European Journal of Histochemistry 2013; volume 57:e17

Immunoreactivity for Introduction Correspondence: Sonia Nemolato, Istituto di beta 4 and thymosin beta 10 Anatomia Patologica, Dipartimento di Scienze in the adult rat oro-gastro- Beta are a versatile family of Chirurgiche, PO S. Giovanni di Dio, Università di intestinal tract small expressed in multiple tissues in Cagliari, via Università 60, 09124 Cagliari, Italy. mammals, that show many intracellular and Tel. +39.070.6092370 - Fax: +39.070.657882. extracellular activities.1,2 These peptides are E-mail: [email protected] S. Nemolato,1 J. Ekstrom,2 T. Cabras,3 named thymosins after their first isolation in C. Gerosa,1 D. Fanni,1 E. Di Felice,1 Key words: rats, thymosin beta 4, thymosin beta the calf .3 Fifteen highly homologous 1 3 4 10, immunohistochemistry, gastrointestinal tract. A. Locci, I. Messana, M. Castagnola, beta thymosin variants, containing 40 to 44 G. Faa1 4 amino acid residues, have been described. Contributions: SN, GF, research design and man- 1Istituto di Anatomia Patologica, Among beta thymosins, thymosin beta 4 uscript writing; JE, CM, IM, critical review; TC, 5,6 7 Dipartimento di Scienze Chirurgiche, PO (Tβ4) and thymosin beta 10 (Tβ10) are the CG, DF, EDF, AL, data analysis and research per- 8 forming. S. Giovanni di Dio, Università di Cagliari, most abundant in human cells and rat tissues. During the years, beta thymosins have been Italy; 2Division of Pharmacology, Institute detected inside of cells of different organs,9-14 Conflict of interests: the authors declare no con- of Neuroscience and Physiology, as well as in human blood,15 in human saliva,16 flict of interests. Sahlgrenska Academy, University in tears17 and in wound fluid after abdominal Acknowledgments: this work has been supported of Gothenburg, Sweden; 18 surgery. Many physiological properties and by “Fondazione Banco di Sardegna”. The authors 3Dipartimento di Scienze della Vita e cellular functions are connected to Tβ4: G- would like to thank Mr. Ignazio Ferru for the sec- dell’Ambiente, Università di Cagliari, -sequestering,1 promotion of cell migra- retarial assistance. The authors also gratefully Italy; 4Istituto di Biochimica e Biochimica tion,8 angiogenesis,9,19 stem cell differentia- acknowledge the Sardinia Regional Government Clinica, Facoltà di Medicina, Università tion,11 modulation of cytokines and for the financial support (P.O.R. Sardegna F.S.E. 20 Cattolica di Roma, Italy chemokines. This is also involved in Operational Programme of the Autonomous lesion-induced neuroplasticity through Region of Sardinia, European Social Fund 2007- microglia upregulation and it participates in 2013 - Axis IV Human Resources, Objective l.3, the growth of neuronal processes.21 Therefore, Line of Activity l.3.1 Avviso di chiamata per il finanziamento di Assegni di Ricerca). Abstract the mRNA encoding for Tβ4 is expressed in mouse embryonic stem cells and in mesoder- Received for publication: 21 January 2013. 22 mal-like cells (cardiac and skeletal muscle). Accepted for publication: 5 April 2013. Thymosin beta 4 (Tβ4) and thymosin beta Moreover, recent studies demonstrated a role 10 (T 10) are two members of the -thymosin This work is licensed under a Creative Commons β β of Tβ4 in inducing the expression of the vascu- family, involved in multiple cellular activities lar endothelial growth factor (VEGF) in colon Attribution NonCommercial 3.0 License (CC BY- in different organs in multiple animal species. 19 NC 3.0). cancer cells in experimental models. Tβ4 also Here we report the expression pattern of T 4 β partecipates to the modulation of human ©Copyright S. Nemolato et al., 2013 23 and Tβ10 in rat tissues, in the gut and in colonic immune system probably through Licensee PAGEPress, Italy annexed glands. The two peptide were differ- degranulation of mucosal mast cells.24 European Journal of Histochemistry 2013; 57:e17 ently expressed: Tβ4 was absent in salivary Contrasting results have been published on doi:10.4081/ejh.2013.e17 glands whereas Tβ10 was expressed in parotid the role of Tβ10 in tumour progression. On and in submandibular glands. Tβ4 was mildly one hand, Tβ10 diminishes tumor growth, 25 expressed in the tongue and in the esophagus, angiogenesis and proliferation and Tβ10 over-expression has been related to the where Tβ10 was absent. A similar expression was found in the , and colon increase of apoptosis in human ovarian cancer Materials and Methods cells.25 On the other hand, T 10 has been asso- mucosa. In Tβ4 reactivity was β restricted to the Langerhans islet cells; T 4 ciated to the progression of papillary In order to test Tβ4 and Tβ10 immunoreac- β 26 was also detected in the exocrine cells. Both carcinoma and over-expression of the peptide tivity in animals, 20 male wistar rats were the object of our study, divided into 10 male and 10 peptide were not expressed in cells. When has been observed in non-small cell lung can- cer27 and in pancreatic cancer. 28 The recent female. Tissues were obtained from each ani- the rat expression pattern in rat organs was report by our group of a strong expression for mal including samples from tongue, esopha- compared to reactivity for T 4 and T 10 in β β T 10 in the human salivary glands during gus, stomach, ileum, colon, parotid gland, sub- humans, marked differences were found. Our β development,29 induced us to better analyse mandibular gland, sublingual gland, liver and data clearly indicate a species-specific expres- the protein expression pattern for Tβ10 in the pancreas. All samples were fixed in 10% forma- sion of Tβ4 and Tβ10, characterized by the oro-gastro-intestinal tract in adult rats, in lin, paraffin-embedded and routinely processed. actual unpredictability of the expression of order to show if T 10 is expressed in the gas- Paraffin sections were immunostained with these peptides in different cells and tissues. β trointestinal tract in adulthood, and to com- anti-Tβ4 and anti-Tβ10 antibodies, using the The common high expression of Tβ4 in mast pare the expression of this peptide with that labeled streptavidin-biotin complex system cells, both in humans and in rats, represents reported in the human digestive tract and in (LSAB2, Dako, Cytomation, Carpinteria, CA, one of the few similarities between these two annexed glands.24,30,31 Moreover, rat tissues USA) in a Dako Autostainer (Dako). Briefly, species. were immunostained for Tβ4, with the aim of samples were deparaffinized, rehydrated, and verifying the reciprocal interactions of Tβ4 endogenous peroxidase activity was quenced and Tβ10 in the oro-gastrointestinal tract. (30 min) by 0.3% hydrogen peroxide in

