thymosin-beta(4) and thymosin-beta(9)

Using a partially purified preparation, thymosin fraction 5, we have documented that thymosin can correct many of the immunological deficiencies resulting from the lack of thymosin function in animal models and in humans. Ongoing studies indicate that there is a family of biologically active peptides within fraction 5 that act on T-cell subpopulations to maintain normal immunological reactivity. Several of these peptides have been purified to homogeneity. Two peptides, thymosin alpha 1 and beta 4, have been sequenced and chemically synthesized. Thymosin fraction 5 has been used in most clinical trials reported to date, including children with immunodeficiency disease and patients with autoimmune diseases and cancer. Most recently, the National Cancer Institute has initiated a number of Phase I and Phase II clinical trials with thymosin fraction 5 and synthetic alpha 1 as part of a new Biological Response Modifier Program. Preliminary results from two of these studies look encouraging.

Topics: Amino Acid Sequence; Animals; Autoimmune Diseases; Carcinoma, Small Cell; Cattle; Clinical Trials as Topic; Isoelectric Focusing; Lung Neoplasms; Macrophage Migration-Inhibitory Factors; Molecular Weight; Peptides; T-Lymphocytes; T-Lymphocytes, Regulatory; Terminology as Topic; Thymalfasin; Thymosin

Using a partially purified preparation, thymosin fraction 5, we have documented that thymosin can correct many of the immunological deficiencies resulting from the lack of thymosin function in animal models and in humans. Ongoing studies indicate that there is a family of biologically active peptides within fraction 5 that act on T-cell subpopulations to maintain normal immunological reactivity. Several of these peptides have been purified to homogeneity. Two peptides, thymosin alpha 1 and beta 4, have been sequenced and chemically synthesized. Thymosin fraction 5 has been used in most clinical trials reported to date, including children with immunodeficiency disease and patients with autoimmune diseases and cancer. Most recently, the National Cancer Institute has initiated a number of Phase I and Phase II clinical trials with thymosin fraction 5 and synthetic alpha 1 as part of a new Biological Response Modifier Program. Preliminary results from two of these studies look encouraging.

Topics: Amino Acid Sequence; Animals; Autoimmune Diseases; Carcinoma, Small Cell; Cattle; Clinical Trials as Topic; Isoelectric Focusing; Lung Neoplasms; Macrophage Migration-Inhibitory Factors; Molecular Weight; Peptides; T-Lymphocytes; T-Lymphocytes, Regulatory; Terminology as Topic; Thymalfasin; Thymosin

We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.. A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.. We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.. In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Drug Storage; Freeze Drying; Humans; Mass Spectrometry; Protein Precursors; Proteomics; Thymosin; Time Factors; Ubiquitin

Thymosin proteins, originally isolated from fractionation of thymus tissue, represent a class of compounds that we now know are present in numerous other tissues, are unrelated to each other in a genetic sense, and appear to have different functions within the cell. Thymosin α1 (generic drug name thymalfasin; trade name Zadaxin) is derived from a precursor molecule, prothymosin, by proteolytic cleavage, and stimulates the immune system. Although the peptide is natively unstructured in aqueous solution, the helical structure has been observed in the presence of trifluoroethanol or unilamellar vesicles, and these studies are consistent with the presence of a dynamic helical structure whose sides are not completely hydrophilic or hydrophobic. This helical structure may occur in circulation when the peptide comes into contact with membranes. In this report, we discuss the current knowledge of the thymosin α1 structure and similar properties of thymosin β4 and thymosin β9, in different environments.

Topics: Animals; Humans; Magnetic Resonance Spectroscopy; Thymalfasin; Thymosin

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.

Topics: Allantois; Animals; Chick Embryo; Chorion; Models, Animal; Peptide Fragments; Protein Precursors; Thymalfasin; Thymosin

Recombinant plant (birch) profilin was analyzed for its ability to promote actin polymerization from the actin:thymosin beta4 and beta9 complex. Depending on the nature of the divalent cation, recombinant plant (birch) profilin exhibited two different modes of interaction with actin, like mammalian profilin. In the presence of magnesium ions birch profilin promoted the polymerization of actin from A:Tbeta4. In contrast, in the presence of calcium but absence of magnesium ions birch profilin was unable to initiate the polymerization of actin from the complex with Tbeta4. However, under these conditions profilin formed a stable stoichiometric complex with skeletal muscle alpha-actin, as verified by its ability to increase the critical concentration of actin polymerization. Chemical cross-linking indicated that birch profilin competes with Tbeta4 for actin binding. Ternary complex formation of birch profilin with actin:DNase I complex was suggested by chemical cross-linking. However, the determination of the critical concentrations of actin polymerization in the simultaneous presence of birch profilin and DNase I indicated that profilin and DNase I did not form a ternary complex. These data indicated a negative co-operativity between the profilin and DNase I binding sites on actin.

Topics: Actins; Animals; Binding Sites; Contractile Proteins; Cross-Linking Reagents; Deoxyribonuclease I; Humans; Microfilament Proteins; Plant Proteins; Polymers; Profilins; Rabbits; Thymosin; Trees

A large-scale method for the isolation of thymosin beta 4 (up to 120 mg) and thymosin beta 9 (up to 40 mg) from bovine lung (up to 2 kg) was developed. The isolation protocol included tissue homogenization in 0.4 M HClO4, centrifugation, solid-phase extraction through LiChroprep RP-18 material, chromatofocusing on polybuffer exchanger PBE 94-modified Sepharose and dialysis against water. The isolated products were characterized by analytical isoelectric focusing, reversed-phase HPLC, electrospray ionization mass spectrometry and amino acid analysis. The method developed is rapid and convenient, requires no expensive equipment and can be used for the isolation of thymosin beta 4 and homologous peptides from various animal tissues.

