively regulate cell cycle progression through Cyclin D1 repression in MCL. We observed that PI3-Ka inhibitor IV decreased Cyclin D1 expression and active pRb which are responsible for G0/G1 cell cycle arrest. The treatment with IC87114 (10 μM) performed moderate decrease of p-Akt, p-S6rp, and p-4E-BP, while no change in the levels of p-GSK3-b, Cyclin D1, or p-pRb was observed in both Granta 519 and Jeko-1 cells. We also tested whether the combination of PI3-Ka inhibitor IV or IC87114 with the proteasome inhibitor bortezomib induces synergistic cytotoxicity in MCL. No synergistic anti-proliferative effect was observed in any of the MCL cell lines analyzed. These findings demonstrate that p110a may be the responsible Class IA PI3K isoform for the development of MCL cell proliferation, and p110a isoform-selective PI3K inhibitor but not p110d or p110b inhibitors may provide a better therapeutic index relative to pan-PI3K inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Slater, Alexandre, Ying Di, Joanne C. Clark, Natalie J. Jooss, Eleyna M. Martin, Fawaz Alenazy, Mark R. Thomas et al. "Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer". Blood 137, n.º 24 (17 de junio de 2021): 3443–53. http://dx.doi.org/10.1182/blood.2020009440.

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Abstract Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C′ loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C′ loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.

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41

Sloand, Elaine M., Katayoun Rezvani, Agnes Yong, Daniel Douek, Roger Kurlander, David Price, John Barrett y Neal S. Young. "Cytotoxic CD8 T Cell Immune Responses to Wilms Tumor Protein (WT-1) Characterizes Immunosuppression-Responsive Myelodysplasia (MDS)." Blood 108, n.º 11 (16 de noviembre de 2006): 849. http://dx.doi.org/10.1182/blood.v108.11.849.849.

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Abstract Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p<0.001). When patient and control CD8 cells were cultured in the presence of WT-1- and cyclin D1-loaded HLA-A2-restricted antigen-presenting cells (using three different HLA-A0201- restricted epitopes of WT-1 and four different epitopes for CD1), eight patients’ CD8 cell tested positive for interferon gamma, indicating a cytotoxic response. Staining with labeled MHC class I A2-restricted tetramers used to measure WT-1-specific cells showed concordant results in all eight patients. Tetramer staining was increased in 12 of 14 trisomy 8 patients examined. Cytotoxic T cells specific for WT-1 were present within the expanded V β subfamilies previously found to suppress trisomy 8 cell proliferation in vitro. CD8 cells from another eight MDS patients without trisomy 8 but who had responded to immunosuppression, sampled prior to treatment, also demonstrated WT1-specific cytotoxic activity, but only two of 18 non-responders showed measurable levels of WT-1-specific cytotoxic T cells (entire treated cohort seen in Fig 1). We infer from these data an autoimmune pathophysiology for trisomy 8 MDS with: 1) over-expression of WT-1 by the aneuploid clone; 2) a specific cytotoxic CD8 T cell immune response to WT-1; 3) subsequent cytotoxic T cell marrow suppression by apoptosis, either by cross reactivity or a bystander effect; and 4) improvement in hematopoiesis after immunosuppression. Other MDS which is responsive to immunosuppression may also be mediated by WT1. Figure Figure

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42

Malkovsky, M., D. G. McNeel, M. Wallace, K. Schell, G. Wilding, J. Eikhoff, M. J. Staab, D. Horvath, J. Straus y G. Liu. "Use of zoledronic acid (ZA) and interleukin-2 (IL-2) to activate and expand Vγ9Vδ2 T cells for therapeutic use in patients with metastatic renal cell carcinoma (mRCC)". Journal of Clinical Oncology 24, n.º 18_suppl (20 de junio de 2006): 14607. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.14607.

