Nitric oxide (NO) regulates placental blood flow and actively participates in trophoblast invasion and placental development. Asymmetric dimethylarginine (ADMA) can inhibit NO synthase, which generates NO. ADMA has been associated with uterine artery flow disturbances such as preeclampsia. Substantial experimental evidence has reliably supported the hypothesis that an adverse in utero environment plays a role in postnatal physiological and pathophysiological programming. Growing evidence suggests that the placental nitrergic system is involved in epigenetic fetal programming. In this review, we discuss the roles of NO and ADMA in normal and compromised pregnancies as well as the link between placental insufficiency and epigenetic fetal programming.
References
[1]
Myatt, L. Placental adaptive responses and fetal programming. J. Physiol 2006, 572, 25–30.
[2]
Reynolds, L.P.; Borowicz, P.P.; Caton, J.S.; Vonnahme, K.A.; Luther, J.S.; Buchanan, D.S.; Hafez, S.A.; Grazul-Bilska, A.T.; Redmer, D.A. Uteroplacental vascular development and placental function: an update. Int. J. Dev. Biol 2010, 54, 355–366.
[3]
Krause, B.J.; Hanson, M.A.; Casanello, P. Role of nitric oxide in placental vascular development and function. Placenta 2011, 32, 797–805.
[4]
Godfrey, M. The role of the placenta in fetal programming—A review. Placenta 2002, 23, S20–S27.
[5]
Reynolds, L.P.; Caton, J.S.; Redmer, D.A.; Grazul-Bilska, A.T.; Vonnahme, K.A.; Borowicz, P.P.; Luther, J.S.; Wallace, J.M.; Wu, G.; Spencer, T.E. Evidence for altered placental blood flow and vascularity in compromised pregnancies. J. Physiol 2006, 572, 51–58.
[6]
Vallance, P.; Leiper, J. Cardiovascular biology of the asymmetric dimethylarginine:dimethylarginine dimethylaminohydrolase pathway. Arterioscler. Thromb. Vasc. Biol 2004, 24, 1023–1030.
[7]
Teerlink, T.; Luo, Z.; Palm, F.; Wilcox, C.S. Cellular ADMA: Regulation and action. Pharmacol. Res 2009, 60, 448–460.
[8]
Sibal, L.; Agarwal, S.C.; Home, P.D.; Boger, R.H. The role of asymmetric dimethylarginine (ADMA) in endothelial dysfunction and cardiovascular disease. Curr. Cardiol. Rev 2010, 6, 82–90.
[9]
Fickling, S.A.; Williams, D.; Vallance, P.; Nussey, S.S.; Whitley, G.S. Plasma of endogenous inhibitor of nitric oxide synthesis in normal pregnancy and pre-eclampsia. Lancet 1993, 342, 242–243.
[10]
Holden, D.P.; Fickling, S.A.; Whitley, G.S.; Nussey, S.S. Plasma concentrations of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia. Am. J. Obstet. Gynecol 1998, 178, 551–556.
[11]
Jansson, T.; Powell, T.L. Role of the placenta in fetal programming: Underlying mechanisms and potential interventional approaches. Clin. Sci. (Lond.) 2007, 113, 1–13.
[12]
Reynolds, L.P.; Caton, J.S. Role of the pre- and post-natal environment in developmental programming of health and productivity. Mol. Cell Endocrinol 2012, 354, 54–59.
[13]
Rugg-Gunn, P.J. Epigenetic features of the mouse trophoblast. Reprod. Biomed. Online 2012, 25, 21–30.
[14]
Illi, B.; Colussi, C.; Grasselli, A.; Farsetti, A.; Capogrossi, M.C.; Gaetano, C. NO sparks off chromatin: Tales of a multifaceted epigenetic regulator. Pharmacol. Ther 2009, 123, 344–352.
[15]
Nott, A.; Riccio, A. Nitric oxide-mediated epigenetic mechanisms in developing neurons. Cell Cycle 2009, 8, 725–730.
[16]
Tain, Y.L.; Huang, L.T. Asymmetric dimethylarginine: Clinical applications in pediatric medicine. J. Formos. Med. Assoc 2011, 110, 70–77.