Topics: Amino Acid Sequence; Animals; Cattle; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Indicators and Reagents; Isoelectric Focusing; Lung; Mass Spectrometry; Microfilament Proteins; Molecular Sequence Data; Thymosin

Thymosin beta 4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin beta 9 and thymosin beta met9 are minor variants of thymosin beta 4. The possible function of these peptides has been investigated by comparing the actin binding properties of these beta-thymosins. Thymosin beta 9 and thymosin beta met9 were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 microM (as compared to 2 +/- 0.3 microM for thymosin beta 4); like thymosin beta 4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin beta 4 and thymosin beta met9 with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta 4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin beta 4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of approximately 1.5 microM-1 s-1 and 2 s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin beta 4 function in vivo is discussed.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Amino Acid Sequence; Animals; Drug Interactions; Fluorescence; Genetic Variation; Mathematics; Methionine; Molecular Sequence Data; Oxidation-Reduction; Polymers; Protein Binding; Rabbits; Thymosin

A thymosin beta 4 ELISA was developed in which thymosin beta 4, absorbed on microwells, competed with thymosin beta 4 in solution for the binding sites of an anti-thymosin beta 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta 4 antibodies were raised in rabbits against intact thymosin beta 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta 4[1-14] and thymosin beta 4[1-11]. The antibody against thymosin beta 4[1-14] was used in the thymosin beta 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta 9, was fast, easy to perform and exhibited good assay characteristics.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Binding, Competitive; Cattle; Chromatography, High Pressure Liquid; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Thymosin

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin alpha and thymosins beta 4 and beta 9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin alpha, rat parathymosin and calf thymosin beta 4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin alpha and rat parathymosin both migrate to the nucleus whilst thymosin beta 4 remains in the cytoplasm. The peptide (1-88) of calf prothymosin alpha is shown not to accumulate in the Xenopus nucleus, demonstrating that the C-terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin beta 4 should be grouped separately from the pro- and parathymosins.

Topics: Amino Acid Sequence; Amino Acids; Animals; Cattle; Cell Nucleus; Circular Dichroism; Cytoplasm; Humans; Isoelectric Focusing; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Oocytes; Protein Precursors; Rats; Thymosin; Thymus Gland; Xenopus

In order to obtain specific antibodies against thymosin beta 9 showing minimal cross-reactivity with the highly homologous peptide thymosin beta 4, the N-terminal fragment 1-14 of thymosin beta 9 was used for immunization. These antibodies have been tested in a competitive ELISA and show less than 1% cross-reactivity with thymosin beta 4. On the other hand, antibodies raised against the native thymosin beta 9 (1-14) cross-react 35% with thymosin beta 4. Specific antibodies against thymosin beta 9 are important for studying the concentration and localization of thymosin beta 9 in thymus and other bovine tissues because thymosin beta 9 is always accompanied by thymosin beta 4. Using N-terminal fragments of thymosin beta 4-like peptides may be a general approach for obtaining specific antibodies since this part of sequence is less conserved in thymosin beta 4-like peptides.

Topics: Amino Acid Sequence; Animals; Cattle; Chromatography, High Pressure Liquid; Cross Reactions; Molecular Sequence Data; Peptide Fragments; Thymosin; Thymus Gland

We have identified a new thymosin beta 4-like peptide in pork spleen. The new peptide (12 mg) and thymosin beta 4 (33 mg) were isolated from 230 g of spleen by solid phase extraction, preparative isoelectric focusing, and HPLC. The new peptide was termed thymosin beta 9 Met to indicate its close relationship to thymosin beta 9 from calf. The only difference from thymosin beta 9 is the substitution of leucine by methionine at position 6. This peptide replaces thymosin beta 10 which is the minor thymosin beta 4-like peptide in most mammals, e.g., in man, rat, mouse, cat, and rabbit. The structure was determined by amino acid analysis, tryptic digestion, and carboxypeptidase digestion. Pork spleen contains 192 micrograms of thymosin beta 4 and 117 micrograms of thymosin beta 9 Met per gram of tissue.

Topics: Amino Acid Sequence; Animals; Chromatography, Ion Exchange; Isoelectric Focusing; Molecular Sequence Data; Peptides; Species Specificity; Spleen; Swine; Thymosin

Thymosin beta 4 has been determined by a simple and fast one-step procedure in different tissues of rats. The tissues (1 to 40 mg) were disintegrated and deproteinized by homogenization in perchloric acid. After neutralization by potassium hydroxide the supernatant solution was used for determining thymosin beta 4 by reverse-phase HPLC without further manipulations. Not only does this procedure avoid artificial proteolysis as effectively as extraction of tissues by guanidinium chloride or boiling buffer, but it offers two further advantages. First, no additional steps--as for example desalting--are necessary prior to HPLC and thus the risk of losing thymosin beta 4 is eliminated. Using this procedure thymosin beta 4 is recovered quantitatively. The method is linear over the range 0.04 to 1.13 nmol and thymosin beta 4 is well separated from other thymosin beta 4-like peptides known to be present in mammals; i.e., thymosin beta Ala4, thymosin beta 9, thymosin beta 10, and thymosin beta Arg10. Second, the acid-insoluble pellet of the same extract can be used to determine the DNA content of the sample. Thus it is possible to relate thymosin beta 4 to DNA, which then allows comparing cells of different tissues and cell lines to one another. This procedure is also applicable to small peptides soluble in perchloric acid.

Topics: Amino Acid Sequence; Animals; Cattle; Chromatography, High Pressure Liquid; DNA; Male; Rats; Rats, Inbred Strains; Sulfoxides; Thymosin