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Identification and distribution of thymosin alpha 1-like immunoreactivity
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 397-402, 1988 0145-305X/88 ~3.00 + .00 Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved
IDENTIFICATION AND DISTRIBUTION OF THYMOSIN ALPHA I-LIKE IMMJNOREACTIVITY Karen K. Oates, Gail T. Ginsburg, 1 Paul H. Naylor, Lewis F. Affronti and Allan L. Goldstein 2
Department Rf.Biology ~eorge Mason unzverszty Fairfax, VA
22030
2 Department of Biochemistry George Washington University Washington, DC 20057
INTRODUCTION The epithelial cells of the thymus gland in mammals secrete a mixture of hormone-like peptides with diverse biologic activities. A well characterized, biologically active thymic extract, called thymosin fraction 5 (TFS) has been shown to contain over 40 chemically distinct peptides. With the purification of several of the peptides found in TF5 the hypoth@sis of a family of peptides with different biological activity profiles has been validated. The most characterized peptide thymosin alpha 1 has been purified to homogeneity, sequenced and is currently under phase II clinical trials as an adjuvent in cancer therapy (i). Recent research has shown that thymosin alpha 1 is derived from a larger thymic precursor molecule called prothymosin alpha (2). Thymosin alpha i has specific immunologic functions. It has been shown to stimulate production of lymphokines such as migration inhibiting factor (MIF), alpha and gamma interferon (3), lymphotoxin and T-cell growth factor (TCGF/IL-2) and IL-2 receptor (4,5). Additional biological activities of thymosin alpha 1 includes the induction of T helper cells and the expression of phenotypic T-cell markers. Pharmacologic studies of the Thymosins along with direct measurements of cyclic nucleotides and calcium have shown a second messenger mechanism for lymphocyte activation and maturation by the thymic hormones. One of the earliest detectable effects of TF5 on thymocytes is an increase in their intracellular cyclic 397
398
THYMOSIN ALPHA 1-LIKE PEPTIDES
Vol. 12, No. 2
GMP levels (6). TF5 causes an influx of calcium into the thymocytes, which is consistent with an increase in c-GMP in a dose dependent manner. TF5 and thymosin alpha 1 have also been shown to increase prostaglandin E2 (PGE2) production in immature lymphocytes (7). This PGE2 synthesis may also be involved in the early activation of T-cells. Recent studies show evidence of mammalian hormones in simple multicellular and unicellular organisms (8). These biochemically active peptides are highly conserved as well as widely distributed. In this paper, we present evidence that thymosin alpha l-like immunoreactive peptides are also found in a broad phylogenetic distribution. Using a protein extraction procedure (Sep-Pak) and specific radioimmunoassay, thymosin alpha l-like immunoreactive peptides were identified in insects, salt-water crabs, fungi, protozoa and bacteria. This data extends earlier observations by Desichaux et al., (9) and Naylor et al., (lO) that thymosin alpha 1 is on an evolutionary scale much more ancient than had been previously thought and indicates that this fundamental element of the cell-mediated immune system exists in unicellular organisms whereas endocrine compartmentalization and the immune system, with its definitive target cells, appear phylogenetically much later.
EXPERIMENTAL Two species of Mycobacteria, M. phlei and M. tuberculosis, were grown in Proskauer-Beck broth. A sample of the spent--medium was set aside for extraction and radioimmunoassay. The organisms were resuspended in physiological saline and repeatedly washed free of all media by gentle mixing and centrifugation before sonication. The fungi, Rhizopus stoloniter was grown to confluence and harvested on thymosin alpha 1-free basal medium before sonication. The insects, Peroplaneta species (milkweed bug) and Oncopelfus fasciatus (cockroach), were grown in darkened barrels with Purina Rodent Chow as the food source. A generous approximation of the amount of Chow consumed in a week is 0.5 gms. Each sample was sonicated and centrifuged as previously described. The salt-water crabs, Callinectes sapidus, were collected in Massawoman Bay, Maryland. The crabs were dissected and samples of viscera tissue and nervous tissue were obtained, which included the ganglions and the optic stalk. Each sample was sonicated and centrifuged as previously described. The protozoan, Tetrahymena pyriformis was grown at 30oc in medium containing glucose, amino acids, minerals, vitamins, and inorganic salts (ll) without serum or other macromolecules, under controlled aeration conditions and harvested at the end of the logarithmic growth phase. The cells were separated from the growth medium by centrifugation at 4°C and stored at -79°C until extracted and lyophilized. The insects, crabs, protozoan, fungi and bacteria were extracted using a Sep-Pak C18 Cartridge (ll, 12). Immunoreactive thymosin alpha 1 fraction was eluted from the Sep-Pak column. This procedure was repeated for each sample. The extractons were frozen and lyophilized overnight in a Flexi-Dry Systems lyophilizer. The lyophilized samples were reconstituted with RIA Buffer. A portion of each reconstituted sample was reserved for a protein determination. The food source for each organism was also extracted and lyophilized in the same manner.
