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Item The elongin B ubiquitin homology domain(Elsevier, 1999) Brower, Christopher S.; Shilatifard, Ali; Mather, Timothy; Kamura, Takumi; Takagi, Yuichiro; Haque, Dewan; Treharne, Annemarie; Foundling, Stephen I.; Conaway, Joan Weliky; Conaway, Ronald C.Mammalian Elongin B is a 118-amino acid protein composed of an 84-amino acid amino-terminal ubiquitin-like domain and a 34-amino acid carboxyl-terminal tail. Elongin B is found in cells as a subunit of the heterodimeric Elongin BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling complexes. As part of our effort to understand how the Elongin BC complex regulates the activity of Elongin A, we are characterizing Elongin B functional domains. In this report, we show that the Elongin B ubiquitin-like domain is necessary and sufficient for interaction with Elongin C and for positive regulation of Elongin A transcriptional activity. In addition, by site-directed mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaction with Elongin C. Finally, we observe that both the ubiquitin-like domain and carboxyl-terminal tail are conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that efficiently substitute for mammalian Elongin B in reconstitution of the transcriptionally active Elongin ABC complex, suggesting that the carboxyl-terminal tail performs an additional function not detected in our assays.Item A molecular basis for stabilization of the von hippel-lindau (VHL) tumor suppressor protein by components of the VHL ubiquitin ligase(Elsevier, 2002) Kamura, Takumi; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan WelikyThe multiprotein von Hippel-Lindau (VHL) tumor suppressor (CBCVHL, Cul2-ElonginBC-VHL) and SCF (Skp1-Cul1/Cdc53-F-box protein) complexes are members of structurally related families of E3 ubiquitin ligases that use a heterodimeric module composed of a member of the Cullin protein family and the RING finger protein Rbx1 (ROC1/Hrt1) to activate ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. VHL and F-box proteins function as the substrate recruitment subunits of CBCVHLand SCF complexes, respectively. In cells, many F-box proteins are short lived and are proposed to be ubiquitylated by an autocatalytic mechanism and destroyed by the proteasome following assembly into SCF complexes. In contrast, the VHL protein is stabilized by interaction with the Elongin B and C subunits of CBCVHL in cells. In this report, we have presented direct biochemical evidence that unlike the F-box protein Cdc4, which is ubiquitylated in vitro by Cdc34 in the context of the SCF, the VHL protein is protected from Ubc5-catalyzed ubiquitylation following assembly into the CBCVHL complex. CBCVHL is continuously required for negative regulation of hypoxia-inducible transcription factors in normoxic cells and of SCF complexes, many of which function only transiently during the cell cycle or in response to cellular signals. Our findings provide a molecular basis for the different modes of cellular regulation of VHL and F-box proteins and are consistent with the known roles of CBCVHL.Item Identification of mammalian mediator subunits with similarities to yeast mediator subunits srb5, srb6, med11, and ROX3(Elsevier, 2003) Sato, Shigeo; Tomomori-Sato, Chieri; Banks, Charles A.S.; Sorokina, Irina; Parmely, Tari J.; Kong, Stephanie E.; Jin, Jingji; Cai, Yong; Lane, William S.; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan WelikyThe Mediator is a multiprotein coactivator required for activation of RNA polymerase II transcription by DNA binding transactivators. We recently identified a mammalian homologue of yeast Mediator subunit Med8 and partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis of proteins present in the most highly purified Med8-containing fractions by tandem mass spectrometry led to the identification of many known mammalian Mediator subunits, as well as four potential Mediator subunits exhibiting sequence similarity to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical evidence that these four proteins are bona fide mammalian Mediator subunits. In addition, we identify direct pairwise binding partners of these proteins among the known mammalian Mediator subunits. Taken together, our findings identify a collection of novel mammalian Mediator subunits and shed new light on the underlying architecture of the mammalian Mediator complex.Item A mammalian homolog of drosophila melanogaster transcriptional coactivator intersex is a subunit of the mammalian Mediator Complex(Elsevier, 2003) Sato, Shigeo; Tomomori-Sato, Chieri; Banks, Charles A.S.; Parmely, Tari J.; Sorokina, Irina; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan WelikyThe multiprotein