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14607 Background: Approximately 3–6% of human peripheral blood lymphocytes express the γδ T cell receptor (TCR). The majority of these cells express the Vδ2 TCR variable segment associated with the Vγ9 segment. Vγ9Vδ2 T cells can recognize phosphorylated nonpeptidic microbial metabolites and alkylamines. This recognition of phosphorylated molecules requires neither antigen uptake/processing, nor MHC class I/II expression, allowing for a rapid response to immune challenge. Aminobisphosphonates are a class of agents capable of activating human Vγ9Vδ2 T cells. Here we conduct a study using ZA with low-dose IL-2 in order to augment Vγ9Vδ2 T cells for therapeutic use in mRCC. Methods: Patients with immunotherapy naïve mRCC were treated with ZA (4 mg IV d1) with IL-2 (ranging from 1–7 MU/m2 SQ days 1–5, repeated weekly × 3, in 4 week cycles). Blood was collected at various timepoints between day 1 and 15 of the first cycle of therapy to assess the relative frequency, proliferative capacity, and cytolytic activity of each subjects’ Vg9Vd2 T cells. Clinical responses were assessed using RECIST criteria. Results: To date, 7 patients have received treatment on protocol. No objective clinical responses were observed by RECIST criteria but prolonged stable disease (with one minor response) was observed in 2 patients (on study for 280 and 224 days respectively) on this trial. A relative increase in Vγ9Vδ2 T cell frequency could be detected by day 8 of therapy including one patient with an increase in Vγ9/Vδ2 positive T cells from 9.6 to 19.4%. Results of the change in Vγ9Vδ2 T cell frequency, proliferative capacity, and cytolytic activity against RCC cell lines will be presented in detail. Conclusions: Modest amplification of Vγ9Vδ2 T cell frequency was seen; however, not to the degree appreciated in preclinical models. At present, we are planning a dose-titration of ZA with fixed dosed IL-2 in order to optimize this therapeutic strategy. The exploitation of Vγ9Vδ2 T cells as a therapy for mRCC is novel, and the current study suggests this approach deserves further evaluation. No significant financial relationships to disclose.

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43

Cichocki, Frank M., Todd Lenvik, Stephen K. Anderson y Jeffrey S. Miller. "STAT5A Overexpression Enhances Killer Immunoglobulin Receptor (KIR) Expression in Developing NK Cells and Is Associated with a Loss of Reverse Transcription from the Proximal KIR Promoter." Blood 110, n.º 11 (16 de noviembre de 2007): 798. http://dx.doi.org/10.1182/blood.v110.11.798.798.

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Abstract Down-regulation of HLA-class I molecules is commonly observed in virally infected and malignantly transformed cells and can contribute to the ability of these cells to escape recognition by the adaptive immune system. NK cells are able to detect the loss of even single HLA alleles on potential target cells through their clonally distributed HLA-class I-specific inhibitory receptors (KIR). While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides in areas surrounding the transcription initiation region, the mechanisms that regulate variegated KIR expression are largely unknown. The manipulation of KIR expression is of considerable interest due to the widely reported correlation between KIR ligand status and patient survival in hematopoietic stem cell transplant settings. Because the transcription factor STAT5 is activated downstream of the BCR/ABL oncogene, which we have previously shown to substantially augment KIR expression levels in primary NK cells, we hypothesized that STAT5 could directly influence KIR expression. To test this hypothesis, we purified CD34+ hematopoietic progenitor cells from umbilical cord blood and retrovirally transduced these cells with either a control murine stem cell virus (MSCV) vector encoding eGFP alone or an MSCV vector encoding both eGFP and a constitutively active form of STAT5A, termed STAT5A 1*6. CD34/eGFP-positive cells were then purified and cultured on the EL08-D1 stroma line, known to support NK cell development. After a period of 28 days in culture, 6.25±2.73% of the NK cells in eGFP control cultures were KIR-positive compared with 29.3±4.27% of STAT5A 1*6 transductants. To more closely examine individual KIR expression at the transcriptional level, we carried out a quantitative RT-PCR analysis with probe sets previously validated for amplification of individual KIR (Cooley et al., Blood). We observed a general increase in the mRNA expression levels of both inhibitory and activating KIR in STAT5A 1*6-transduced cells. In order to investigate the mechanism underlying the increased KIR expression observed in the STAT5A 1*6-transductants, we performed an RT-PCR specific for the reverse transcript originating from the proximal promoter of the KIR3DL1 gene. In a model proposed by Anderson et al., reverse transcription from the bidirectional proximal promoter of variegated KIR genes may be responsible for the silencing of the KIR locus through the production of dsRNA, which then induces DNA methylation in a siRNA-dependent manner. Our RT-PCR results from multiple donors clearly show an absence of reverse proximal transcript in cells transduced with the STAT5A 1*6 construct in contrast to high levels of transcript in eGFP controls. These results provide evidence for a role of STAT5A in the induction of KIR expression and support a model of KIR regulation involving intergenic transcription and possibly siRNA-mediated gene silencing. A better understanding of how to manipulate these KIR expression patterns may be of benefit to exploit the potential of NK cell therapy to improve transplant outcomes.

