Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

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.     

Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.     

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.     

Thymosin beta4 and thymosin beta4-derived peptides induce mast cell exocytosis

The peptide thymosin beta4 (Tbeta4) promotes angiogenesis and wound healing. Mast cells are involved in these processes as well and therefore we investigated the effect of Tbeta4 on mast cells. Exposure to 0.2-2000nM Tbeta4 induced mediator release (up to 23%) in murine peritoneal and human HMC-1 mast cells in a concentration-dependent manner. While the peptide AcSDKP, matching the 4 N-terminal amino acid residues of Tbeta4, mediated low but detectable mediator release, peptides corresponding to the Tbeta4 amino acid sequences 16-38 and 17-23 stimulated mast cells mediator release on a level equal to or higher than that observed with native Tbeta4. These observations and certain characteristics of Tbeta4-mediated mast cell activation suggest that the actin-binding motif LKKTET present in Tbeta4 (amino acid 17-22) might be implicated in this process. Thus, Tbeta4 activates mediator release in mast cells by a process that possibly involves an actin-binding motif and this could be important for understanding the mechanisms of Tbeta4-mediated effects in vivo.     

Thymosin beta 4Xen: a new thymosin beta 4-like peptide in oocytes of Xenopus laevis

Two new thymosin beta 4-like peptides have been detected in ovaries of Xenopus laevis and Rana esculenta. Previously, it was reported that thymosin beta 4 can be found in various species, from mammals to amphibians, e.g., in X. laevis [S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576]. However, oocytes and spleen from R. esculenta contain no thymosin beta 4 but a similar peptide without methionine. The peptide from R. esculenta elutes from a reversed-phase column about 5 min later than thymosin beta 4. The peptide from X. laevis, referred to as thymosin beta 4Xen, can hardly be distinguished from thymosin beta 4 by its retention time on HPLC, by amino acid analysis, its isoelectric point, or tryptic fingerprinting. Amino acid analyses of the tryptic fragments, however, have revealed that thymosin beta 4 and beta 4Xen are different. The amino acid sequence of thymosin beta 4Xen is reported. Thymosin beta 4 and beta 4Xen differ in the amino acid residues at positions 15, 40, and 41. At position 15 serine is replaced by alanine and at 41-42 the sequence is Thr-Ser instead of Ala-Gly. Depending on their size, defolliculated oocytes contain between 2.7 and 52.6 ng thymosin beta 4Xen which is comparable to the amount of histones in oocytes.     

Immunohistochemical localization of thymosin beta 9 in bovine tissues

Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin beta 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 beta 9 in order to minimize the cross-reactivity with thymosin beta 4 which was found to be also present in bovine tissues. The specific antibodies against thymosin beta 9 raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin beta 4 in different tissues. Although thymosin beta 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 sarcolemma.     

Thymosin beta4 promotes matrix metalloproteinase expression during wound repair

Immobilized patients, diabetics, and the elderly suffer from impaired wound healing. The 43-amino acid angiogenic peptide thymosin beta4 (Tbeta4) has previously been found to accelerate dermal wound repair in rats, aged mice, and db/db diabetic mice. It also promotes corneal repair in both normal rats and mice. Because proteinases are important in wound repair, we hypothesized that Tbeta4 may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Analysis by RT-PCR of whole excised mouse dermal wounds on days 1, 2, and 3 after wounding showed that Tbeta4 increased several metalloproteinases, including MMP-2 and -9 expression by several-fold over control on day 2 after wounding. We further analyzed the metalloproteinases secreted in response to exogenous Tbeta4 by cells normally present in the wound. Western blot analysis of cultured keratinocytes, endothelial cells, and fibroblasts that were treated with increasing concentrations of Tbeta4 showed increases in the levels of MMP-1, -2, and -9 in a cell-specific manner. Tbeta4 also enhanced the secretion of MMP-1 and MMP-9 by activated monocytes. The central actin-binding domain, amino acids 17-23, had all of the activity for metalloproteinase induction. We conclude that part of the wound healing activity of Tbeta4 resides in its ability to increase proteinase activity via its central actin-binding domain. Thus, Tbeta4 may play a pivotal role in extracellular matrix remodeling during wound repair.     