Mediator complex is a coactivator required for transcriptional activation of RNA polymerase II transcribed genes by DNA binding transcription factors. We previously partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits. Here we identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. The AK000411 protein, which we designate hIntersex (human Intersex), shares significant sequence similarity with the Drosophila melanogaster intersex protein, which has functional properties expected of a transcriptional coactivator specific for the Drosophila doublesex transactivator. In addition, we show that hIntersex assembles into a subcomplex with Mediator subunits p28b and TRFP. Taken together, our findings identify a new subunit of the mammalian Mediator and shed new light on the architecture of the mammalian Mediator complex.Item Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure(2003-07-01) Sheikh-Hamad, David; Bick, Roger; Gang-Yi, Wu; Monster Christensen, Birgitte; Razeghi, Peter; Poindexter, Brian; Taegtmeyer, Heinrich; Wamsley, Ann; Padda, Ranjit; Entman, Mark; Nielsen, Soren; Youker, KeithCardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart. Stanniocalcin-1 (STC1) is a homodimeric glycoprotein hormone involved in calcium regulation in bony fish (8), where elevation of serum calcium triggers the release of STC1 from the corpuscles of Stannius (23), organs associated with the kidneys (26). On circulation in the gill and intestine, STC1 inhibits calcium flux from the aquatic environment through these organs, thus maintaining normal calcium concentrations in the blood (15, 24). In mammals, STC1 is expressed in multiple organs, including the heart, skeletal muscle, brain, thyroid, spleen, thymus, parathyroid, lung, kidney, pancreas, small intestine, colon, placenta, ovary, testes, and prostate (4, 5, 22). The wide expression of STC1 suggested that it might function in an autocrine and/or paracrine manner, whereas its localization to the heart and skeletal muscle suggested a role in myocyte function. Through the evolutionary process from fish to mammals, STC1 appears to have maintained its functional role in calcium regulation, because mammalian STC1 appears to be involved in calcium homeostasis in the normal physiology of the gut (16) and in the adaptive response of brain cells to ischemic injury (28). Because cardiomyocyte calcium homeostasis demonstrates a wide range of abnormalities in patients with heart failure (2, 9, 11, 13, 17, 21), we hypothesized that myocardial expression of STC1 may be relevant to calcium homeostasis in the failing heart. Our current data suggest differential expression of STC1 protein in cardiomyocytes and blood vessel walls of failing hearts and are consistent with a potential role for STC1 in cardiomyocyte calcium homeostasis.Item A mammalian mediator subunit that shares properties with saccharomyces cerevisiae mediator subunit CSE2(Elsevier, 2004) Tomomori-Sato, Chieri; Sato, Shigeo; Parmely, Tari J.; Banks, Charles A.S.; Sorokina, Irina; Florens, Laurence; Zybailov, Boris; Washburn, Michael P.; Brower, Christopher S.; Conaway, Ronald C.; Conaway, Joan WelikyThe multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353–10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.Item AT1A-mediated activation of kidney JNK1 and SMAD2 in obstructive uropathy: Preservation of kidney tissue mass using candesartan(American Physiological Society, 2004-09-01) Wamsley-Davis, Ann; Padda, Ranjit; Truong, Luan D.; Tsao, Chun Chui; Zhang, Ping; Sheikh-Hamad, DavidLiterature suggests the involvement of the renin-angiotensin system and transforming growth factor (TGF)-β in the renal injury that follows chronic ureteric obstruction. SMAD proteins and the JNK1 cascade are essential components of TGF-β signaling machinery, and recent data suggest cooperative interaction between JNK1 and SMAD proteins in TGF-β-mediated gene expression. We used a rat model of chronic unilateral ureteric obstruction to study the effects of candesartan, an AT1A-receptor blocker, on tissue morphology and the activities of JNK1 and SMAD2 protein in the kidney. Ureteric obstruction for 28 days leads to interstitial fibrosis, tubule atrophy, and marked activation of SMAD2 and JNK1, without significant change in p38 kinase or ERK. Candesartan treatment, however, attenuated the chronic tubulointerstitial injury in obstructed kidneys and was associated with significant preservation of kidney tissue mass. Furthermore, treatment with candesartan diminished JNK1 activity and downregulated SMAD2 protein and activity in obstructed kidneys. In conclusion, obstructed kidneys showed chronic tubulointerstitial injury, which was associated with JNK1 and SMAD2 activation. The renoprotective