The identification of prothymosin α-like material in vertebrate lymphoid organs by a radioimmunoassay for the N-terminal decapeptide

Yialouris, P.P.; Evangelatos, Gregory P.; Soteriadis-Vlahos, C.; Heimer, Edgar P.; Félix, Arthur; Tsitsiloni, O.E.; Haritos, A.A.

doi:10.1016/0022-1759(88)90207-4

Cited by 24 publications

(23 citation statements)

References 27 publications

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“…Characterization of antibodies G and Y raised against ProTa/KLH in ProTa-ELISAs and dotblots has been recently reported and commented on by our group (Costopoulou et al 1998), showed moderate titers for the ProTa molecule, which are, however, at least comparable to those thus far reported in the literature (Yialouris et al 1988;Loidi et al 1997). On the con- trary, the antibody Y raised against ProTa[1-28]/KLH, ProTa[87-109]/KLH, and ProTa[101-109]/KLH showed a very low specific signal during titer determination.…”

Section: Discussionsupporting

confidence: 79%

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Klimentzou

Drougou

Fehrenbacher

et al. 2008

J Histochem Cytochem.

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Prothymosin α (ProTα) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProTα immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProTα or the synthetic fragments ProTα[1-28], ProTα[87-109], and ProTα[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProTα[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProTa in tumorigenesis.

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Section: Discussionsupporting

confidence: 79%

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Klimentzou

Drougou

Fehrenbacher

et al. 2008

J Histochem Cytochem.

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“…In the HPLC gel filtration of a thymic extract ( Fig. 2A) higher crossreactivity with thymosin al than with prothymosin a (on an equimolar basis) (14,18), it is assumed that the thymosin al-like lower molecular weight immunocrossreactive material is representing less than 20% of the whole immunocrossreactivity. No significant change in the gel filtration profile was noticed in old thymus samples (results not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The radioimmunoassay for the N terminus of prothymosin a was carried out as described by Yialouris et al (14). The radioimmunoassays for the C terminus of prothymosin a and the N terminus of parathymosin a were developed by generating rabbit antisera against the synthetic peptides human prothymosin-a-(90-109) and human parathymosin-a-(1-30), both with an N-terminal extension of Cys-Aca (Aca, aminocaproic acid) and coupled to keyhole limpet hemocyanin (KLH).…”

Section: Methodsmentioning

confidence: 99%

Expression of alpha-thymosins in human tissues in normal and abnormal growth.

Tsitsiloni

Stiakakis

Koutselinis

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Radioimmunoassays specific for the N and C termini of human prothymosin a and the N terminus of human parathymosin a were employed for the measurement of the levels of a-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two a-thymosins was observed in thymus (prothymosin a-rich) and liver (parathymosin a-rich). A decline in the levels of both a-thymosins was found with age, with prothymosin a in thymus showing the sharpest change (15-to 30-fold). The levels of both a-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin a and parathymosin a was 17.9-and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin a and parathymosin a. These results suggest an in vivo relationship of the expression of a-thymosins with the human tissue cell proliferation activity.Human prothymosin a (1) and parathymosin a (2) are 109 and 101 residues long and partly homologous, particularly at their N termini. They are ubiquitous in mammalian tissues (1, 3).Existing literature for the two a-thymosins points toward an extracellular role of immunologic nature and an intracellular role related to cell growth. In the immunologic direction, thymosin al (the N-terminal fragment 1-28 of prothymosin a) and/or prothymosin a have been reported to increase the expression of interleukin 2 (IL-2) and IL-2 high-affinity receptors in normal human lymphocytes (4,5), cooperate with a,B-interferon in boosting natural killer (NK) activity in immunosuppressed and tumor-bearing mice (6), and restore deficient autologous and allogeneic mixed lymphocyte responses in lymphocytes from patients with clinically active multiple sclerosis (7) and systemic lupus erythematosus (8). In cell growth, increased levels of mRNA for prothymosin a have been observed in lymphocytes stimulated with mitogens and serum reconstitution (1, 9), in lymphocytes of leukemic patients (10), and in regenerating liver (9). The activation of the protooncogene MYC was also reported to lead to rapid increase in transcription of the prothymosin a gene (11), and antisense oligomers for prothymosin a were found to inhibit myeloma cell division (12). J.G. and Ch.M.) from breast and (by D.P.) from colon cancer patients. Tissues were stored at -45°C. Extraction was carried out according to Tsitsiloni et al. (13). MATERIALS AND METHODSThe radioimmunoassay for the N terminus of prothymosin a was carried out as described by Yialouris et al. (14). The radioimmunoassays for the C terminus of prothymosin a and the N terminus of parathymosin a were developed by generating rabbit antisera against the synthetic peptides human prothymosin-a-(90-109) and human parathymosin-a-(1-30), both with an N-terminal extension of Cys-Aca (Aca, aminocaproic acid) and coupled to keyhole limpet hemocyanin (KLH). The synthetic peptides human prothymosin-a-(90-109) and human parathy...

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“…Peptide fragment thymosin t4 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and the analog [Tyr12]-thymosin t4 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were prepared by solid phase synthesis on an automatic Synostat-peptide synthesizer (Biotronik, Maintal, Germany) using 4-(hydroxymethyl)-phenoxymethyl-copoly-(styrene-1% divinyl-benzene)-resin preloaded with FMOC-Ser (tBu)-OH (for each synthesis 0.33 g = 0.26 mmol). The synthetic analog [TyrlZ]-thymosin /34 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) was radioiodinated by the chloramine T method as previously reported for tracers prepared for e-thymosin radioimmunoassays 15'16. All amino acids were coupled twice in DMF in threefold excess in the presence of 0.35 g BOP/0.11 g HOBt and 1.6 ml diisopropylethylamine (25% in DMF).…”

Section: Methodsmentioning

confidence: 99%

Evidence for the extranuclear localization of thymosins in thymus

et al. 1992

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A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.

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The identification of prothymosin α-like material in vertebrate lymphoid organs by a radioimmunoassay for the N-terminal decapeptide

Cited by 24 publications

References 27 publications

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin α, a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Expression of alpha-thymosins in human tissues in normal and abnormal growth.

Evidence for the extranuclear localization of thymosins in thymus

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