Vol. 12, No. 2
THYMOSIN ALPHA 1-LIKE PEPTIDES
399
RADIOI~UNOASSAY RESULTS Thymosin alpha i concentrations in solution were measured using a modification of the original radioimmunoassay (13) as described previously by McClure et al. (15). Using a Sep-Pak separation and radio immunoassay, we report the presence of thymosin alpha-1 like reactivity in cell extracts from insects, craOs, fungi and protozoans (Table 1). This data supplements findings of thymosin alpha-1 reactivity in fish (9), earthworms, and tunicates (i0). TABLE 1 Thymosin Alpha l-like immunoreactivity Samples Tested Insects: Peroplaneta sp. Oncopelfus fasciatus
pg/ml 10,600 3,354
231 ~71
Crab: Nervous Visceral
7,220 14,920
225 272
6,592
555
23,923
16,000
Myceteae (fungi): Rhizopus stolonifer Protozoan: Tetrahymena pyriformis
pg/mg protein
The unicellular bacteria were grown in a defined medium, the spent medium was assayed (free of cells) to determine if thymosin alpha l-like immunoreactive material was secreted into the media by the organisms during log phase. These results are shown in Table 2.
TABLE 2 Thymosin alpha l-like Immunoreactive Material in Bacterial Culture Supernatant
Bacteria
Thymosin alpha 1 in pg/ml M. phlei M. tuberculosis
2,209 1,121
m
Controls
Bacteria free media
undetected
400
THYMOSIN ALPHA 1-LIKE PEPTIDES
Vol. 12, No, 2
The starting material and food source for each of the organisms was assayed after extraction for thymosin alpha l-like reactivity which could have contributed to the concentration found in the cell homogenates. These results are summarized in Table 5. TABLE 5 Thymosin alpha l-like Immunoreactive Material in the Various Food Sources Starting Food Source
Thymosin alpha l-like immunoactivity pglml
Proskauer-Beck broth (extracted)
Undetected
Fungi growth media (extracted)
Undetected
Purina Rodent chow (extracted)
900 pg/ml or 900 pgthymosin alpha 1 per 500 mg Of chow
Tetrahymena growth media (extracted)
Undetected
DISCUSSION We have identified the presence of thymosin alpha 1 in salt-water crabs, insects, fungi, protozoa and bacteria. The data suggests that thymosin alpha l, which has multiple known immunologic functions, may have originated in unicellular organisms. Although organisms have evolved in complexity and become compartmentalized, thymosin alpha l-like immunoreactive material is present and remains highly conserved. Roth et al, 1982, suggests that many hormones may originate as factors that act upon local tissue and in more complex organisms evolve into hormones or neurotransmitters which remained conserved and phylogenetically old whereas the anatomic structure of the endocrine system has evolved to newer and more complex forms. The thymosin alpha 1 data provides additional support for this hypothesis. Computer matching of thymosin alpha i peptide shows no homology to any of the protein sequence listed in the peptide protein catalog. The occurrence of thymosin alpha l-like immunoreactive material in unicellular organisms can also be explained as the result of a late recombination event (i.e., that these mammalian genes are not native to the unicellular organism but were introduced later by viruses or other vectors of DNA recombination (7). The widespread identification of thymosin alpha l-like material in this diverse group of species suggest this is unlikely. Sequence analysis of the material along with biological function assays may help resolve this issue. With evidence of a widely distributed and highly conserved biochemical compound, it is felt that thymosin alpha 1 may have functions in addition to those of the immune system. Recently, high concentrations of thymosin alpha l-like peptides have been identified in discrete locations of the rat brain and pituitary gland (15). Possibly thymosin alpha 1 functions as an immunoregulatory peptide in the CNS. The presence