Thymosin beta 4 and its N-terminal tetrapeptide, AcSDKP, inhibit proliferation, and induce dysplastic, non-apoptotic nuclei and degranulation of mast cells

Thymosin beta4 (Tbeta4), a 5 kDa polypeptide, is a member of the beta-thymosin family. It acts as the principal intracellular G-actin sequestering peptide and exhibits extracellular functions in angiogenesis and wound healing. The N-terminus of Tbeta4 contains a bioactive tetrapeptide, acSDKP, a negative regulator of hematopoietic stem-cell proliferation. Here, we show that both peptides inhibit mast-cell proliferation over the concentration range of 10(-6) to 10(-17) M with the maximum effect of both at 10(-14) M. Both Tbeta4 and acSDKP caused dysplastic mast-cell nuclei that were confirmed by DAPI fluorescent staining. Flow-cytometric analysis of ploidy revealed that the dysplastic nuclei were not multinucleated, but fragmented nuclei in G2 growth arrest. We could further demonstrate that 10(-8) or 10(-14) M Tbeta4 or acSDKP induce mast-cell degranulation. A concentration of 10(-8) M Tbeta4 or acSDKP caused 57 or 89% degranulation, respectively. A number of tryptic fragments of Tbeta4 were assayed beside intact Tbeta4 and the tetrapeptide, and found to be inactive.     

The effect of anti-beta 43-47 Fab fragments on expression of the polymerization sites in the E domain of fibrinogen

In order to assess the significance of the aminoterminal residues of the B beta chain in expression of polymerization sites of the E domain, we have prepared polyclonal antiserum against a synthetic peptide corresponding to beta 43-47 of human fibrinogen. Affinity purified immunoglobulin IgG and Fab prepared from these antibodies reacted strongly and specifically with the synthetic pentapeptide and intact fibrinogen molecule. This specificity was determined both by radioimmunoassay and Western blot analysis of fibrinogen and its plasmic fragments and described in our previous paper (Cierniewski et al., 1986, Biochim. Biophys. Acta, 884, 594-597). Immunochemically purified anti beta 43-47 antibodies and their Fab fragments were strong inhibitors of the fibrin monomer polymerization. Our results imply that amino acid sequence beta 43-47 recognized by these antibodies may be in a close vicinity to the contact sites of the E domain.     

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.     

Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Primary structure of thymosin beta 12, a new member of the beta-thymosin family isolated from perch liver.

A new polypeptide termed thymosin beta 12 has been isolated from perch liver and its primary structure elucidated. This polypeptide contains 43 amino acid residues with a molecular weight of 4822 Da. The content of thymosin beta 12 from perch liver has been determined as 43 micrograms/g of tissue. The amino-terminal end of this polypeptide is blocked by an acetyl group as deciphered by fast-atom bombardment mass spectrometric analysis. Sequence analysis reveals that thymosin beta 12 is 79% homologous to thymosin beta 4, an immunomodulator which was originally isolated from calf thymus. Thymosin beta 12 also shows 84% sequence homology to thymosin beta 11, a beta 4 analog which replaces beta 4 in two species of bony fish, oscar and rainbow trout. The evolutionary implication of such results will be discussed. The isolation of a new beta 4-related peptide from perch liver which differs from beta 11 indicates that beta-thymosin peptides are widely distributed in lower vertebrate classes.     

Primary antisera against selected steroids or proteins and secondary antisera against gamma-globulins--an available tool for studies of reproductive processes

This paper presents characteristics of different polyclonal antisera raised against several steroid and protein antigens: 1/ primary antisera against steroid hormones: estradiol-17beta (anti-E2), estrone (anti-E1), testosterone (anti-T), androstendione (anti-A4), cortisol (anti-F) and corticosterone (anti-B); 2/ primary antisera against porcine luteinizing hormone (anti-pLH) and against different forms of porcine pregnancy associated glycoproteins (anti-pPAG) - proteins produced by chorionic tissue; 3/ secondary monovalent antisera raised against rabbit gamma-globulins (Sm-r); 4/ secondary polyvalent antisera against rabbit, pig and quinea pig gamma-globulins mixed at a ratio 1:1:1 (Sp-rpq). All antisera described in the paper present sufficient quality to be routinely used in various RIA, ELISA or Western determinations in physiological and clinical studies of reproductive processes. The antisera against steroid hormones and pLH are available on request.     