[page 106] [European Journal of Histochemistry 2013; 57:e17] Original Paper

methanol. Slides were then subjected to heat- Table 1. Immunoreactivity for thymosin beta 4 (Tβ4) and thymosin beta 10 (Tβ10) in induced antigen retrieval by steaming rat tissues and humans. unstained sections in a Target Retrieval Organ Tβ4 Rat Tβ10 Rat Tβ4 Human Tβ10 Human Solution (Dako TRS pH 6.1) for 30 min. Slides were then incubated with 10% normal goat Parothyd - +++ ++ serum in phosphate-buffered saline (PBS) for Submandibular gland - +++ ++ 60 min to block non-specific binding, followed Sublingual gland --++ by incubation (60 min at room temperature) Tongue +-+++ with a monoclonal anti-Thymosin Beta 4 anti- Oesofagus --+- body (Bachem-Peninsula Lab, San Carlos, CA, Stomach +++- USA) and with a monoclonal anti-Thymosin Ileum +++- Beta 10, respectively diluted 1:600 and 1:500 in Colon ++++ - the blocking solution. Slides were extensively washed with PBS containing 0.01% Triton X- Pancreas + ++ ++ + 100 and incubated with a secondary reagent Liver --+++ +++

Figure 1. Parotid. a) No immunoreactivity for T 4 is detected in Figure 2. Submandibular glands. a) T 4 is not expressed in the the parotid gland; scattered mast cells are stronglyβ immunoreac- structures of the submandibular glands;β only a fine positivity tive for the peptide; OM 250x. b) A granular and diffuse positiv- could be detectable in the surrounding stroma; mast cells show a ity for T 10 is observed in all the acinar structures; ductal cells strong granular cytoplasmic positivity (yellow arrows); OM 400x. show an βapical and homogeneous reactivity for the peptide; OM b) Coarse granules of T 10 are observed in the acini of the sub- 400x. mandibular gland (yellowβ arrows); ductal cells present a fine cyto- plasmic immunoreactivity for the peptide mainly localized in the lumen (red arrows); OM 400x.