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Duhoux, Francois P., Agnes Jager, Luc Dirix, Manon Thirza Huizing, Guy Heinrich Maria Jerusalem, Peter Vuylsteke, Eveline De Cuypere et al. "Combination of paclitaxel and LAG3-Ig (IMP321), a novel MHC class II agonist, as a first-line chemoimmunotherapy in patients with metastatic breast carcinoma (MBC): Interim results from the run-in phase of a placebo controlled randomized phase II." Journal of Clinical Oncology 35, n.º 15_suppl (20 de mayo de 2017): 1062. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.1062.

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1062 Background: IMP321 is a recombinant soluble LAG3-Ig fusion protein that binds to MHC class II molecules and mediates antigen-presenting cell (APC) activation followed by CD8 T-cell activation. The activation of the dendritic cell network with IMP321 the day after a low dose injection of a single agent chemotherapy may lead to stronger anti-tumor CD8 T-cell responses. We report initial results of the safety run-in of a randomized, placebo-controlled phase IIb trial in patients (pts) with hormone receptor positive (HR+) MBC receiving first-line weekly paclitaxel. Methods: In the safety run-in phase 15 pts with MBC received weekly paclitaxel (80 mg/m2; D1, D8, D15) in a four week cycle in conjunction with either 6 mg (n = 6; cohort 1) or 30 mg (n = 9, cohort 2) IMP321 injections s.c. (D2 and D16) for 6 cycles. Patients without progressive disease could continue with a maintenance phase of 12 additional injections of IMP321 every 4 weeks. Blood samples for pharmacokinetics and immuno-monitoring were taken in cycle 1 and 4 just before and after IMP321 injection. Results: In total 15 pts (median age 53 years) were enrolled between Jan 2016 and Oct 2016. No dose limiting toxicities have been reported. Cytokine release syndrome grade 1 was the only serious adverse event (SAE) related to IMP321 and occurred twice in the same patient. Grade 1 and 2 injection site reactions were the most common related AEs and occurred in 14 pts (93 %). A dose-dependent increase in serum IMP321 concentration was observed among the two dose levels with a Cmaxbetween 4 and 24 hours. Increased number of circulating monocytes, dendritic cells and increased activation were observed at both dose levels of IMP321, supporting the working hypothesis. Conclusions: The 30 mg s.c. IMP321 given every two weeks in combination with weekly paclitaxel is the recommended phase 2 dose which is used in the ongoing randomized placebo controlled phase II part of the study. Clinical trial information: NCT02614833.

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45

Meachem, Sarah J., Saleela M. Ruwanpura, Jessica Ziolkowski, Jacquelyn M. Ague, Michael K. Skinner y Kate L. Loveland. "Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation". Journal of Endocrinology 186, n.º 3 (septiembre de 2005): 429–46. http://dx.doi.org/10.1677/joe.1.06121.