effects afforded by AT1A-receptor blockade in obstructive uropathy are consistent with attenuation of JNK1- and SMAD2-mediated renal injury. Obstruction of urine flow complicates many human diseases, such as prostate hypertrophy, abdominopelvic malignancies, and retroperitoneal fibrosis. It is characterized by infiltration of mononuclear inflammatory cells, predominantly macrophages, and is accompanied by activation of cytokines, growth factors, and mediators of apoptosis, the net result of which is the deletion of tubular cells by apoptosis and replacement of renal parenchyma with fibrous tissue. If left untreated, chronic kidney obstruction leads to loss of functional renal parenchyma, and ultimately, the development of kidney failure. Numerous reports implicate the renin-angiotensin system in the pathogenesis of ureteric obstruction, and inhibition of the rennin-angiotensin system, using either angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers, has been shown to attenuate the renal injury that follows ureteric obstruction (19, 23, 37). Activation of the kidney renin-angiotensin system occurs immediately after urteric obstruction, with resultant vasoconstriction and salt and water retention (14). These hemodynamic changes are followed by the activation of a number of cytokines (6, 9, 20, 21, 36, 50), some of which have been directly linked to angiotensin II, such as monocyte chemotactic protein-1 (MCP-1) (20) and transforming growth factor-β (TGF-β) (22), which appears to play a key role in the genesis of the ensuing fibrosis (32). TGF-β signaling is initiated after ligand binding to the TGF-β receptor (15, 33), leading to phosphorylation of SMAD2 or SMAD3 (1, 47, 53). SMAD proteins represent a group of transcription factors involved in gene regulation downstream of the TGF-β receptor. A heteromeric SMAD proteins complex is formed (7), resulting in translocation of the complex to the nucleus (1, 31, 47), where it can interact with transcription factors directly (7), or indirectly (27), to regulate gene expression. It was recently reported that JNK pathway (25, 51), which targets the activation of c-Jun and activated transcription factor-2 (ATF-2) (8, 12, 16, 26), is stimulated rapidly by TGF-β in human fibrosarcoma cells and that JNK activity is essential for TGF-β-induced fibronectin production (17). Furthermore, it was also suggested that SMAD proteins and the JNK1 pathway interact cooperatively in TGF-β signaling (17). These observations assume particular importance, in light of recent data that implicate JNK1 in the development of cell apoptosis after ischemia and UV irradiation in a variety of tissues (2, 3). Apoptosis is a crucial component in the pathogenesis of ureteric obstruction (5, 49). Based on these observations, we hypothesized that JNK1 may be a key contributor to the development of renal injury and fibrosis in obstructive uropathy, and thus renal protection after AT1A-receptor blockade in obstructive uropathy may be related to the downregulation of JNK1 and SMAD proteins. Consistent with this hypothesis, our data suggest activation of SMAD2 and JNK1 signaling in obstructed kidneys in a manner that is AT1A dependent. AT1A- receptor blockade using candesartan downregulates JNK1 and SMAD2, decreases tissue fibrosis, and leads to tissue preservation in obstructed kidneys.Item MEKK3-mediated signaling to p38 kinase and TonE in hypertonically stressed kidney cells(American Physiological Society, 2006-10-01) Padda, Ranjit; Wamsley-Davis, Ann; Gustin, Michael C.; Ross, Rebekah; Yu, Christina; Sheik-Hamad, DavidMitogen-activated protein kinase (MAPK) cascades contain a trio of kinases, MAPK kinase kinase (MKKK) → MAPK kinase (MKK) → MAPK, that mediate a variety of cellular responses to different signals including hypertonicity. The signaling response to hypertonicity is conserved across evolution from yeast to mammals in that it involves activation of p38/SAPK. However, very little is known about which upstream protein kinases mediate activation of p38 by hypertonicity in mammals. The MKKKs, MEKK3 and MEKK4, are upstream regulators of p38 in many cells. To investigate these signaling proteins as potential activators of p38 in the hypertonicity response, we generated stably transfected MDCK cells that express activated versions of MEKK3 or MEKK4, utilized RNA interference to deplete MEKK3, and employed pharmacological inhibition of p38 kinase. MEKK3-transfected cells demonstrated increased betaine transporter (BGT1) mRNA levels and upregulated tonicity enhancer (TonE)-driven luciferase activity under isotonic (basal) and hypertonic conditions compared with empty vector-transfected controls; small-interference RNA-mediated depletion of MEKK3 downregulated the