[European Journal of Histochemistry 2013; 57:e17] [page 107] Original Paper

(En Vision kit) according with the manufactur- er instructions (Dako, Glostrup, Denmark). Diaminobenzidine (DAB) was used as chro- mogen. After additional washes, colour was developed using the AEC reagent (Dako), sec- tions were counterstained with Mayer’s hema- toxylin and mounted. Sections of reactive lymph nodes with Tβ4-immunoreactive histio- cytes were utilized as a positive control. As a negative control, the same procedure was applied omitting the primary antibody.

Results

Parotid

Tβ4 immunoreactivity was completely Figure 3. Tongue. A weak immunoreactivity for T 4 is observed only in the superficial absent both in acini and in ducts (Figure 1a). layers of the tongue’s epithelium; a weak immunostainingβ for the peptide was observed in cell membranes of muscle cells (see inset, yellow arrows); OM 250x. Tβ10 was expressed both in acinar serous

Figure 4. Stomach. a) Immunoreactivity for T 4 is expressed in Figure 5. Ileum. a,b) T 4 and T 10 are detected in the surface scattered foveolar cells (see inset, yellow arrows)β and in the lumi- epithelium of enterocytesβ coveringβ the villi of the ileum (see nal surface of the stomach; OM 400x. b) T 10 is mainly localized inset, black arrows); OM 400x. in the luminal border of the foveolar cellsβ of the stomach; OM 250x.

[page 108] [European Journal of Histochemistry 2013; 57:e17] Original Paper cells, showing a granular pattern, as well as in Tongue Ileum ductal cells, in which a homogeneous cytoplas- A mild immunoreactivity for Tβ4 was detect- Tβ4 was maily expressed in the cytoplasm of mic staining was detected (Figure 1b). ed in the superficial layers of the stratified enterocytes covering ileal villi (Figure 5a). A Submandibular gland epithelium. Moreover, a weak immunostaining similar pattern characterized immunoreactivi- Fine T 4-immunoreactive granules were for the peptide was observed in muscle cells, ty for Tβ10 (Figure 5b). β mainly localized at the cell membrane (Figure observed in the periglandular stroma and in 3). No reactivity for T 10 was found. scattered mast cells, in the absence of any β Colon reactivity inside the salivary gland cells Tβ4 was maily expressed at the apical pole Esophagus of enterocytes, and in fine granular deposits (Figure 2a). A strong reactivity for Tβ10 was observed in acinar serous cells, appearing as Scattered Tβ4-immunoreactive granules inside the intestinal lumen (Figure 6a), paral- were detected inside the esophageal lumen. leling the expression pattern observed for coarse granules and in mucous cells appearing No reactivity for T 10 was observed. T 10 (Figure 6b). as fine granules. The ducts show a homoge- β β neous cytoplasmic staining and intraluminal Stomach Pancreas granular deposits (Figure 2b). Immunoreactivity for Tβ4 was restricted to Tβ4 immunoreactivity was restricted to Sublingual glands scattered foveolar cells and to intraluminal Langherans islets, in the absence of any sig- granular deposits (Figure 4a). A similar expres- nificant immunostaining in the esocrine pan- No reactivity was detected for Tβ4 and for Tβ10. sion pattern was observed for Tβ10 (Figure 4b). creas (Figure 7a). On the contrary, Tβ10 was

Figure 6. Colon. a,b) A fine granular positivity for T 4 is Figure 7. Pancreas. a) T 4 is diffusely immunoexpressed in the observed in the brush border of the enterocytes in the surfaceβ islets of Langherans; no βimmunoreactivity is observed in the rest epithelium of the colon and in the intestinal lumen; T 10 paral- of pancreatic parenchima; OM 400x. b) Immunoreactivity for lels the immunoreactivity of T 4; OM 400x. β T 10 is localized both in islets of Langherans and in the acini β (seeβ inset); the latest show a fine granular positivity in the absence of ductal reactivity; OM 400x.