Vol. 12, No. 2
THYMOSIN ALPHA I-LIKE PEPTIDES
401
of thymosin alpha 1 in simpler organisms may reflect a phylogenetically older function of this peptide as a neurotransmitter in local cell tissue. This neurotransmitter function may supplement as well as predate thymosin alpha l's known role in the immune system of higher multicellular organisms. These findings suggest that thymosin alpha i did not arise evolutionarily at the time of the thymus gland development but rather has molecular origins, possibly as far back as procaryotic organisms. Thymosin alpha-1 may act as an immune modulator in fish, earthworms, and tunicates, since each have rudimentary immune systems. The reason for thymosin alpha-1 like material in unicellular organisms is not clear. In man the occurance of thymosin alpha-1 in non-thymic sites (ie. spleen and brain) may suggest either endocrine or autocrine roles for this peptide. As a result of this study, tbymosin alpha 1 can now be added to the increasing list of hormones such as insulin (16), catecholamines (17), B-HCG (18), B-endorphin (19) and other biochemically related elements found to exist and possibly originate in unicellular organisms.
REFERENCES
.
Goldstein, A.L., Low, T.L.K. McAdoo, M., McClure, Thurman, J.B., Rossio, J., Lai, C.Y., Chang, D., Wang, S.S., Harvey, C., Ramel, A.H. and Meienhoffer, Jr. Thymosin alpha l: Isolation and Sequence analysis of an Immunologically active Thymic Polypeptide. Proc. Natl. Acad. Sci. USA. 74, 1977, 725-729.
.
Haritos, A.A., Blacker, R., Stin, S., Calderella, J., and Horecker, B.L. The Primary Structure of Rat Thymus Prothymosin Alpha. Proc. Natl. Acad. Sci. USA. 82, 1985, 343:346.
3.
Huang, K., Kind, P.D., Jagoda, E.M., and Goldstein, A.L. Thymosin Treatment Modulates Production of Interferon. O. Interferon Research. l, 1981, 411-420.
4.
Zatz, M.M. Low, T.L.K., and Goldstein, A.L. Role of thymosin and other thymic hormones in T-cell differentiation. In: Biological Responses in Cancer. (Plenm Publishing Corp.) Vol. l, 1982, 219-246.
5.
Sztein, M.B., Sekkate, S.A., and Goldstein, A. L. (1986). Modulation of Interleukin-2 Receptor expression on Normal Human lymphocytes by Thymic Hormones. Proc. Nalt. Acad. Sci. (U.S.A.). 83, 1986, 6107-6111.
6.
Naylor, P.H., and Goldstein, A.L. Thymosin: Cyclic Nucleotides and T-cell Differentiation. Life Science, 25, 1979, 301-309.
7.
Garaci, C.R., Favalli, C., Delgobbo, Y. Garaci, E., and Jaffe, B.M. Thymosin Action mediated by Prostaglandin Release. Science, 220, 1983, I163-1165.
402
THYMOSIN ALPHA I-LIKE PEPTIDES
.
9.
Vol. 12, No. 2
LeRoith, Shiloach, D., Roth, J., and Lesniak, M.A. Evolutionary Origins of Vertebrate hormones: Substances similar mammalian insulins are native to unicellular eukaryotes. Proc. Natl. Acad. Sci. USA. Vol. 77:10, 1908, 6184-6188. Deschaux, P., Peres. G., Naylor, P. and Goldstein A. Evidence for alpha-l-Thymosin in the blood plasma of fish; difference between a fresh-water fish and a salt-water fish. IRCS Ned. Sci. 12, 1984, 977-978.
lO.
Oates, K.K., Affronti, A.F. Naylor, P.H. and Goldstein A. Identification of Thymosin-alpha-1 like material in unicellular organisms. In: Neural and endocrine Peptides and Receptors. Vth International Washington Spring Symposium. May 28-31 (1985). Abstract and Poster.
ll.