Lucke, T.; Kanzelmeyer, N.; Kemper, M.J.; Tsikas, D.; Das, A.M. Developmental changes in the l-arginine/nitric oxide pathway from infancy to adulthood: Plasma asymmetric dimethylarginine levels decrease with age. Clin. Chem. Lab. Med 2007, 45, 1525–1530.
[19]
Horowitz, J.D.; Heresztyn, T. An overview of plasma concentrations of asymmetric dimethylarginine (ADMA) in health and disease and in clinical studies: Methodological considerations. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci 2007, 851, 42–50.
[20]
Rossmanith, W.G.; Hoffmeister, U.; Wolfahrt, S.; Kleine, B.; McLean, M.; Jacobs, R.A.; Grossman, A.B. Expression and functional analysis of endothelial nitric oxide synthase (eNOS) in human placenta. Mol. Hum. Reprod 1999, 5, 487–494.
[21]
Ariel, I.; Hochberg, A.; Shochina, M. Endothelial nitric oxide synthase immunoreactivity in early gestation and in trophoblastic disease. J. Clin. Pathol 1998, 51, 427–431.
[22]
Suzuki, T.; Ikeda, Y.; Yoshikawa, H.; Tanaka, K.; Morita, H.; Yamamoto, M.; Takizawa, T. Gestational changes in production of NO and expression of NOS mRNA isoforms in the rat placenta. J. Vet. Med. Sci 2009, 71, 495–498.
[23]
Suzuki, T.; Mori, C.; Yoshikawa, H.; Miyazaki, Y.; Kansaku, N.; Tanaka, K.; Morita, H.; Takizawa, T. Changes in nitric oxide production levels and expression of nitric oxide synthase isoforms in the rat uterus during pregnancy. Biosci. Biotechnol. Biochem 2009, 73, 2163–2166.
[24]
Purcell, T.L.; Given, R.; Chwalisz, K.; Garfield, R.E. Nitric oxide synthase distribution during implantation in the mouse. Mol. Hum. Reprod 1999, 5, 467–475.
[25]
Seligman, S.P.; Buyon, J.P.; Clancy, R.M.; Young, B.K.; Abramson, S.B. The role of nitric oxide in the pathogenesis of preeclampsia. Am. J. Obstet. Gynecol 1994, 171, 944–948.
[26]
Xiao, D.; Pearce, W.; Zhang, L. Pregnancy enhances endothelium-dependent relaxation of ovine uterine artery: Role of NO and intracellular Ca2+. Am. J. Physiol. Heart Circ. Physiol 2001, 281, H183–H190.
[27]
Kaufmann, P.; Mayhew, T.M.; Charnock-Jones, D.S. Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy. Placenta 2004, 25, 114–126.
[28]
Demir, R.; Kayisli, U.A.; Cayli, S.; Huppertz, B. Sequential steps during vasculogenesis and angiogenesis in the very early human placenta. Placenta 2006, 27, 535–539.
[29]
Shizukuda, Y.; Tang, S.; Yokota, R.; Ware, J.A. Vascular endothelial growth factor-induced endothelial cell migration and proliferation depend on a nitric oxide-mediated decrease in protein kinase C activity. Circ. Res 1999, 85, 247–256.
[30]
Nath, A.K.; Enciso, J.; Kuniyasu, M.; Hao, X.Y.; Madri, J.A.; Pinter, E. Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy. Development 2004, 131, 2485–2496.
[31]
Frank, S.; Stallmeyer, B.; Kampfer, H.; Schaffner, C.; Pfeilschifter, J. Differential regulation of vascular endothelial growth factor and its receptor fms-like-tyrosine kinase is mediated by nitric oxide in rat renal mesangial cells. Biochem. J 1999, 338, 367–374.
[32]
Han, R.N.; Stewart, D.J. Defective lung vascular development in endothelial nitric oxide synthasedeficient mice. Trends Cardiovasc. Med 2006, 16, 29–34.