activity of p38 kinase and decreased the expression of BGT1 mRNA. p38 Kinase inhibition abolished the effects of MEKK3 activation on BGT1 induction. In contrast, the response to hypertonicity in MEKK4-kA-transfected cells was similar to that observed in empty vector-transfected controls. Our data are consistent with the existence of an input from MEKK3 →→ p38 kinase →→ TonE. Many organisms, including bacteria, yeast, plants, and animals, adapt to sustained hypertonic stress by the preferential accumulation of compatible organic osmolytes (56). In water-deprived mammals, for example, the extracellular osmolality of the kidney medulla may exceed 4,000 mosmol/kgH2O (38). Roughly one-half of the prevailing medullary interstitial solutes consist of urea, whereas the other half is composed of NaCl (15). Urea easily equilibrates across biological membranes and does not cause water shift between the intracellular and extracellular compartments. However, NaCl remains confined to the extracellular space, owing to the action of the Na-K-ATPase. An increase in the extracellular concentration of NaCl contributes to dehydration of the intracellular milieu (hypertonic stress), and restoration of intracellular volume in hypertonically stressed kidney cells requires the induction of a group of genes that lead to the accumulation of organic osmolytes intracellulary [BGT1 for betaine transporter (54), SMIT for inositol transporter (55), taurine transporter (49), and the aldose reductase enzyme (AR), which catalyze the reduction of d-glucose to the organic solute sorbitol (3)]. The transcriptional “machinery” that drives the expression of these genes [SMIT, BGT1, taurine transporter, and AR] under hypertonic conditions is similar (13, 19, 36, 44) and involves interaction between the cis-element [tonicity enhancer (TonE) (36), also known as osmotic response element (ORE) (13); referred to herein as TonE] and transcription factor TonE binding protein [TonEBP; (37), also known also as ORE binding protein (OREBP) (24) as well as NFAT5 (34); referred to herein as TonEBP]. Activation of TonE-mediated gene expression by hypertonicity is not unique to kidney cells [Madin-Darby canine kidney (MDCK) (36); rabbit kidney papillary epithelial cells (PAP-HT25) (25); mouse inner medullar collecting duct cells (mIMCD) (48)], as it has been shown to occur in neurons (35), human liver-derived HepG2 (41), Chang liver, Cos-7, and HeLa cells (24). Deletion of the TonEBP gene in mice blocks the expression of TonE-mediated gene expression in the kidney medulla almost completely, as evidenced by the diminished expression of the BGT1, SMIT, and AR genes. Remarkably, mice lacking TonEBP show atrophy of the renal medulla, which contains smaller cells and displays increased apoptosis (33). While transcriptional control of hypertonicity-induced genes in mammalian cells is reasonably well characterized, the signaling pathways leading to TonE-mediated gene expression need further delineation. In yeast, the adaptation to osmotic stress is dependent on the p38 MAPK homolog high-osmolarity glycerol 1 (HOG1) (7). Similarly, the induction of TonE-mediated gene expression in mammalian cells is p38 kinase dependent (41, 46) but requires cooperative action of Fyn, the catalytic subunit of PKA and the DNA damage-inducible kinase ATM (reviewed in Ref. 47). While ERK and JNK are induced by hypertonicity, the significance of their activation is not clear, as JNK and ERK do not appear to have an effect on TonE-mediated gene expression (reviewed in Ref. 47). In the current experiments, we sought to determine upstream signaling molecules in the p38 kinase cascade that “drive” the expression of hypertonicity-induced genes (represented by the betaine transporter BGT1) and affect TonE-mediated gene expression in kidney cells. The activity of p38 kinase is dependent on MAPK kinases (MKKs) and their activators, the MAPK kinase kinases (MKKKs; see review in Ref. 52). MEK kinase 1 (MEKK1; 1 of the MKKKs) is linked to JNK activation, whereas MEKK2 is linked to JNK and ERK activation (reviewed in Ref. 27). On the other hand, MEKK3 may activate ERK, JNK, and p38, whereas MEKK4 may activate JNK and p38 kinase (27). Hence, we hypothesized that MEKK3 and/or MEKK4 are likely mediators of p38 kinase activation in kidney cells under hypertonic conditions. Our data are consistent with the existence of MEKK3 → → → p38 kinase input to drive TonE-mediated gene expression.Item Sex differences in μ-opioid receptor expression in the rat midbrain periaqueductal gray are essential for eliciting sex differences in morphine analgesia(Society for Neuroscience, 2008) Loyd, Dayna R.; Wang, Xioaya; Murphy, Anne Z.Opioid-based narcotics are the most widely prescribed