Rapid induction of thymosin beta 4 in concanavalin A-stimulated thymocytes by translational control

The expression of thymosin beta 4, an ubiquitous peptide of high cellular content, was studied in concanavalin A-stimulated rat thymocytes within the first 3 h after activation of the cells. An early 6.3-fold increase of the peptide occurred after 1 h of stimulation amounting to 0.4% of the total cellular protein. This increase coincided with that of thymosin beta 4 biosynthesis measured by [35S]methionine incorporation. The share of thymosin beta 4 synthesis in total protein synthesis 1 h after addition of concanavalin A amounts to 1% but no elevation of the corresponding mRNA was observed. These data suggest that a translational control mechanism is involved in this rapid induction. Consequently, actinomycin D did not inhibit thymosin beta 4 induction in contrast to cycloheximide. The peaks of maximal thymosin beta 4 levels and biosynthesis were followed by rapid decreases of these parameters suggesting a function of thymosin beta 4 in the early phase of T cell activation.     

Local photorelease of caged thymosin beta4 in locomoting keratocytes causes cell turning

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin beta4 (Tbeta4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tbeta4 was produced in an inactive form by "caging" with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tbeta4 did not change the normal actin polymerization kinetics, whereas photoactivated Tbeta4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tbeta4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tbeta4 and the consequent reduction in the diffusional spread of the Tbeta4-actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tbeta4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tbeta4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tbeta4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tbeta4.     

The Capsid Gene of Feline Calicivirus Contains Linear B-Cell Epitopes in both Variable and Conserved Regions

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.     

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.     

Effect of thymosin beta15 on the branching of developing neurons

The thymosin betas (Tbetas) are polypeptide regulators of actin dynamics that are critical for the growth and branching of neurites in developing neurons. We found that mRNAs for Tbeta4, Tbeta10, and Tbeta15 were highly expressed in the developing rat brain during neuritogenesis, supporting a role for the Tbetas in this process. Overexpression of the Tbetas increased the number of neurite branches per neuron in cultured hippocampal and cerebral cortex neurons, and Tbeta15 had the greatest effect. Actin binding activity appears to be essential for the branch-promoting activity of Tbetas because two mutants of Tbeta15 lacking monomeric actin binding activity failed to stimulate branch formation. We also found that transfection of siRNA against Tbeta15 reduced branching. Taken together, these data suggest that the three Tbetas, and especially Tbeta15, stimulate neurite branching during brain development.     

HTLV-II-specific antisera raised in rabbits immunized with a synthetic peptide of HTLV-II envelope protein.

In order to discriminate HTLV-II from HTLV-I, HTLV-II-specific polyclonal antibodies against a synthetic peptide of HTLV-II envelope sequence were raised in rabbits. We immunized two adult rabbits with a KLH-conjugated synthetic peptide corresponding to the amino acid sequence 171-196 of the HTLV-II envelope sequence, which is a specific region for HTLV-II as evaluated with an ELISA method. The resulting rabbit antisera to the synthetic peptide reacted with gp46 of HTLV-II lysates in Western blot analysis but not with that of HTLV-I. Flow cytometric analysis and immunohistochemical study revealed that these affinity purified antisera recognized some HTLV-II-producing cell lines examined, but not HTLV-I-producing cell lines or other cell lines uninfected by HTLV. These findings indicate that these antisera specifically recognized the envelope glycoprotein (gp46) of HTLV-II and suggest the specificity of this region in the immune response to HTLV-II. Such antisera are useful in distinguishing between HTLV-I and HTLV-II infection and in determining the presence of individual HTLV-II-infected cells both in vivo and in vitro, including non-lymphoid cells. They may also assist in the elucidation of the pathogenesis of HTLV-II.