Kon, K.; Fujii, S.; Kosaka, H.; Fujiwara, T. Nitric oxide synthase inhibition by N(G)-nitro-l-arginine methyl ester retards vascular sprouting in angiogenesis. Microvasc. Res 2003, 65, 2–8.
[35]
Dulak, A.; Jozkowicz, J. Regulation of vascular endothelial growth factor synthesis by nitric oxide: Facts and controversies. Antioxid. Redox. Signal 2003, 5, 123–132.
[36]
Vida, G.; Sulyok, E.; Ertl, T.; Martens-Lobenhoffer, J.; Bode-B？ger, S.M. Birth by cesarean section is associated with elevated neonatal plasma levels of dimethylarginines. Pediatr. Int 2012, 54, 476–479.
Myatt, L. Review: Reactive oxygen and nitrogen species and functional adaptation of the placenta. Placenta 2010, 24, S66–S69.
[39]
Vonnahme, K.A.; Wilson, M.E.; Li, Y.; Rupnow, H.L.; Phernetton, T.M.; Ford, S.P.; Magness, R.R. Circulating levels of nitric oxide and vascular endothelial growth factor throughout ovine pregnancy. J. Physiol 2005, 565, 101–109.
[40]
Reynolds, L.P.; Redmer, D.A. Angiogenesis in the placenta. Biol. Reprod 2001, 64, 1033–1040.
[41]
Bird, I.M.; Zhang, L.; Magness, R.R. Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function. Am. J. Physiol. Regul. Integr. Comp. Physiol 2003, 284, R245–R258.
[42]
Khullar, S.; Greenwood, S.L.; McCord, N.; Glazier, J.D.; Ayuk, P.T. Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: Implications for fetal growth. Free Radic. Biol. Med 2004, 36, 271–277.
[43]
Kossenjans, W.; Eis, A.; Sahay, R.; Brockman, D.; Myatt, L. Role of peroxynitrite in altered fetal-placental vascular reactivity in diabetes or preeclampsia. Am. J. Physiol. Heart Circ. Physiol 2000, 278, H1311–H1139.
[44]
Roberts, V.H.; Webster, R.P.; Brockman, D.E.; Pitzer, B.A.; Myatt, L. Post-translational modifications of the P2X(4) purinergic receptor subtype in the human placenta are altered in preeclampsia. Placenta 2007, 28, 270–277.
[45]
Webster, R.P.; Macha, S.; Brockman, D.; Myatt, L. Peroxynitrite treatment in vitro disables catalytic activity of recombinant p38 MAPK. Proteomics 2006, 6, 4838–4844.
[46]
Savvidou, M.D.; Hingorani, A.D.; Tsikas, D.; Frolich, J.C.; Vallance, P.; Nicolaides, K.H. Endothelial dysfunction and raised plasma concentrations of asymmetric dimethylarginine in pregnant women who subsequently develop pre-eclampsia. Lancet 2003, 361, 1511–1517.
[47]
McCarthy, A.L.; Woolfson, R.G.; Evans, B.J.; Davies, D.R.; Raju, S.K.; Poston, L. Functional characteristics of small placental arteries. Am. J. Obstet. Gynecol 1994, 170, 945–951.
[48]
Chang, J.K.; Roman, C.; Heymann, M.A. Effect of endothelium-derived relaxing factor inhibition on the umbilical-placental circulation in fetal lambs in utero. Am. J. Obstet. Gynecol 1992, 166, 727–734.
[49]
Suzuki, T.; Nagamatsu, C.; Kushima, T.; Miyakoshi, R.; Tanaka, K.; Morita, H.; Sakaue, M.; Takizawa, T. Apoptosis caused by an inhibitor of NO production in the decidua of rat from mid-gestation. Exp. Biol. Med. (Maywood) 2010, 235, 455–462.
[50]
Lowe, D.T. Nitric oxide dysfunction in the pathophysiology of preeclampsia. Nitric Oxide 2000, 4, 441–458.
[51]
Baylis, C.; Beinder, E.; Suto, T.; August, P. Recent insights into the roles of nitric oxide and renin-angiotensin in the pathophysiology of preeclamptic pregnancy. Semin. Nephrol 1998, 18, 208–230.