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The critical influence of follicle stimulating hormone (FSH) on male fertility relates both to its impact on Sertoli cell proliferation in perinatal life and to its influence on the synthesis of Sertoli cell-derived products essential for germ cell survival and function in the developing adult testis. The nature and timing of this shift of germ cells to their reliance on specific Sertoli cell-derived products are not defined. Based on existing data, it is apparent that the dominant function of FSH shifts between 9 and 18 day postpartum (dpp) during the first wave of spermatogenesis from driving Sertoli cell proliferation to support germ cells. To enable comprehensive analysis of the impact of acute in vivo FSH suppression on Sertoli and germ cell development, FSH was selectively suppressed in Sprague–Dawley rats by passive immunisation for 2 days and/or 4 days prior to testis collection at 3, 9 and 18 dpp. The 3 dpp samples displayed no measurable changes, while 4 days of FSH suppression decreased Sertoli cell proliferation and numbers in 9 dpp, but not 18 dpp, animals. In contrast, germ cell numbers were unaffected at 9 dpp but decreased at 18 dpp following FSH suppression, with a corresponding increase in germ cell apoptosis measured at 18 dpp. Sixty transcripts were measured as changed at 18 dpp in response to 4 days of FSH suppression, as assessed using Affymetrix microarrays. Some of these are known as Sertoli cell-derived FSH-responsive genes (e.g. StAR, cathepsin L, insulin-like growth factor binding protein-3), while others encode proteins involved in cell cycle and survival regulation (e.g. cyclin D1, scavenger receptor class B 1). These data demonstrate that FSH differentially affects Sertoli and germ cells in an age-dependent manner in vivo, promoting Sertoli cell mitosis at day 9, and supporting germ cell viability at day 18. This model has enabled identification of candidate genes that contribute to the FSH-mediated pathway by which Sertoli cells support germ cells.

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46

Sloand, Elaine M., Katayoun Rezvani, John Barrett, Lori Mainwaring, Roger Kurlander, Emma Gostick, Shakti Ramkissoon et al. "Myelodysplasia with Trisomy 8 Is Associated with a Cytotoxic CD8 T Cell Immune Response to Wilms Tumor-1 Protein (WT1)." Blood 104, n.º 11 (16 de noviembre de 2004): 474. http://dx.doi.org/10.1182/blood.v104.11.474.474.

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Abstract Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated autoimmune process in some patients. Among 133 patients treated at NIH, predominantly with antithymocyte globulin, trisomy 8 as the sole karyotypic abnormality was specifically and significantly associated with favorable hematologic response to immunosuppresssion, as compared to patients with other cytogenetic abnormalities or with a normal karyotype. Trisomy 8 MDS patients show stable increases in the proportion of aneuploid bone marrow cells following immunosupppression (IST). We have reported that all of thirty patients with trisomy 8 had significant CD8 T cell expansion of one or more T cell receptor (TCR) Vβ subfamilies as measured by flow cytometry, and expanded subfamilies showed complimentary determining region 3 (CDR3) skewing by spectratyping. Sorted T cells of the expanded Vβ subfamilies, but none of the remaining subfamilies, specifically inhibit hematopoietic colony formation by trisomy 8 cell progenitors. Colony formation of cytogenetically normal cells from the same individuals was less or not inhibited. Here, we examined protein expression levels and measured the response of cytotoxic T cells to two antigens which we had found to be overexpressed in trisomy 8 CD34 cells by microarray analysis (Chen G et al, Blood In press): cyclin D1 (CD1) and Wilms tumor-1 antigen (WT1). Peripheral blood and bone marrow granulocytes of six trisomy 8 patients with refractory anemia all demonstrated WT1 expression levels 100–1000 x normal (N=38) by real time PCR; the three patients with normal cytogenetics had levels comparable to normal. Immunoblotting confirmed massively increased WT1 peptide in all six trisomy 8 patients tested, with five MDS patients of normal karyotype showing normal levels. Patient and control CD8 cells were cultured for 6 hours with WT1 and CD1-loaded HLA-A2-restricted antigen-presenting cells using three different peptides for WT1 and four for CD1. Cytotoxic CD8 T cells responses were identified by flow cytometric analysis of intracellular interferon-γ. Eight of 17 trisomy 8 patients showed significant responses (>8% CD8 cells activated compared to unstimulated samples; mean =5%) to WT1 but not to CD1 or non-peptide loaded antigen presenting cells. CD8 cell responses to WT1 measured using MHC class I A2-restricted tetramers were concordant with the results by intracellular staining in all 15 patients tested. In contrast, no responses to WT1 were seen in normal controls and five MDS patients lacking cytogenetic abnormalities. While PB of one of the three monosomy 7 patients showed upregulation of WT1, there was no cytotoxic lymphocyte response against the peptide; unlike trisomy 8 CD34 cells those from monosomy 7 patients did not express co-stimulatory molecules, HLA class I and B7.1. These data suggest an autoimmune pathophysiology for the cytopenia of trisomy 8 myelodysplasia with the following scenerio: WT1 is overexpressed by the trisomy 8 clone, resulting in a specific cytotoxic CD8 T cell immune response to WT1. Apoptotis of marrow cells results, either by cross reactivity or as a bystander effect. Improvement in hematopoiesis is seen following IST.