therapeutic agent for the alleviation of persistent pain; however, it is becoming increasingly clear that morphine is significantly less potent in women compared with men. Morphine primarily binds to μ-opioid receptors (MORs), and the periaqueductal gray (PAG) contains a dense population of MOR-expressing neurons. Via its descending projections to the rostral ventromedial medulla and the dorsal horn of the spinal cord, the PAG is considered an essential neural substrate for opioid-based analgesia. We hypothesized that MOR expression in the PAG was sexually dimorphic, and that these sex differences contribute to the observed sex differences in morphine potency. Using immunohistochemistry, we report that males had a significantly higher expression of MOR in the ventrolateral PAG compared with cycling females, whereas the lowest level of expression was observed in proestrus females. CFA-induced inflammatory pain produced thermal hyperalgesia in both males and females that was significantly reversed in males with a microinjection of morphine into the ventrolateral PAG; this effect was significantly greater than that observed in proestrus and estrus females. Selective lesions of MOR-expressing neurons in the ventrolateral PAG resulted in a significant reduction in the effects of systemic morphine in males only, and this reduction was positively correlated with the level of MOR expression in the ventrolateral PAG. Together, these results provide a mechanism for sex differences in morphine potency.Item Androgen and estrogen (α) receptor localization on periaqueductal gray neurons projecting to the rostral ventromedial medulla in the male and female rat(Elsevier, 2008-12) Lloyd, Dayna L.; Murphy, Anne Z.The periaqueductal gray (PAG) is involved in many gonadal steroid-sensitive behaviors, including responsiveness to pain. The PAG projects to the rostral ventromedial medulla (RVM), comprising the primary circuit driving pain inhibition. Morphine administered systemically or directly into the PAG produces greater analgesia in male compared to female rats, while manipulation of gonadal hormones alters morphine potency in both sexes. It is unknown if these alterations are due to steroidal actions on PAG neurons projecting to the RVM. The expression of androgen (AR) and estrogen (ERα) receptors in the PAG of female rats and within this descending inhibitory pathway in both sexes is unknown. The present study used immunohistochemical techniques (1) to map the distribution of AR and ERα across the rostrocaudal axis of the PAG; and (2) to determine whether AR and/or ERα were colocalized on PAG neurons projecting to the RVM in male and female rats. AR and ERα immunoreactive neurons (AR-IR, ERα-IR) were densely distributed within the caudal PAG of male rats, with the majority localized in the lateral/ventrolateral PAG. Females had significantly fewer AR-IR neurons, while the quantity of ERα was comparable between the sexes. In both sexes, approximately 25–50% of AR-IR neurons and 20–50% of ERα-IR neurons were retrogradely labeled. This study provides direct evidence of the expression of steroid receptors in the PAG and the descending pathway driving pain inhibition in both male and female rats and may provide a mechanism whereby gonadal steroids modulate pain and morphine potency.Item Reciprocal responsiveness to interleukin-12 and interferon-specifies human CD8 effector versus central memory T-cell fates(Ash Publications, 2009) Ramos, Hilario J.; Davis, Ann M.; Cole, Alexander G.; Schatzle, John D.; Forman, James; Farrar, J. DavidMultiple innate signals regulate the genesis of effector and memory CD8+ T cells. In this study, we demonstrate that the innate cytokines interleukin (IL)–12 and interferon (IFN)–α/β regulate distinct aspects of effector and memory human CD8+ T-cell differentiation. IL-12 exclusively promoted the development of IFN-γ– and tumor necrosis factor (TNF)–α–secreting T effector memory (TEM) cells, whereas IFN-α drove the development of T central memory (TCM) cells. The development of TEM and TCM was linked to cell division. In rapidly dividing cells, IL-12 programmed TEM through induction of the IL-12 receptor β2. In contrast, IFN-α regulated TCM development by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN-α/β receptor-2. The strength of signal delivered through T-cell receptor (TCR) engagement regulated the responsiveness of cells to IL-12 and IFN-α. In the presence of both IL-12 and IFN-α, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCM and TEM. Together, our results support a novel model in which IL-12 and IFN-α act in a nonredundant manner to regulate the colinear generation of both effector and memory cells.Item Ablation of arginylation in the mouse N-end