[52]
Noris, M.; Todeschini, M.; Cassis, P.; Pasta, F.; Cappellini, A.; Bonazzola, S.; Macconi, D.; Maucci, R.; Porrati, F.; Benigni, A.; et al. l-arginine depletion in preeclampsia orients nitric oxide synthase toward oxidant species. Hypertension 2004, 43, 614–622.
[53]
Myatt, L.; Eis, A.L.; Brockman, D.E.; Kossenjans, W.; Greer, I.A.; Lyall, F. Endothelial nitric oxide in placental villous tissue from normal, pre-eclamptic and intrauterine restricted pregnancies. Hum. Reprod 1997, 12, 714–718.
[54]
Leiper, J.; MacAllister, R.; Whitley, G.; Santa Maria, J.; Chubb, A.; Charles, I.; Vallance, P. Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology to microbial arginine deiminases. Biochem. J 1999, 343, 209–214.
[55]
Anderssohn, M.; Maass, L.M.; Diemert, A.; Lüneburg, N.; Atzler, D.; Hecher, K.; B？ger, R.H. Severely decreased activity of placental dimethylarginine dimethylaminohydrolase in pre-eclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol 2012, 161, 152–156.
[56]
Akbar, F.; Heinonen, S.; Pirskanen, M.; Uimari, P.; Tuomainen, T.P.; Salonen, J.T. Haplotypic association of DDAH1 with susceptibility to pre-eclampsia. Mol. Hum. Reprod 2005, 11, 73–77.
[57]
Kim, Y.J.; Park, B.H.; Park, H.; Jung, S.C.; Pang, M.G.; Ryu, H.M.; Lee, K.S.; Eom, S.M.; Park, H.Y. No association of the genetic polymorphisms of endothelial nitric oxide synthase, dimethylarginine dimethylaminohydrolase, and vascular endothelial growth factor with preeclampsia in Korean populations. Twin Res. Hum. Genet 2008, 11, 77–83.
[58]
Sobrevia, L.; Mann, G.E. Dysfunction of the endothelial nitric oxide signalling pathway in diabetes and hyperglycaemia. Exp. Physiol 1997, 82, 423–452.
[59]
Kucuk, M.; Doymaz, F. Placental weight and placental weight-to-birth weight ratio are increased in diet- and exercise-treated gestational diabetes mellitus subjects but not in subjects with one abnormal value on 100-g oral glucose tolerance test. J. Diabetes Complicat 2009, 23, 25–31.
[60]
Daskalakis, G.; Marinopoulos, S.; Krielesi, V.; Papapanagiotou, A.; Papantoniou, N.; Mesogitis, S.; Antsaklis, A. Placental pathology in women with gestational diabetes. Acta. Obstet. Gynecol. Scand 2008, 87, 403–407.
Figueroa, R.; Martinez, E.; Fayngersh, R.P.; Tejani, N.; Mohazzab, H.K.M.; Wolin, M.S. Alterations in relaxation to lactate and H2O2 in human placental vessels from gestational diabetic pregnancies. Am. J. Physiol 2000, 278, H706–H713.
[63]
Farías, M.; Puebla, C.; Westermeier, F.; Jo, M.J.; Pastor-Anglada, M.; Casanello, P.; Sobrevia, L. Nitric oxide reduces SLC29A1 promoter activity and adenosine transport involving transcription factor complex hCHOP-C/EBPα in human umbilical vein endothelial cells from gestational diabetes. Cardiovas. Res 2010, 86, 45–54.
[64]
Vásquez, G.; Sanhueza, F.; Vásquez, R.; González, M.; San Martín, R.; Casanello, P.; Sobrevia, L. Role of adenosine transport in gestational diabetes-induced l-arginine transport and nitric oxide synthesis in human umbilical vein endothelium. J. Physiol 2004, 560, 111–122.
[65]
Stojanovic, N.; Lewandowski, K.; Salata, I.; Bienkiewicz, M.; Tuck, S.; Prelevic, G.; Press, M. Serum levels of matrix metalloproteinases MMP-2 and MMP-9 and their inhibitors in women with glucose intolerance in pregnancy and normal controls. Gynecol. Endocrinol 2010, 26, 201–207.