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47

Moratalla, Rosario, Mario Vallejo, Bulent Elibol y Ann M. Graybiel. "D1-class dopamine receptors influence cocaine-induced persistent expression of Fos-related proteins in striatum". NeuroReport 8, n.º 1 (diciembre de 1996): 1–5. http://dx.doi.org/10.1097/00001756-199612200-00001.

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Smith, Porsha L., Fengting Yan, John T. Patton, Lapo Alinari, Vrajesh Karkhanis, Leona Ayers, Natarajan Muthusamy et al. "PRMT5 Transgenic Mice Develop Aggressive Lymphoblastic Lymphomas". Blood 128, n.º 22 (2 de diciembre de 2016): 2936. http://dx.doi.org/10.1182/blood.v128.22.2936.2936.

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Abstract Introduction: Emerging data collected from whole genome and epigenomic studies in solid and blood cancers has pointed toward dysregulation of chromatin remodelers as a unique class of cancer drivers. Next generation sequencing of lymphoma has identified several mutations affecting enzymes that regulate epigenetic control of gene expression. The epigenetic modifier protein arginine methyltransferase 5 (PRMT5) that has been shown to be essential for Epstein-Barr virus-driven B-cell transformation, is overexpressed in several histologic subtypes of B-cell non-Hodgkin's lymphomas (NHL) and is required for the driver activity of oncogenes such as MYC and NOTCH. While these findings suggest that PRMT5 may act as a driver of lymphomagenesis, definitive experiments to address its driver activity have yet to be performed. To address this question, we developed a transgenic mouse model by immunoglobulin m heavy chain enhancer/promoter (Em)-driven PRMT5 over expression in the lymphoid compartment of FVB/N mice. Methods: Eµ-hPRMT5 transgenic mice were created by injecting a vector containing floxed human PRMT5 under the control of the Eµ enhancer/promoter into FVB/N pronuclei that were implanted into pseudo-pregnant FVB/N mice. We obtained 5 founder lines demonstrating the presence of transgene construct by genotype PCR analysis of tail snip DNA. Founder mice were crossed with wild type FVB/N mice to obtain a F1 generation. Mice were followed clinically in standard pathogen-free housing until exhibiting phenotypic features at which time necropsy was performed. Immunophenotypic analysis was performed by flow cytometry, clonality by T cell receptor (TCR) Vb PCR, and pathology by hematoxylin-eosin staining and tissue micro-arrays developed for immunohistochemical staining (IHCS). Statistical significance was determined using a two-tail t-test and survival analysis conducted using Kaplan Meier curves. Results: F1 generation Eµ-hPRMT5 mice significantly overexpressed PRMT5 mRNA in unpurified splenocytes or bone marrow relative to non-transgenic mice (p-value < 0.001). Sorting B (CD19), NK (NK1.1) and T-cell (CD3) mononuclear subsets from splenocytes collected from Eµ-hPRMT5 mice (n=3/group) revealed PRMT5 mRNA to be overexpressed 37-fold (p-value <0.01), 7-fold (p-value <0.01) and 6-fold (p-value <0.05), respectively compared to WT FVB/N mice. All 5 founder lines were found to develop aggressive lymphomas at a statistically significant higher incidence compared to wild type (WT) FVB/N mice (range 10.7-34.6% lymphomagenesis). Gross anatomical characterization of Lymphoma bearing mice demonstrated focal lymphoid tumors, lymphadenopathy, organomegaly (liver, spleen, kidney), and malignant atypical lymphocytosis. Flow cytometric and IHCS studies showed features consistent with immature pre B and T lymphoblastic lymphomas (LL). Pre B LLs were characterized by high surface IgM, TdT and CD19 expression as analyzed by flow cytometry. Pre T LL demonstrated cytoplasmic CD3, TdT, and CD43 expression. We successfully developed a T LL cell line (Tg813) from a pre T-LL tumor isolated from a thymic tumor. Tg813 was clonal (Vb-17), demonstrated complex cytogenetic features, and over-expressed PRMT5, CYCLIN D1, CYCLIN D3, C-MYC transcript and protein, and the PRMT5 histone mark, symmetric (Me2)-H4R3. Inhibition of PRMT5 with a small molecule inhibitor, shRNA or genetic deletion using CRISPR/CAS9 PRMT5-specific gRNA (targeting exon 2) led to reduced proliferation, apoptosis and loss of CYCLIN D1 and C-MYC expression in Tg813. Engraftment of the Tg813 LL into both SCID and immunocompetent FVB/N mice led to disseminated lymphomas 21 days post-engraftment. In vivo induced expression of PRMT5 gRNA in CAS9+ Tg813 tumors is currently underway. Conclusions:The spontaneous lymphomagenesis observed in the Eµ-hPRMT5 transgenic mouse model supports the hypothesis that PRMT5 over-expression can provide sufficient driver activity for this disease. We describe a novel in vivo and in vitro model of PRMT5-driven LL that provides a useful platform for studying the biologic role of this epigenetic modifier in cancer and for development of PRMT5 targeted therapeutic approaches for lymphoma. Disclosures Baiocchi: Essanex: Research Funding.