rule pathway: Loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations(Public Library of Science, 2009) Brower, Christopher S.; Varshavsky, AlexanderIn the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway’s ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ,15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1- deficient males are also infertile, owing to defects in Ate12/2 spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.Item Anti-hyperalgesic effects of anti-serotonergic compounds on serotonin- and capsaicin-evoked thermal hyperalgesia in the rat(Elsevier, 2012) Lloyd, Dayna R.; Chen, P.B.; Hargreaves, K.M.The peripheral serotonergic system has been implicated in the modulation of an array of pain states, from migraine to fibromyalgia; however, the mechanism by which serotonin (5HT) induces pain is unclear. Peripherally released 5HT induces thermal hyperalgesia, possibly via modulation of the transient receptor potential V1 (TRPV1) channel, which is gated by various noxious stimuli, including capsaicin. We previously reported in vitro that 5HT increases calcium accumulation in the capsaicin-sensitive population of sensory neurons with a corresponding increase in proinflammatory neuropeptide release, and both are antagonized by pretreatment with 5HT2A and 5HT3 antagonists, as well as the anti-migraine drug sumatriptan. In the current study, we extended these findings in vivo using the rat hind paw thermal assay to test the hypothesis that peripheral 5HT enhances TRPV1-evoked thermal hyperalgesia that can be attenuated with 5HT2A and 5HT3 receptor antagonists, as well as sumatriptan. Thermal hyperalgesia and edema were established by 5HT injection (0.1–10 nmol/100 μl) into the rat hind paw, and the latency to paw withdrawal (PWL) from noxious heat was determined. Rats were then pretreated with either 5HT before capsaicin (3 nmol/10 μl), the 5HT2A receptor antagonist ketanserin or the 5HT3 receptor antagonist granisetron (0.0001–0.1 nmol/100 μl) before 5HT and/or capsaicin, or the 5HT1B/1D receptor agonist sumatriptan (0.01–1 nmol/100 μl) before capsaicin, and PWL was determined. We report that 5HT pretreatment enhances TRPV1-evoked thermal hyperalgesia, which is attenuated with local pretreatment with ketanserin, granisetron, or sumatriptan. We also report that peripheral 5HT induced a similar magnitude of thermal hyperalgesia in male and female rats. Overall, our results provide in vivo evidence supporting an enhancing role of 5HT on TRPV1-evoked thermal hyperalgesia, which can be attenuated by peripheral serotonergic intervention.Item Sex differences in serotonin enhancement of capsaicin-evoked calcitonin gene-related peptide release from human dental pulp(Lippincott, 2012-10) Lloyd, Dayna R.; Sun, Xiaoling X.; Locke, Erin E.; Salas, Margaux M.; Hargreaves, Kenneth M.Serotonin (5HT) enhances transient receptor potential V1 channel (TRPV1)–evoked calcitonin gene-related peptide (CGRP) release from female, but not male, human dental pulp. This enhancement occurs greatest during the luteal phase of the menstrual cycle. Serotonin (5HT) is a pronociceptive mediator in the periphery, and evidence implicates involvement in trigeminal pain processing. However, the mechanism(s) by which 5HT modulates trigeminal nociceptors remains unclear. Trigeminal pain can be evoked by the transient receptor potential V1 channel (TRPV1), which is expressed by nociceptive trigeminal neurons and induces release of proinflammatory calcitonin gene-related peptide (CGRP). In our preclinical models, 5HT evoked thermal hyperalgesia and enhanced calcium influx and CGRP release from the TRPV1 population of trigeminal nociceptors. Whether this occurs in humans is unknown. As dental pulp is densely innervated by trigeminal nociceptors, routine tooth extractions offer a unique opportunity to examine whether 5HT enhances CGRP release from human nociceptors. Pulpal tissue was collected from 140 extracted molar teeth from men and women, and basal release samples were collected before treatment with saline or 5HT 100 μmol/L. CGRP release was then stimulated with the TRPV1 agonist capsaicin 1 μmol/L and quantitated by enzyme immunoassay. Additional samples were collected for Western blots to examine 5HT receptor expression. We report that 5HT induced a significant increase in capsaicin-evoked CGRP release, and that this enhancement was observed only in female dental pulp, with no effect of 5HT on male dental pulp. The greatest amount of CGRP release occurred in dental pulp from women in the luteal phase of the menstrual cycle. These results indicate that 5HT enhances capsaicin-evoked CGRP release from human trigeminal nociceptors in a sexually dimorphic manner providing a mechanistic basis