[European Journal of Histochemistry 2013; 57:e17] [page 109] Original Paper detected both in the exocrine and in the in human tissues, significant differences were mosins: intracellular and extracellular endocrine pancreas. Endocrine cells of the evidenced, supporting the hypothesis that activities of a versatile actin binding pro- Langherans islets showed a strong cytoplasmic beta-thymosin expression in cells and tissues tein family. Cell Motil Cytoskeleton reactivity, whereas in acinar cells Tβ10 was is species-specific. The most striking differ- 2009;66:839-51. mainly detected in granular deposits. No ences were found in liver specimens, charac- 3. Klein JJ, Goldstein AL, White A. immunostaining was found in ductal cells nor terized by the complete absence of both thy- Enhancement of in vivo incorporation of inside the tubular lumen (Figure 7b). mosins in rat, contrasting with the strong and labeled precursors into DNA and total pro- diffuse reactivity for Tβ4 and Tβ10 previously tein of mouse lymph nodes after adminis- Liver reported in humans.34 However, which is the tration of thymic extracts. Proc Natl Acad role of T 4 and T 10 in adult rat tissues? The Sci USA 1965;53:812-7. No reactivity for Tβ4 and Tβ10 was observed β β in the liver samples. Data regarding detailed mechanisms of the action of Tβ4 and 4. Hannappel E, Davoust S, Horecker BL. immunoreactivity for both thymosins in the Tβ10 in different mammalian cells and tissues Isolation of peptides from calf thymus. different rat organs are summarized in Table 1. are not fully understood, as well as the similar- Biochem Biophys Res Commun. 1982;104: ities and differences between these isoforms.35 266-71. Previous studies evidenced that activities of 5. Low TL, Goldstein AL. Chemical character- these two beta-thymosins are paradoxically ization of thymosin β4. J Biol Chem 1982; Discussion different, Tβ4 promoting cell migration and 257:1000-6. angiogenesis, and Tβ10 inhibiting angiogene- 6. Hannappel E, Xu GJ, Morgan J, Hemstead sis.35 Opposing effects on angiogenesis are The role of T 4 and T 10, the beta-thy- J, Horecker BL. Thymosin β4: a ubiquitous β β likely to be mediated via T 4 stimulation and mosins expressed virtually in all mammalian β peptide in rat and mouse tissues. Proc Natl T 10 inhibition of VEGF production.36 tissues and cells, has not been completely clar- β Acad Sci USA 1982;79:2172-5. ified yet. Previous studies on their expression Moreover, in contrast to Tβ4, Tβ10 has been 7. Ericksson-Viitanen S, Ruggieri S, Natalini in the rat central nervous system evidenced shown to be a negative regulator of tumor P, Horecker BL. Thymosin Beta 10, a new 37 that temporal and cellular patterns of their development and progression. In short, Tβ4 analogue of thymosin beta 4 in mam- expression are different, suggesting that each might promote cell survival by blocking apopto- malian tissues. Arch Biochem Biophys 38 beta-thymosin could play a specific physiologi- sis, whereas Tβ10 might exert a proapoptotic 1983;225:407-13. 39 cal function during development and in adult- activity, by accelerating apoptotic cell death. 8. Yu FX, Lin SC, Morrison-Bogoard M, hood.32 Our study confirms the existence of According with these data, we may speculate Atkinson MA, Yin HL. Thymosin beta 10 marked differences in the distribution of T 4 that the complex and pleiotropic expression of and thymosin beta 4 both actin sequester- β T 4 and T 10 here reported in different tis- ing proteins. J Biol Chem 1993;268:502-9. and Tβ10 in the oro-gastro-intestinal tract of β β the adult rat. The most striking differences sues and cell types might reflect different 9. Philp D, Goldstein AL, Kleinman HK. were found in the parotid and submandibular