Holz, G.G., Erwin, J., Rosenbaum, N. and Aaronson, S. Triparanol Inhibition of Tetrahymena, and Its Prevention by Lipids Arch. Biochem. Biophys. 98, 1962, 312-322.
12.
Naylor, P.H., McClure Y.E., Spangelo B.L., Low T.L.K. and Goldstein, A.L. Immunochemical Studies on thymosin: Radioimmunoassay of Thymosin B4. Immunopharmacology, 7, 1984, 9-16.
13.
Low, T.K.L., McClure, 3.E., Naylor, P.H., Spangelo, B.L., and Goldstein, A.L. Isolation of Thymosin Alpha 1 From Thymosin Fraction Five of Different Species by HPLC. JR of Chromatography 266, 1983, 533-544.
14.
McClure, J.E., Lameris, N., Wara, D.W. and Goldstein, A.L. Immunochemical Studies on Thymosin: Radioimmunoassay of thymosin alpha -1. Jr. of Immunology. 128:1, 1982, 368-375.
15.
Palaszymski, E.W., Moody, T.W., O'Donahue, T.L., and Goldstein, A.L. Thymosin alpha 1 - like peptides: localization and biochemcial characterization in the rat brain and pituitary. Peptides, 4, 1983, 463-467.
16.
Uvnas, B. and Uvnas-Wallenstein, K. "Insulinergic" nerves to the skeletal muscle of the cat? Acta. Physiol. Scand. 103, 1978, 345-348.
17.
Csaba, G. and Lantos, T. Effects of epinephrine on glucose metabolism in tetrahymena. Endokrinologie, 68, 1976, 239-240.
18.
Cohen, H. and Strampp, A. Bacterial Synthesis of a substance similar to human chorionicgonadotropin. Proc. Soc. Expt. Biol. and Med. 152, 1976, 408-410.
19.
LeRoith, D., Liotta, A.S. Rotn J., et al. Corticotropin and B-endorphin-like materials are native to unicellular organisms. Proc. Nat'l Acad. Sci. USA. 79, 1982, 2086-2090.
Received: October, 1986 Accepted: November, 1987
IDENTIFICATION AND DISTRIBUTION OF THYMOSIN ALPHA I-LIKE IMMJNOREACTIVITY Karen K. Oates, Gail T. Ginsburg, 1 Paul H. Naylor, Lewis F. Affronti and Allan L. Goldstein 2
Department Rf.Biology ~eorge Mason unzverszty Fairfax, VA
22030
2 Department of Biochemistry George Washington University Washington, DC 20057
INTRODUCTION The epithelial cells of the thymus gland in mammals secrete a mixture of hormone-like peptides with diverse biologic activities. A well characterized, biologically active thymic extract, called thymosin fraction 5 (TFS) has been shown to contain over 40 chemically distinct peptides. With the purification of several of the peptides found in TF5 the hypoth@sis of a family of peptides with different biological activity profiles has been validated. The most characterized peptide thymosin alpha 1 has been purified to homogeneity, sequenced and is currently under phase II clinical trials as an adjuvent in cancer therapy (i). Recent research has shown that thymosin alpha 1 is derived from a larger thymic precursor molecule called prothymosin alpha (2). Thymosin alpha i has specific immunologic functions. It has been shown to stimulate production of lymphokines such as migration inhibiting factor (MIF), alpha and gamma interferon (3), lymphotoxin and T-cell growth factor (TCGF/IL-2) and IL-2 receptor (4,5). Additional biological activities of thymosin alpha 1 includes the induction of T helper cells and the expression of phenotypic T-cell markers. Pharmacologic studies of the Thymosins along with direct measurements of cyclic nucleotides and calcium have shown a second messenger mechanism for lymphocyte activation and maturation by the thymic hormones. One of the earliest detectable effects of TF5 on thymocytes is an increase in their intracellular cyclic 397
398
THYMOSIN ALPHA 1-LIKE PEPTIDES
Vol. 12, No. 2
GMP levels (6). TF5 causes an influx of calcium into the thymocytes, which is consistent with an increase in c-GMP in a dose dependent manner. TF5 and thymosin alpha 1 have also been shown to increase prostaglandin E2 (PGE2) production in immature lymphocytes (7). This PGE2 synthesis may also be involved in the early activation of T-cells. Recent studies show evidence of mammalian hormones in simple multicellular and unicellular organisms (8). These biochemically active peptides are highly conserved as well as widely distributed. In this paper, we present evidence that thymosin alpha l-like immunoreactive peptides are also found in a broad phylogenetic distribution. Using a protein extraction procedure (Sep-Pak) and specific radioimmunoassay, thymosin alpha l-like immunoreactive peptides were identified in insects, salt-water crabs, fungi, protozoa and bacteria. This data extends earlier observations by Desichaux et al., (9) and Naylor et al., (lO) that thymosin alpha 1 is on an evolutionary scale much more ancient than had been previously thought and indicates that this fundamental element of the cell-mediated immune system exists in unicellular organisms whereas endocrine compartmentalization and the immune system, with its definitive target cells, appear phylogenetically much later.