for prevalence of trigeminal pain disorders in women.Item Preemptive perineural bupivacaine attenuates the maintenance of mechanical and cold allodynia in a rat spinal nerve ligation model(BMC Anesthesiology, 2015) Clifford, John L.; Mares, Alberto; Hansen, Jacob; Averitt, Dayna L.Background: Neuropathic pain is evasive to treat once developed, however evidence suggests that local administration of anesthetics near the time of injury reduces the development of neuropathic pain. As abnormal electrical signaling in the damaged nerve contributes to the initiation and maintenance of neuropathic pain, local administration of anesthetics prior to injury may reduce its development. We hypothesized that local treatment with bupivacaine prior to nerve injury in a rat model of spinal nerve ligation (SNL) would attenuate the initiation and/or maintenance of neuropathic pain behaviors. Methods: On the day prior to SNL, baseline measures of pre-injury mechanical, thermal, and/or cold sensitivity were recorded in adult male Sprague–Dawley rats. Immediately prior to SNL or sham treatment, the right L5 nerve was perineurally bathed in either 0.05 mL bupivacaine (0.5 %) or sterile saline (0.9 %) for 30 min. Mechanical allodynia, thermal hyperalgesia, and/or cold allodynia were then examined at 3, 7, 10, 14 and 21 days following SNL. Results: Rats exhibited both mechanical and cold allodynia, but not thermal hyperalgesia, within 3 days and up to 21 days post-SNL. No significant pain behaviors were observed in sham controls. Preemptive local bupivacaine significantly attenuated both mechanical and cold allodynia as early as 10 days following SNL compared to saline controls and were not significantly different from sham controls. Conclusions: These data indicate that local treatment with bupivacaine prior to surgical manipulations that are known to cause nerve damage may protect against the maintenance of chronic neuropathic pain.Item Mitotic activation of a novel histone deacetylase 3-linker histone H1.3 protein complex by protein kinase CK2(Elsevier, 2015-12-09) Bergel, Michael; Hemangi, Patil; Wilks, Carrie; Wold Gonzalez, Rhiannon; Dhanireddy, Sudheer; Conrad-Webb, HeatherHistone deacetylase 3 (HDAC3) and linker histone H1 are involved in both chromatin compaction and the regulation of mitotic progression. However, the mechanisms by which HDAC3 and H1 regulate mitosis as well as the factors controlling HDAC3 and H1 activity during mitosis are unclear. Furthermore, as of now, no association between class I, II or IV (non-sirtuin) HDACs and linker histones has been reported. Here we describe a novel HDAC3-H1.3 complex containing SMRT and N-CoR which accumulated in synchronized HeLa cells in late G2 and mitosis. Nonetheless, the deacetylation activity by the HDAC3 in the complex was evident only in mitotic complexes. HDAC3 associated to H1.3 was highly phosphorylated on S424 only during mitosis. Isolation of inactive HDAC3-H1.3 complexes from late-G2 cells, and phosphorylation of HDAC3 in the complexes at serine 424 by protein kinase CK2 (also known as casein kinase 2), activated the HDAC3 in vitro. In vivo, CK2α and CK2α ′ double knockdown cells demonstrated a significant decrease in HDAC3 S424 phosphorylation during mitosis. HDAC3 and H1.3 colocalized in between the chromosomes, with polar microtubules and spindle poles during metaphase through telophase, and partially colocalized with chromatin during prophase and interphase. H1 was previously reported to associate with microtubules; thus could potentially function in targeting HDAC3 to the microtubules. We suggest, that phosphorylation of HDAC3 in the complex by CK2 during mitosis activates the complex for a dual role: compaction of the mitotic chromatin as well as regulation of the polar microtubules dynamic instability during mitosis.Item Polyphenols extracted from grape powder induce lipogenesis and glucose uptake during differentiation of murine preadipocytes(Experimental Biology and Medicine, 2016) Torabi, Sheida; DiMarco, Nancy M.Assessing the effects of grapes and grape powder extracted polyphenols on lipogenesis and glucose uptake in adipocytes may clarify the risk/benefit of recommending them to individuals with obesity and insulin resistance. We investigated the effect of grape powder extracted polyphenols (GPEP) on intracellular fat accumulation and glucose uptake during differentiation of 3T3-F442A preadipocytes. Total polyphenols were extracted and measured based on gallic acid equivalents (GAE). There were 2167mg of GAE polyphenols in 100 g of grape powder. 