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Vanover, Kimberly, Steven Glass, Susan Kozauer, Jelena Saillard, Juan Sanchez, Michal Weingart, Sharon Mates, Andrew Satlin y Robert Davis. "30 Lumateperone (ITI-007) for the Treatment of Schizophrenia: Overview of Placebo-Controlled Clinical Trials and an Open-label Safety Switching Study". CNS Spectrums 24, n.º 1 (febrero de 2019): 190–91. http://dx.doi.org/10.1017/s1092852919000245.

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AbstractBackgroundLumateperone is a first-in-class agent in development for schizophrenia that acts synergistically through serotonergic, dopaminergic and glutamatergic systems. Lumateperone is a potent 5-HT2A antagonist, a mesolimbic/mesocortical dopamine phosphoprotein modulator (DPPM) with pre-synaptic partial agonist and post-synaptic antagonist activity at D2, a glutamate GluN2B receptor phosphoprotein modulator with D1-dependent enhancement of both NMDA and AMPA currents via the mTOR protein pathway and an inhibitor of serotonin reuptake.MethodsLumateperone was evaluated in 3 controlled clinical trials to evaluate efficacy in patients with acute schizophrenia. The primary endpoint was change from baseline on the PANSS total score compared to placebo. In Study ‘005, 335 patients were randomized to receive ITI-007 60mg or 120mg , risperidone 4mg (active control) or placebo QAM for 4weeks. In Study ‘301, 450 patients were randomized to receive ITI-007 60mg or 40mg , or placebo QAM for 4weeks. In Study ‘302, 696 patients were randomized to receive ITI-007 60mg or 20mg , risperidone 4mg (active control) or placebo QAM for 6weeks. Also, an open-label safety switching study was conducted in which 302 patients with stable schizophrenia were switched from standard-of-care (SOC) antipsychotics and treated for 6weeks with lumateperone QPM and then switched back to SOC.ResultsIn Studies ‘005 and ‘301, lumateperone (60mg ITI-007) met the primary endpoint with statistically significant superior efficacy over placebo at Day 28. In Study ‘302, neither dose of lumateperone separated from placebo on the primary endpoint; a high placebo response was observed in this study. Across all 3 efficacy trials, lumateperone improved symptoms of schizophrenia with the same trajectory and same magnitude of improvement from baseline to endpoint on the PANSS total score.Lumateperone was well-tolerated with a favorable safety profile in all studies. In the two studies with risperidone included as an active control, lumateperone was statistically significantly better than risperidone on key safety and tolerability measures. In the open-label safety switching study statistically significant improvements from SOC were observed in body weight, cardiometabolic and endocrine parameters worsened again when switched back to SOC medication. In this study, symptoms of schizophrenia generally remained stable or improved. Greater improvements were observed in subgroups of patients with elevated symptomatology (comorbid symptoms of depression and those with prominent negative symptoms).DiscussionLumateperone represents a novel approach to the treatment of schizophrenia with a favorable safety profile in clinical trials. The lack of cardiometabolic and motor safety issues presents a safety profile differentiated from standard-of-care antipsychotic therapy.Funding Acknowledgements: Intra-Cellular Therapies, Inc.