EXPERIMENTAL Two species of Mycobacteria, M. phlei and M. tuberculosis, were grown in Proskauer-Beck broth. A sample of the spent--medium was set aside for extraction and radioimmunoassay. The organisms were resuspended in physiological saline and repeatedly washed free of all media by gentle mixing and centrifugation before sonication. The fungi, Rhizopus stoloniter was grown to confluence and harvested on thymosin alpha 1-free basal medium before sonication. The insects, Peroplaneta species (milkweed bug) and Oncopelfus fasciatus (cockroach), were grown in darkened barrels with Purina Rodent Chow as the food source. A generous approximation of the amount of Chow consumed in a week is 0.5 gms. Each sample was sonicated and centrifuged as previously described. The salt-water crabs, Callinectes sapidus, were collected in Massawoman Bay, Maryland. The crabs were dissected and samples of viscera tissue and nervous tissue were obtained, which included the ganglions and the optic stalk. Each sample was sonicated and centrifuged as previously described. The protozoan, Tetrahymena pyriformis was grown at 30oc in medium containing glucose, amino acids, minerals, vitamins, and inorganic salts (ll) without serum or other macromolecules, under controlled aeration conditions and harvested at the end of the logarithmic growth phase. The cells were separated from the growth medium by centrifugation at 4°C and stored at -79°C until extracted and lyophilized. The insects, crabs, protozoan, fungi and bacteria were extracted using a Sep-Pak C18 Cartridge (ll, 12). Immunoreactive thymosin alpha 1 fraction was eluted from the Sep-Pak column. This procedure was repeated for each sample. The extractons were frozen and lyophilized overnight in a Flexi-Dry Systems lyophilizer. The lyophilized samples were reconstituted with RIA Buffer. A portion of each reconstituted sample was reserved for a protein determination. The food source for each organism was also extracted and lyophilized in the same manner.
Vol. 12, No. 2
THYMOSIN ALPHA 1-LIKE PEPTIDES
399
RADIOI~UNOASSAY RESULTS Thymosin alpha i concentrations in solution were measured using a modification of the original radioimmunoassay (13) as described previously by McClure et al. (15). Using a Sep-Pak separation and radio immunoassay, we report the presence of thymosin alpha-1 like reactivity in cell extracts from insects, craOs, fungi and protozoans (Table 1). This data supplements findings of thymosin alpha-1 reactivity in fish (9), earthworms, and tunicates (i0). TABLE 1 Thymosin Alpha l-like immunoreactivity Samples Tested Insects: Peroplaneta sp. Oncopelfus fasciatus
pg/ml 10,600 3,354
231 ~71
Crab: Nervous Visceral
7,220 14,920
225 272
6,592
555
23,923
16,000
Myceteae (fungi): Rhizopus stolonifer Protozoan: Tetrahymena pyriformis
pg/mg protein
The unicellular bacteria were grown in a defined medium, the spent medium was assayed (free of cells) to determine if thymosin alpha l-like immunoreactive material was secreted into the media by the organisms during log phase. These results are shown in Table 2.