[66]
Novaro, V.; Colman-Lerner, A.; Ortega, F.V.; Jawerbaum, A.; Paz, D.; Lo Nostro, F.; Pustovrh, C.; Gimeno, M.F.; González, E. Regulation of metalloproteinases by nitric oxide in human trophoblast cells in culture. Reprod. Fertil. Dev 2001, 13, 411–420.
[67]
Akturk, M.; Altinova, A.; Mert, I.; Dincel, A.; Sargin, A.; Buyukkagnici, U.; Arslan, M.; Danisman, N. Asymmetric dimethylarginine concentrations are elevated in women with gestational diabetes. Endocrine 2010, 38, 134–141.
[68]
Wu, G.; Pond, W.G.; Flynn, S.P.; Ott, T.L.; Bazer, F.W. Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation. J. Nutr 1998, 128, 2395–2402.
[69]
Kwon, H.; Ford, S.P.; Bazer, F.W.; Spencer, T.E.; Nthanielsz, P.W.; Nijland, M.J.; Hess, B.W.; Wu, G. Maternal undernutrition reduces concentrations of amino acids and polyamines in ovine fetal plasma and fluids. Biol. Reprod 2004, 71, 901–908.
[70]
Ozaki, T.; Hawkins, P.; Nishina, H.; Steyn, C.; Poston, L.; Hanson, M.A. Effects of undernutrition in early pregnancy on systemic small artery function in late-gestation fetal sheep. Am. J. Obstet. Gynecol 2000, 183, 1301–1307.
[71]
Redmer, D.A.; Wallace, J.M.; Reynolds, L.P. Effect of nutrient intake during pregnancy on fetal and placental growth and vascular development. Domest. Anim. Endocrinol 2004, 27, 199–217.
[72]
Hood, J.D.; Meininger, C.J.; Ziche, M.; Granger, H.J. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am. J. Physiol 1998, 274, H1054–H1058.
[73]
Babaei, S.; Teichert-Kuliszewska, K.; Monge, J.C.; Mohamed, F.; Bendeck, M.P.; Stewart, D.J. Role of nitric oxide in the angiogenic response in vitro to basic fibroblast growth factor. Circ. Res 1998, 18, 1007–1015.
[74]
Rutland, C.S.; Latunde-Dada, A.O.; Thorpe, A.; Plant, R.; Langley-Evans, S.; Leach, L. Effect of gestational nutrition on vascular integrity in the murine placenta. Placenta 2007, 28, 734–742.
[75]
Tain, Y.L.; Hsieh, C.S.; Lin, I.C.; Chen, C.C.; Sheen, J.M.; Huang, L.T. Effects of maternal l-citrulline supplementation on renal function and blood pressure in offspring exposed to maternal caloric restriction: The impact of nitric oxide pathway. Nitric Oxide 2010, 23, 34–41.
[76]
Polyakov, A.; Cohen, S.; Baum, M.; Trickey, D.; Jolley, D.; Wallace, E.M. Patterns of antenatal corticosteroid prescribing 1998–2004. Aust. N. Z. J. Obstet. Gynaecol 2007, 47, 42–45.
[77]
Huang, L.T. The link between perinatal glucocorticoids exposure and psychiatric disorders. Pediatr. Res 2011, 69, 19R–25R.
[78]
Lui, C.C.; Wang, J.Y.; Tain, Y.L.; Chen, Y.C.; Chang, K.A.; Lai, M.C.; Huang, L.T. Prenatal stress in rat causes long-term spatial memory deficit and hippocampus MRI abnormality: Differential effects of postweaning enriched environment. Neurochem. Int 2011, 58, 434–441.
[79]
Hewitt, D.P.; Mark, P.J.; Waddell, B.J. Glucocorticoids prevent the normal increase in placental vascular endothelial growth factor expression and placental vascularity during late pregnancy in the rat. Endocrinology 2006, 147, 5566–5574.