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Kapuria, Vaibhav, Geoffrey Bartholomeusz, Ling-Yuan Kong, William Bornmann, Zhenghong Peng, Ashutosh Pal, David Maxwell, Moshe Talpaz y Nicholas Donato. "A Novel Small-Molecule Approach To Inhibit Jak2 Tyrosine Kinase Signaling." Blood 110, n.º 11 (16 de noviembre de 2007): 1556. http://dx.doi.org/10.1182/blood.v110.11.1556.1556.

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Abstract Jak kinases are non-receptor protein tyrosine kinases that play a pivotal role in cytokine/growth factor signaling through phosphorylation of specific proteins such as the Stat molecules. Activated Stats translocate to the nucleus where they mediate transcription of several target genes involved in cell cycle progression and survival (Bcl-xL, cyclin D1, c-myc, survivin. Many tumors have highly activated Stats that are associated with aberrant Jak2 regulation and recent studies have shown that activating mutations in Jak2 (V617F) play a key role in many myeloproliferative disorders such as polycythemia vera and essential thrombocythemia. Jak inhibitors may be useful in treating many diseases with aberrant Jak2/Stat signaling. The most commonly used inhibitor of Jak2 is the tyrphostin AG490, which inhibits Stat3 activation by preventing its tyrosine phosphorylation. However AG490 has limited in vivo efficacy and must be administered at high concentrations (>50 μM) for anti-tumor effects. We describe here a new class of compounds, termed degrasyns, that block Jak2 mediated activation of Stat3 in intact cells at high nM to low μM concentrations. Degrasyns (WP1130/CP2005) did not directly inhibit Jak2 tyrosine kinase activity but suppressed Stat3 activation by reducing the cytoplasmic levels of Jak2. Degrasyn-mediated Jak2 down-regulation was rapid (complete in 2 hrs) and not inhibited by proteasomal, lysosomal, or serine/threonine protease inhibitors. Biochemical studies and confocal microscopy show that degrasyn induces translocation of Jak2 from the plasma membrane/cytosolic fraction into the cytoskeletal fraction and this altered partitioning of Jak2 was associated with loss of cytokine-mediated Stat activation by degrasyn. Jak2 translocation was associated with tyrosine phosphorylation of specific proteins which complex with Jak2. Lyn kinase in the cytoskeletal fraction was highly activated by degrasyn in multiple hematopoetic tumors (multiple myeloma, mantle cell lymphoma, leukemias). Jak2 translocation and Stat inhibition by degrasyn is mechanistically distinct from “classical” Jak2 inhibitors and is not associated with a translocation of other kinases or cytokine signaling molecules in the Jak2 cascade (IL-6R, gp130, Lyn, Btk, Hck, Akt, PI-3K, Erk, Src, Jak1). Degrasyn induces cytoskeletal translocation of both wild-type and mutant (V617F) Jak2 and was associated with induction of apoptosis in HEL cells expressing the Jak2 V617F mutation. These results suggest that degrasyn suppresses Jak/Stat signaling through a unique mechanism involving translocation of Jak2 into a signal transduction incompetent compartment and may be used to investigate a novel form of Stat suppression. Degrasyn may also have anti-tumor effects on cells with aberrant activation of Jak/Stat signaling.

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