TABLE 2 Thymosin alpha l-like Immunoreactive Material in Bacterial Culture Supernatant
Bacteria
Thymosin alpha 1 in pg/ml M. phlei M. tuberculosis
2,209 1,121
m
Controls
Bacteria free media
undetected
400
THYMOSIN ALPHA 1-LIKE PEPTIDES
Vol. 12, No, 2
The starting material and food source for each of the organisms was assayed after extraction for thymosin alpha l-like reactivity which could have contributed to the concentration found in the cell homogenates. These results are summarized in Table 5. TABLE 5 Thymosin alpha l-like Immunoreactive Material in the Various Food Sources Starting Food Source
Thymosin alpha l-like immunoactivity pglml
Proskauer-Beck broth (extracted)
Undetected
Fungi growth media (extracted)
Undetected
Purina Rodent chow (extracted)
900 pg/ml or 900 pgthymosin alpha 1 per 500 mg Of chow
Tetrahymena growth media (extracted)
Undetected
DISCUSSION We have identified the presence of thymosin alpha 1 in salt-water crabs, insects, fungi, protozoa and bacteria. The data suggests that thymosin alpha l, which has multiple known immunologic functions, may have originated in unicellular organisms. Although organisms have evolved in complexity and become compartmentalized, thymosin alpha l-like immunoreactive material is present and remains highly conserved. Roth et al, 1982, suggests that many hormones may originate as factors that act upon local tissue and in more complex organisms evolve into hormones or neurotransmitters which remained conserved and phylogenetically old whereas the anatomic structure of the endocrine system has evolved to newer and more complex forms. The thymosin alpha 1 data provides additional support for this hypothesis. Computer matching of thymosin alpha i peptide shows no homology to any of the protein sequence listed in the peptide protein catalog. The occurrence of thymosin alpha l-like immunoreactive material in unicellular organisms can also be explained as the result of a late recombination event (i.e., that these mammalian genes are not native to the unicellular organism but were introduced later by viruses or other vectors of DNA recombination (7). The widespread identification of thymosin alpha l-like material in this diverse group of species suggest this is unlikely. Sequence analysis of the material along with biological function assays may help resolve this issue. With evidence of a widely distributed and highly conserved biochemical compound, it is felt that thymosin alpha 1 may have functions in addition to those of the immune system. Recently, high concentrations of thymosin alpha l-like peptides have been identified in discrete locations of the rat brain and pituitary gland (15). Possibly thymosin alpha 1 functions as an immunoregulatory peptide in the CNS. The presence
Vol. 12, No. 2
THYMOSIN ALPHA I-LIKE PEPTIDES
401
of thymosin alpha 1 in simpler organisms may reflect a phylogenetically older function of this peptide as a neurotransmitter in local cell tissue. This neurotransmitter function may supplement as well as predate thymosin alpha l's known role in the immune system of higher multicellular organisms. These findings suggest that thymosin alpha i did not arise evolutionarily at the time of the thymus gland development but rather has molecular origins, possibly as far back as procaryotic organisms. Thymosin alpha-1 may act as an immune modulator in fish, earthworms, and tunicates, since each have rudimentary immune systems. The reason for thymosin alpha-1 like material in unicellular organisms is not clear. In man the occurance of thymosin alpha-1 in non-thymic sites (ie. spleen and brain) may suggest either endocrine or autocrine roles for this peptide. As a result of this study, tbymosin alpha 1 can now be added to the increasing list of hormones such as insulin (16), catecholamines (17), B-HCG (18), B-endorphin (19) and other biochemically related elements found to exist and possibly originate in unicellular organisms.
REFERENCES
.
Goldstein, A.L., Low, T.L.K. McAdoo, M., McClure, Thurman, J.B., Rossio, J., Lai, C.Y., Chang, D., Wang, S.S., Harvey, C., Ramel, A.H. and Meienhoffer, Jr. Thymosin alpha l: Isolation and Sequence analysis of an Immunologically active Thymic Polypeptide. Proc. Natl. Acad. Sci. USA. 74, 1977, 725-729.
.
Haritos, A.A., Blacker, R., Stin, S., Calderella, J., and Horecker, B.L. The Primary Structure of Rat Thymus Prothymosin Alpha. Proc. Natl. Acad. Sci. USA. 82, 1985, 343:346.
3.