direct and indirect effects on the actin Thymosin Tβ4 promotes angiogenesis, glands, in which T 4 was absent whereas cytoskeleton, as well as modulation of signal- wound healing, and hair follicle develop- β ing pathways that impact on different cellular ment. Mech Ageing Dev 2004;125:113-5. immunostaining for Tβ10 was strong and dif- fuse. On the contrary, no reactivity for both functions. In particular, over-expression of Tβ4 10. Bock-Marquette I, Saxena A, White MD, beta-thymosins was detected in sublingual could be related to prevention of apoptosis by Dimaio JM, Srivastava D. Thymosin Tβ4 40 glands. These differences in beta-thymosin blocking early apoptotic signals, whereas activates integrin-linked kinase and pro- expression between different salivary glands overexpression of Tβ10 might be related to its motes cardiac cell migration, survival and confirm that each beta-thymosin probably pro-apoptotic activity and to a down-regulation cardiac repair. Nature 2004;432:466-72. plays a specific role in each salivary gland, of cell growth35 and of angiogenesis.36 11. Smart N, Risebro CA, Melville AA, Moses K, irrespectively of their common embryogenesis. Finally, this study, one of the few in which Schwartz RJ, Chien KR, et al. Thymosin the immunohistochemical expression pattern T 4 induces adult epicardial progenitor No significant difference in Tβ4 and Tβ10 β expression was present in the gastrointestinal of Tβ4 and Tβ10 has been paralleled in the mobilization and neovascularization. tract: the absence of reactivity for both beta- same tissues, evidenced that detailed mecha- Nature 2007;445:177-82. thymosin in the oesofagus contrasts with the nisms of the action of beta-thymosins in differ- 12. Hall AK. Differential expression of thy- presence of both peptides in the enterocytes of ent cells and tissues are not fully understood, mosin genes in human tumors and in the remaining gastrointestinal tract, confirm- and show that our lack of knowledge is partic- developing human . Int J Cancer ing that a patchy distribution of these peptides ularly evident regarding mature adult tissues, 1991;48:672-7. should be expected, even in different parts of the vast majority of studies on the role of beta- 13. Sun W, Kim H. Neurotrophic roles of the the same system. thymosins having been carried out in fetal or beta-thymosins in the development and The peculiar pattern for beta thymosins tumoral tissues. Further studies exploring the regeneration of the nervous system. Ann detected in pancreas deserves some consider- molecular events that are associated with Tβ4 NY Acad Sci 2007;1112:210-8. and T 10 overexpression or down-regulation ations: Tβ10 was strongly expressed both in β 14. Nemolato S, Cabras T, Fanari MU, Cau F, the exocrine and in the endocrine cells, where- are required, in order to give a solution to the Fanni D, Gerosa C, et al. Immunoreactivity thymosin enigma. as Tβ4 reactivity was restricted to the of Thymosin beta 4 in human foetal and Langerhans islet cells. These findings taken adult genitourinary tract. Eur J Histochem together clearly indicate the presence of a 2010;54:e43. complex modulation in the expression of beta- 15. Naylor PH, McClure JE, Spangelo BL, Low thymosins inside the same organ, each thy- References TL, Goldstein AL. Immunochemical studies mosin playing different functions in different on thymosin: radioimmunoassay of thy- 33 cell types, confirming the β-thymosin enigma. 1. Hannappel E. β-Thymosins. Ann NY Acad mosin beta 4. Immunopharmacology 1984; When data obtained in rat tissues were com- Sci 2007;1112:21-37. 7:9-16. pared with immunoreactivity for Tβ4 and Tβ10 2. Mannherz HG, Hannappel E. The β-thy- 16. Inzitari R, Cabras T, Pisano E, Fanali C,

