Immunohistochemical localization of thymosin β9 in bovine tissues

Summary Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin ₉ was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin ₉ in order to minimize the cross-reactivity with thymosin ₄ which was found to be also present in bovine tissues. The specific antibodies against thymosin ₉ raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin ₄ in different tissues. Although thymosin ₉ was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.     

Structure and immunological properties of thymosin beta 9 Met, a new analog of thymosin beta 4 isolated from porcine thymus

During the course for the studies of thymosin beta 4 and prothymosin alpha from porcine thymus, a new analog of thymosin beta 4 has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin beta 9 except that leucine at position 6 in beta 9 is substituted by methionine. Thus, this new peptide has been termed thymosin beta 9 Met. The recoveries of beta 9 Met, beta 4, and prothymosin alpha in porcine tissues have been determined (in micrograms/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin beta 9 Met was found to be more active than beta 4 in enhancing gamma-interferon production in cord blood lymphocytes. However, beta 4 appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Tissue distribution of high molecular weight calmodulin-binding protein

Polyclonal antibodies raised against bovine heart high molecular weight calmodulin-binding protein were used to study the distribution of this protein in diverse bovine tissues. The high molecular weight calmodulin-binding protein, in addition to bovine heart, is also present in lung and brain at much lower levels, but not in skeletal muscle, spleen, kidney or uterus.     

Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.     

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.     

Isolation and characterization of thymosin beta 9 Met from pork spleen

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.     

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.     

One-step procedure for the determination of thymosin beta 4 in small tissue samples and its separation from other thymosin beta 4-like peptides by high-pressure liquid chromatography

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.     

Simultaneous isolation and determination of prothymosin alpha, parathymosin alpha, thymosin beta 4, and thymosin beta 10

A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.     

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.     

Immunological demonstration of a calcium-unresponsive protein kinase C of the delta-type in different species and murine tissues. Predominance in epidermis

An antiserum raised against a delta-protein kinase C (delta-PKC)-specific peptide recognized the purified calcium-unresponsive 76-kDa protein kinase of porcine spleen in the native and the denatured form. This antiserum was used to demonstrate the delta-PKC-like enzyme in spleen of different species, in various cell types and in murine tissues by immunoblotting of the respective extracts. Due to species differences, delta-PKC-like kinases with slightly different molecular weights were observed. The enzyme was found to be present in primary murine keratinocytes, primary bovine endothelial cells, and many cell lines originating from human, rat, and murine tissues. It was present also in all murine tissues tested, predominantly in epidermis, uterus, placenta, lung, brain, spleen, and kidney. In contrast to the conventional alpha, beta, gamma-PKC, it was located almost exclusively in the particulate fraction. The delta-like PKC could be demonstrated in the epidermis and brain of newborn mice, and in both tissues its concentration increased dramatically between day 7 and 14 after birth. The delta-PKC-like kinase of mouse epidermis (p82-kinase) was down-regulated after topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin. The amount of the enzyme decreased to less than 20% of the controls within 16 h and recovered almost completely within 72 h after TPA. The existence of the delta-PKC-like kinase in mouse skin, papillomas, and carcinomas could also be demonstrated by immunocytochemical staining of the respective sections. The enzyme was observed predominantly in epithelial layers. A remarkable immunostaining of nuclei in skin sections disappeared after TPA treatment of the animals.     

Solid phase synthesis of thymosin beta 9

Thymosin beta 9, a 41 residue thymic polypeptide, has been synthesized by a solid phase method. A modification of the low HF method was used to deprotect and cleave the peptide from the resin. Thymosin beta 9 was then obtained in analytically pure form by a one-step purification procedure in 32% yield. The activity of thymosin beta 9 in the terminal deoxynucleotidyl transferase assay was greater than calf thymus fraction 5, but comparable to thymosin beta 4. In contrast to thymosin alpha 1, neither beta 4 nor beta 9 was active in the rosette inhibition assay.     

Immunological characterization of thioltransferase from pig liver

Polyclonal antibodies against pig liver thioltransferase were raised in a New Zealand rabbit. These antibodies completely neutralized the thioltransferase activity of the homogeneous enzyme and that in the crude cytosolic homogenate at an equivalent titer. The antibodies also cross-reacted equally with calf thymus glutaredoxin and calf liver thioltransferase, but not with Escherichia coli thioredoxin, suggesting that thioltransferase and glutaredoxin from the same species are identical. Immunoblotting analysis of the cytosolic proteins from 14 different pig tissues revealed that most pig tissues contain a 12-kDa protein which reacts with these antibodies. This protein is found in greater abundance in stomach, small intestine, liver, skeletal muscle, kidney, heart, lung, and cerebral cortex, whereas retina, cerebellum, spleen, pancreas, and thymus have low levels of the protein. No reactive protein was detected in the lens. The tissue distribution of the protein was also determined by assay of the enzyme activity and was generally in good agreement with that obtained from the immunoblotting survey. Pig liver thioltransferase was cleaved by trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide. The selected peptides purified by reversed phase high performance liquid chromatography or ion exchange fast protein liquid chromatography were subjected to reaction with the polyclonal antibodies against pig liver thioltransferase. Four antigenically reactive fragments were detected by dot-blotting analysis. These peptides are located in the first 30-amino acid residues from the NH2 terminus and the sequence from amino acid residues 39-67, indicating that the active site of the enzyme, Cys22 and Cys25, is located on one of the antigenic determinant domains.     

Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen

A monoclonal antibody (MAb), BLT-1, with specificity for bovine mature T cells was prepared by somatic cell hybridization of myeloma NS-1 and spleen cells from BALB/c mice hyperimmunized with bovine T lymphocytes. The MAb reacted with over 92% of nylon wool-nonadherent lymphocytes (T cells) but not with nylon wool-adherent EAC-positive lymphocytes (B cells) in the indirect immunofluorescence assay. It is an IgM, with kappa-light chains, which fixed complement well and killed over 95% of mature T cells in complement-mediated cytotoxicity assays. It reacted with the same proportions of peripheral lymphoid cells (peripheral blood, lymph nodes, and spleen) as the polyclonal goat anti-bovine thymocyte serum (GABTS), but only with 25% of GABTS-positive thymocytes. Immunoperoxidase staining of frozen tissue sections showed that the BLT-1-positive cells were located in the medulla of the thymus and in the T lymphocyte areas of lymph nodes. Western immunoblotting assays showed that the BLT-1-reactive membrane antigen is a 22,000 m.w. protein which was inducible in bovine thymocytes with bovine thymic hormones, thymosin fraction 5, thymosin alpha 1, and thymopentin ORF-18150, indicating that it is a mature T lymphocyte differentiation antigen. The thymosin alpha 1 and thymopentin were found to show additive effects on mature T cell antigen expression by bovine thymocytes.     

Cloning and characterization of a testis-specific thymosin beta 10 cDNA. Expression in post-meiotic male germ cells.

Thymosin beta 10 is one of a small family of proteins closely related in sequence to thymosin beta 4, recently identified as an actin-sequestering protein. A single molecular weight species of thymosin beta 10 mRNA is present in a number of rat tissues. In adult rat testis, an additional thymosin beta 10 mRNA of higher molecular weight was identified. Nucleotide sequencing of cDNA clones complementary to the testis-specific thymosin mRNA indicated that this mRNA differed from the ubiquitous thymosin beta 10 mRNA only in its 5'-untranslated region, beginning 14 nucleotides upstream of the translation initiation codon. These results, together with primer extension experiments, suggest that the two thymosin beta 10 mRNAs are transcribed from the same gene through a combination of differential promoter utilization and alternative splicing. The novel thymosin beta 10 mRNA could be detected only in RNA isolated from sexually mature rat testis. Both mRNAs were present in pachytene spermatocytes; only the testis-specific mRNA was detected in postmeiotic haploid spermatids. Immunoblot analysis using specific antibodies showed that the thymosin beta 10 protein synthesized in adult testis was identical in size to that synthesized in brain. Immunohistochemical analysis showed that the protein was present in differentiating spermatids, suggesting that the testis-specific thymosin beta 10 mRNA is translated in haploid male germ cells.     

Thymosin beta 4 induced phenotypic changes in Molt-4 leukemic cell line

Thymosin beta 4 was tested for its ability to induce phenotypic changes in the human T-cell line Molt-4. Cells were cultured with nanogram concentrations of thymosin beta 4 for up to 16 days and were analyzed with a panel of monoclonal antibodies, sheep erythrocyte rosetting, peanut agglutinin binding (PNA) and an antibody to the enzyme, terminal deoxynucleotidyl transferase (TdT). Thymosin beta 4 induced Molt-4 cells to reduce the expression of a T-cell lineage specific antigen, with preferential expression on T blast-cells, detected by WT 1 monoclonal antibody. Thymosin beta 4 also induced an increase in sheep erythrocyte rosettes and PNA binding as well as an increased expression of OKT 11 A and OKT 8 in Molt-4 cells. TdT was found to be unchanged, however. Analysis of thymosin beta 4-treated cells with other monoclonal antibodies (OKT 3, OKT 6, OKT 9) showed no change when compared to controls. These results showed that thymosin beta 4 is capable of inducing phenotypic changes in Molt-4 cells. Such changes may represent a differentiation process of these cells through the early stages of the maturation process of thymus-dependent lymphocytes, albeit not to the stage of mature T cells.     

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues

Summary Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouuse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.     

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues

Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.     

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.     

Demonstration of calmodulin-stimulated phosphatase isozymes by monoclonal antibodies

A procedure combining immunoprecipitation and immunotransblot employing subunit-specific monoclonal antibodies of the brain phosphatase, VJ6 and VA1, was used on tissues including heart, muscle, lung, spleen, pancreas, uterus, and liver. The various tissue extracts were subjected to immunoprecipitation by the beta subunit-specific VA1-immunoabsorbant, the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunotransblot, using both the alpha and beta subunit-specific antibodies VJ6 and VA1, respectively. Protein bands corresponding to alpha and beta subunits and the immunostain of beta subunit were detected in all samples, whereas alpha subunit was strongly stained only in the brain extract, weakly in heart and muscle extracts, and essentially negatively in all the other samples. In contrast, a polyclonal antiserum of bovine brain calmodulin-stimulated phosphatase could immunostain both alpha and beta subunits from all tissues. Calmodulin-binding protein fractions from a number of bovine tissues were all shown to contain the immunoprecipitable alpha subunit, as well as calmodulin-stimulated p-nitrophenylphosphatase activity. Micropeptide mapping showed that alpha subunits of bovine brain and bovine lung calmodulin-stimulated phosphatase isozymes were distinct molecular species. These results provide direct evidences for the existence of calmodulin-stimulated phosphatase isozymes in mammalian tissues.