Huang, K., Kind, P.D., Jagoda, E.M., and Goldstein, A.L. Thymosin Treatment Modulates Production of Interferon. O. Interferon Research. l, 1981, 411-420.
4.
Zatz, M.M. Low, T.L.K., and Goldstein, A.L. Role of thymosin and other thymic hormones in T-cell differentiation. In: Biological Responses in Cancer. (Plenm Publishing Corp.) Vol. l, 1982, 219-246.
5.
Sztein, M.B., Sekkate, S.A., and Goldstein, A. L. (1986). Modulation of Interleukin-2 Receptor expression on Normal Human lymphocytes by Thymic Hormones. Proc. Nalt. Acad. Sci. (U.S.A.). 83, 1986, 6107-6111.
6.
Naylor, P.H., and Goldstein, A.L. Thymosin: Cyclic Nucleotides and T-cell Differentiation. Life Science, 25, 1979, 301-309.
7.
Garaci, C.R., Favalli, C., Delgobbo, Y. Garaci, E., and Jaffe, B.M. Thymosin Action mediated by Prostaglandin Release. Science, 220, 1983, I163-1165.
402
THYMOSIN ALPHA I-LIKE PEPTIDES
.
9.
Vol. 12, No. 2
LeRoith, Shiloach, D., Roth, J., and Lesniak, M.A. Evolutionary Origins of Vertebrate hormones: Substances similar mammalian insulins are native to unicellular eukaryotes. Proc. Natl. Acad. Sci. USA. Vol. 77:10, 1908, 6184-6188. Deschaux, P., Peres. G., Naylor, P. and Goldstein A. Evidence for alpha-l-Thymosin in the blood plasma of fish; difference between a fresh-water fish and a salt-water fish. IRCS Ned. Sci. 12, 1984, 977-978.
lO.
Oates, K.K., Affronti, A.F. Naylor, P.H. and Goldstein A. Identification of Thymosin-alpha-1 like material in unicellular organisms. In: Neural and endocrine Peptides and Receptors. Vth International Washington Spring Symposium. May 28-31 (1985). Abstract and Poster.
ll.
Holz, G.G., Erwin, J., Rosenbaum, N. and Aaronson, S. Triparanol Inhibition of Tetrahymena, and Its Prevention by Lipids Arch. Biochem. Biophys. 98, 1962, 312-322.
12.
Naylor, P.H., McClure Y.E., Spangelo B.L., Low T.L.K. and Goldstein, A.L. Immunochemical Studies on thymosin: Radioimmunoassay of Thymosin B4. Immunopharmacology, 7, 1984, 9-16.
13.
Low, T.K.L., McClure, 3.E., Naylor, P.H., Spangelo, B.L., and Goldstein, A.L. Isolation of Thymosin Alpha 1 From Thymosin Fraction Five of Different Species by HPLC. JR of Chromatography 266, 1983, 533-544.
14.
McClure, J.E., Lameris, N., Wara, D.W. and Goldstein, A.L. Immunochemical Studies on Thymosin: Radioimmunoassay of thymosin alpha -1. Jr. of Immunology. 128:1, 1982, 368-375.
15.
Palaszymski, E.W., Moody, T.W., O'Donahue, T.L., and Goldstein, A.L. Thymosin alpha 1 - like peptides: localization and biochemcial characterization in the rat brain and pituitary. Peptides, 4, 1983, 463-467.
16.
Uvnas, B. and Uvnas-Wallenstein, K. "Insulinergic" nerves to the skeletal muscle of the cat? Acta. Physiol. Scand. 103, 1978, 345-348.
17.
Csaba, G. and Lantos, T. Effects of epinephrine on glucose metabolism in tetrahymena. Endokrinologie, 68, 1976, 239-240.
18.
Cohen, H. and Strampp, A. Bacterial Synthesis of a substance similar to human chorionicgonadotropin. Proc. Soc. Expt. Biol. and Med. 152, 1976, 408-410.
19.
LeRoith, D., Liotta, A.S. Rotn J., et al. Corticotropin and B-endorphin-like materials are native to unicellular organisms. Proc. Nat'l Acad. Sci. USA. 79, 1982, 2086-2090.
Received: October, 1986 Accepted: November, 1987