[page 110] [European Journal of Histochemistry 2013; 57:e17] Original Paper

Manconi B, Scarano E, et al. HPLC-ESI-MS modulate human colonic lamina propria Fanni D, Manconi B, et al. Different thy- analysis of oral human fluid reveals that lymphocyte function. Immunopharmaco- mosin Beta 4 immunoreactivity in foetal gingival crevicular fluidi s the main source logy 1990;20:89-96. and adult gastrointestinal tract. PLoS One of oral thymosin Tβ4 and β10. J Sep Sci 24. Nemolato S, Cabras T, Fanari MU, Cau F, 2010;5:e9111. 2009;32:57-63. Fraschini M, Manconi B, et al. Thymosin 32 Gomez-Marquez J, Anadon R. The beta-thy- 17. Badamchian M, Fagarasan MO, Danner beta 4 expression in normal skin, colon mosins, small actin-binding peptides wide- RL, Suffredini AF, Damavandy H, Goldstein mucosa and in tumor infiltrating mast ly expressed in the developing and adult AL. Thymosin beta(4) reduces lethality cells. Eur J Histochem 2010;54:e3. cerebellum. Cerebellum 2002;1:95-102. and down regulates inflammatory media- 25. Kim YC, Kim BG, Lee JH. Thymosin 10 β 33. Sun HQ, Yin HL. The β-thymosin enigma. tors in endotoxin-induced septic shock. Int expression driven by the human TERT pro- Ann NY Acad Sci 2007;1112:45-55. Immunopharmacol 2003;3:1225-33. moter induces ovarian cancer-specific 34. Nemolato S, Van Eyken P, Cabras T, Cau F, 18. Bodendorf S, Born G, Hannappel E. apoptosis through ROS production. PLoS Fanari MU, Locci A, et al. Expression pat- Determination of thymosin beta4 and pro- One 2012;7:e35399. tern of thymosin beta 4 in the adult human tein in human wound fluid after abdomi- 26. Fehér LZ, Pocsay G, Krenács L, Zvara A, liver. Eur J Histochem 2011;55:e25. nal surgery. Ann NY Acad Sci 2007;1112: Bagdi E, Pocsay R, et al. Amplification of 35. Sribenja S, Li M, Wongkham S, Wongkham 418-24. thymosin beta 10 and AKAP13 genes in C, Yao Q, Chen C. Advances in thymosin 19. Jo JO, Kim SR, Bae MK, Kang YJ, Ock MS, metastatic and aggressive papillary thy- beta 10 research: differential expression, Kleinman HK, et al. Thymosin 4 induces roid carcinomas. Pathol Oncol Res 2012; β molecular mechanisms, and clinical impli- the expression of vascular endothelial 18:449-58. cations in cancer and other conditions. growth factor (VEGF) in a hypoxia- 27. Lee SM, Na YK, Hong HS, Jang EJ, Yoon Cancer Invest 2009; 27:1016-22. inducible factor (HIF)-1-dependent man- GS, Park JY, et al. Hypomethylation of the 36. Lee SH, Son MJ, Oh SH, Rho SB, Park K, ner. Biochim Biophys Acta 2010;1803: thymosin (10) gene is not associated β Kim YJ, et al. Thymosin beta 10 inhibits 1244-51. with its overexpression in non-small cell angiogenesis and tumor growth by inter- 20. Crockford D, Turjman N, Allan C, Angel J. lung cancer. Mol Cells 2011;32:343-8. Thymosin beta4: structure, function, and 28. Li M, Zhang Y, Zhai Q, Feurino LW, Fisher fering with Ras function. Cancer Res biological properties supporting current WE, Chen C, et al. Thymosin beta-10 is 2005;65:137-48. and future clinical applications. Ann NY aberrantly expressed in pancreatic cancer 37. Freeman KW, Banyard J. Beta-thymosin in Acad Sci 2010;1194:179-89. and induces JNK activation. Cancer Invest cancer: implications for the clinic. Future 21. Paulussen M, Landuyt B, Schoofs L, Luyten 2009; 27:251-6. Oncol 2009;5:755-8. W, Arckens L. Thymosin beta 4 mRNA and 29. Fanni D, Gerosa C, Nemolato S, Locci A, 38. Sosne G, Qui P, Goldstein AL, Wheater M. peptide expression in phagocytic cells of Marinelli V, Cabras T, et al. Thymosin beta Biological activities of thymosin beta4 different mouse tissues. Peptides 2009;30: 10 expression in developing human salivary defined by actives sites in short peptides 1822-32. glands. Early Hum Dev 2011;87:779-83. sequences. FASEB J 2010;24:2144-51. 22. Gómez-Márquez J, Franco del Amo F, 30. Nemolato S, Messana I, Cabras T, Manconi 39. Hall AK. Thymosin beta-10 accelerates Carpintero P, Anadón R. High levels of B, Inzitari R, Fanali C, et al. Thymosin apoptosis. Cell Mol Biol Res 1995;41:167- mouse thymosin beta4 mRNA in differen- beta(4) and beta(10) levels in pre-term 80. tiating P19 embryonic cells and during newborn oral cavity and foetal salivary 40. Choi SY, Kim DK, Eun B, Kim K, Sun W, development of cardiovascular tissues. glands evidence a switch of secretion dur- Kim H. Anti-apoptotic function of thy- Biochim Biophys Acta 1996;1306:187-93. ing foetal development. PLoS One 2009; mosin-beta in developing chick spinal 23. Elitsur Y, Mutchnick MG, Sakr WA, Luk GD. 4:e5109. motoneurons. Biochem Biophys Res Thymosin alpha 1 and thymosin beta 4 31. Nemolato S, Cabras T, Cau F, Fanari MU, Commun 2006;346:872-8.

[European Journal of Histochemistry 2013; 57:e17] [page 111]