[80]
Parenti, A.; Morbidelli, L.; Cui, X.L.; Douglas, J.G.; Hood, J.D.; Granger, H.J.; Ledda, F.; Ziche, M. Nitric oxide is an upstream signal of vascular endothelial growth factor-induced extracellular signal-regulated kinase1/2 activation in postcapillary endothelium. J. Biol. Chem 1998, 273, 4220–4226.
[81]
Fukumura, D.; Gohongi, T.; Kadambi, A.; Izumi, Y.; Ang, J.; Yun, C.O.; Buerk, D.G.; Huang, P.L.; Jain, R.K. Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability. Proc. Natl. Acad. Sci. USA 2001, 98, 2604–2609.
[82]
Bussolati, B.; Dunk, C.; Grohman, M.; Kontos, C.D.; Mason, J.; Ahmed, A. Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am. J. Pathol 2001, 159, 993–1008.
[83]
Kroll, J.; Waltenberger, J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR). Biochem. Biophys. Res. Commun 1998, 252, 743–746.
[84]
Ribatti, D.; Nico, B.; Crivellato, E. Morphological and molecular aspects of physiological vascular morphogenesis. Angiogenesis 2009, 12, 101–111.
[85]
R？ssig, L.; Li, H.; Fisslthaler, B.; Urbich, C.; Fleming, I.; F？rstermann, U.; Zeiher, A.M.; Dimmeler, S. Inhibitors of histone deacetylation downregulate the expression of endothelial nitric oxide synthase and compromise endothelial cell function in vasorelaxation and angiogenesis. Circ. Res 2002, 91, 837–844.
[86]
Zeng, L.; Xiao, Q.; Margariti, A.; Zhang, Z.; Zampetaki, A.; Patel, S.; Capogrossi, M.C.; Hu, Y.; Xu, Q. HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells. J. Cell Biol 2006, 174, 1059–1069.
[87]
Kuhn, P.; Xu, W. Protein arginine methyltransferases: Nuclear receptor coregulators and beyond. Prog. Mol. Biol. Transl. Sci 2009, 87, 299–342.
[88]
Chen, W.; Cao, M.; Yang, Y.; Nagahama, Y.; Zhao, H. Expression pattern of prmt5 in adult fish and embryos of medaka, Oryzias latipes. Fish Physiol. Biochem 2009, 35, 325–332.
[89]
Tain, Y.L.; Huang, L.T.; Lin, I.C.; Lau, Y.T.; Lin, C.Y. Melatonin prevents hypertension and increased asymmetric dimethylarginine in young spontaneous hypertensive rats. J. Pineal Res 2010, 49, 390–398.
Tain, Y.L.; Freshour, G.; Dikalova, A.; Griendling, K.; Baylis, C. Vitamin E reduces glomerulosclerosis, restores renal neuronal NOS, and suppresses oxidative stress in the 5/6 nephrectomized rat. Am. J. Physiol. Renal Physiol 2007, 292, F1404–F1410.
Germain, A.M.; Valdés, G.; Romanik, M.C.; Reyes, M.S. Evidence supporting a beneficial role for long-term l-arginine supplementation in high-risk pregnancies. Hypertension 2004, 44, e1.
[94]
Cynober, L.; Moinard, C.; De Bandt, J.P. The 2009 ESPEN Sir David Cuthbertson. Citrulline: A new major signaling molecule or just another player in the pharmaconutrition game? Clin. Nutr 2010, 29, 545–551.
[95]
Prieto, C.P.; Krause, B.J.; Quezada, C.; San Martin, R.; Sobrevia, L.; Casanello, P. Hypoxia-reduced nitric oxide synthase activity is partially explained by higher arginase-2 activity and cellular redistribution in human umbilical vein endothelium. Placenta 2011, 32, 932–940.
[96]
Miller, S.L.; Wallace, E.M.; Walker, D.W. Antioxidant Therapies: A potential role in perinatal medicine. Neuroendocrinology 2012, 96, 13–23.
[97]
Dennery, P.A. Oxidative stress in development: nature or nurture? Free Radic. Biol. Med 2010, 49, 1147–1151.