3T3-F442A cells were incubated with GPEP, extracted from 125–500 mg GP/mL of media, until day 8 of differentiation when the cells were collected for different assays. AdipoRedTM assay and Oil Red O staining showed that GPEP induced, in a dose-dependent manner, an increase in intracellular triacylglycerol (TAG) content of adipocytes. Concomitantly, grape powder extracted polyphenols increased, in a dose-dependent manner, glucose uptake by 3T3-F442A cells, and there was a strong positive correlation between glucose uptake and the amount of TAG accumulation (r¼0.826, n¼24, P 0.001). No changes in cell viability was measured by Trypan Blue staining, suggesting that these effects were independent of cytotoxicity. Western-blot showed that GPEP upregulated protein level of glucose transport protein 4 (GLUT4), p-PKB/Akt, and p- AMPK in 3T3-F442A adipocytes. LY294002 (10 mmol/L), a phosphatidyl-inositol 3 kinase inhibitor (PI3K), reversed the effects of grape powder extracted polyphenols on cellular lipid content and glucose uptake. Furthermore, quantitative real-time polymerase chain reaction showed that GPEP increased mRNA expression of GLUT4, fatty acid synthase, lipoprotein lipase, adiponectin, and peroxisome proliferator-activated receptor g, while it decreased mRNA expression of leptin and Insig-1. Our results indicate that GPEP may induce adipocyte differentiation via upregulation of GLUT4, PI3K and adipogenic genes. Future research may be directed toward obese individuals with insulin resistance or individuals with diabetes.Item Analyzing N-terminal arginylation through the use of peptide arrays and degradation assays(Elsevier, 2016) Wadas, Brandon; Piatkov, Konstantin I.; Brower, Christopher S.; Varshavsky, AlexanderNα-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (“canonical”) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only α-amino groups of N-terminal residues but also γ-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.Item Degradation of the separase-cleaved REC8, a meiotic cohesin subunit, by the N-end rule pathway(Elsevier, 2016) Liu, Yu-Jiao; Chang, ZeNan; Wadas, Brandon; Brower, Christopher S.; Song, Zhen-Hua; Xu, Zhi-Liang; Shang, Yong-Liang; Liu, Wei-Xiao; Wang, Li-Na; Dong, Wen; Varshavsky, Alexander; Hu, Rong-Gui; Li, WeiThe Ate1 arginyltransferase (R-transferase) is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N-terminal Asp, Glu, or (oxidized) Cys. The resulting N-terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeast Saccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/Mcd1 cohesin subunit generates a C-terminal fragment that bears N-terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N-terminal Glu, a substrate of the Ate1 R-transferase. Here we constructed and used a germ cell-confined Ate1−/− mouse strain to analyze the separase-generated C-terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N-terminal arginylation, and that male Ate1−/− mice are nearly infertile, due to massive apoptotic death of Ate1−/− spermatocytes during the metaphase of meiosis I. These effects of Ate1 ablation are inferred to be caused, at least in part, by the failure to destroy the C-terminal fragment of Rec8 in the absence of N-terminal arginylation.Item Alleviation of drought stress in Arabidopsis thaliana by 17β-estradiol application(Scientific Research Publishing, 2016-10) Upadhyay, Pallavi; Maier, CameliaAnimal steroidal hormones, including estrogens, are being introduced into the agricultural soil and water supply from increased pharmaceutical and farm waste. Considering the current levels of xenoestrogen contamination of plant environments in view of the climate change induced drought conditions, this study was designed to understand the effect of estradiol (ES) application on Arabidopsis drought stress responses. Estradiol treatment (10 nM, 100 nM) of plants subjected to drought stress conditions by withholding water for 7 days resulted in increased tolerance to drought stress reflected in the significantly higher plant survival rates of 74% and 78%, respectively compared to control plants’ survival rates of 36% (no treatment) and 40% (mock treatment). Estradiol application significantly increased the content of glutathione, proline and H2O2 and significantly enhanced the transcription of the stress responsive genes GSTU3, GER5, HSP101, and HSP70b. A high concentration of ES (10 μM) did not protect plants against drought stress and proved to be toxic. These results provide new insight into the effect of ES on drought-stress responses in Arabidopsis with possible practical agricultural applications regarding the effect of environmental estrogens on crop plants. *This article was published with the assistance of the Texas Woman's University Libraries Open Access Fund. The original article can be found at:http://dx.doi.org/10.4236/ajps.2016.714186
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