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It is important to establish an efficient vascularization for the long-term acceptance of bioengineered skin equivalents treating the cutaneous wounds of diabetic rats with hindlimb ischemia. This study investigates the possible use of a collagen-chitosan sponge scaffold encapsulated with thymosin beta 4 (CCSS-eTβ4), an angiogenic factor, to accelerate cutaneous wound healing in streptozotocin (STZ)-induced diabetic rats with hindlimb ischemia. CCSSs-eTβ4 was fabricated using a freeze-drying method. The scaffolds were analyzed by scanning electron microscopy, swelling and degradation assays, mechanical properties, and scaffolds of 50:50 collagen-chitosan were selected and applied. The controlled release of Tβ4 from the scaffolds elicited localized and prolonged effects over 12 days, as shown by an enzyme-linked immunosorbent assay (ELISA). In vivo, CCSSs-eTβ4 improved diabetic cutaneous wound healing, with faster wound reepithelialization, better dermal reorganization, and higher wound vascularization. Furthermore, CCSSs-eTβ4 downregulated inflammatory genes and upregulated angiogenic genes in the wound tissue. Significant increases in CD31-positive endothelial cells and new vessel density were also observed. In vitro, Tβ4 increased the migratory and proliferative activity of high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). Meanwhile, we found that Tβ4 could promote HG-treated HUVECs migration and improve angiogenesis by activation of the VEGF/AKT pathway. Overall, these findings demonstrated the promising potential of CCSSs-eTβ4 to promote more effective wound healing and suggest its possible application for diabetic cutaneous wound treatment.

Background: The actin cytoskeleton is a hallmark of eukaryotic cells. Its regulation as well as its interaction with other proteins is carefully orchestrated by actin interaction domains. One of the key players is the WH2 motif, which enables binding to actin monomers and filaments and is involved in the regulation of actin nucleation. Contrasting conserved domains, the identification of this motif in protein sequences is challenging, as it is short and poorly conserved., Findings: To identify divergent members, we combined Hidden-Markov-Model (HMM) to HMM alignments with orthology predictions. Thereby, we identified nearly 500 proteins containing so far not annotated WH2 motifs. This included shootin-1, an actin binding protein involved in neuron polarization. Among others, WH2 motifs of 'proximal to raf' (ptr)-orthologs, which are described in the literature, but not annotated in genome databases, were identified., Conclusion: In summary, we increased the number of WH2 motif containing proteins substantially. This identification of candidate regions for actin interaction could steer their experimental characterization. Furthermore, the approach outlined here can easily be adapted to the identification of divergent members of further domain families.

Objective: Thymosin α1 (Tα1) is a peptide hormone whose therapeutic application has been approved in several diseases, but the description of a precise receptor for its therapeutic action still remains elusive and some knowledge of the mechanism of interaction with the cell membrane still needs to be clarified. This work is aimed at studying the folding and interaction of Tα1, which is completely unstructured in water solution, with model membranes., Methods: The folding and interaction of Tα1 with sodium dodecyl sulfate micelles was monitored by NMR and CD spectroscopy techniques., Results: Tα1 assumes a helical conformation in the presence of sodium dodecyl sulfate micelles, showing a helical fold with a structural break around residues 9 and 14. These results were confirmed by circular dichroism and NMR spectroscopy. Moreover, by paramagnetic NMR relaxation it was found that Tα1 is inserted in the hydrophobic region of the micelles by the residues 1 - 5 of the N-terminal end. This result clarifies the modality of insertion that was not obtained in previous NMR studies in trifluoroethanol., Conclusions: These findings suggest that Tα1 folds on the membrane and, when inserted, may be able to interact with nearby proteins and/or receptors acting as an effector and causing a biological signaling cascade.

Introduction: Thymosin α 1 (Tα1) is a peptidic biological response modifier, which plays a significant role in activating and regulating various cells of the immune system. For the above-mentioned activities it is expected to exert a clinical benefit in the treatment of diseases where the immune system is altered., Areas Covered: Several clinical trials have been carried out with Tα1 for treatment or prevention of many different infectious diseases such as hepatitis B and C, sepsis and Aspergillosis in bone marrow-transplanted patients. Data available on the use of Tα1 in infectious disease as well as a vaccine enhancer will be reviewed to possibly generate new working hypothesis., Expert Opinion: Tα1 has been widely used in thousands of patients. Nevertheless, there are some issues that have not yet been properly addressed (i.e., dose, schedule, combination treatments, end-points to be evaluated in clinical trials). In the most recent clinical trials Tα1 has been used at higher doses than those commonly used in the past showing a direct proportionality between the dose and the effect. The safety profile of Tα1 is excellent and it is virtually devoid of toxicity.

Introduction: Thymosin β4 (Tβ4), a multifunctional peptide, has been used successfully in several clinical trials involving tissue repair and regeneration. The review will first update the current information on the common underlying cellular cascades and pathways that are basic to Tβ4's regenerative activity and second, on the current and potential uses of this protein in the clinic., Areas Covered: Significant advances in our understanding of the actions of Tβ4 have occurred in directing stem cell maturation and in regeneration and repair of injuries. Many of its activities directly affect the repair cascade following injury. Using PubMed, we summarize the discovery and isolation of Tβ4 as well as the studies on tissue repair, which have provided the scientific foundation for ongoing and projected trials in the treatment of eye injuries, dermal wounds, repair of the heart following myocardial infarction and healing of the brain following stroke, trauma or neurological diseases., Expert Opinion: Based on its multifunctional activities during tissue regeneration in various animal studies, Tβ4 has the potential for new clinical applications such kidney and liver disease, as well as repair of spinal cord, bone and ligament damage. In addition, it may be useful in the treatment of a wide range of other applications, including the consequences of aging and viral infections.

Introduction: The purpose of our work was to identify unknown interaction partners of thymosin β4 (Tβ4). It was suggested that Tβ4 could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ4 prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ4 counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tβ4 could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-β receptor., Methods: To prove our suggestion of a direct interaction between Tβ4 and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro., Results: We identified an interaction between Tβ4 and PDGF-BB by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking as well as through biotin label transfer using a bifunctional photoactivatable derivative of Tβ4. In an in vitro system, PDGF-BB was identified as the first extracellular partner interacting with Tβ4. This interaction could influence PDGF-BB binding to its receptor and abolish PDGF-BB-related effects., Conclusion: Direct interaction of Tβ4 with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of β-thymosins.

Objective: During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF-IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied., Research Design and Methods: Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics., Results: Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 - 111) superficially interacts with similar TLR4/MD-2 (KD = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 - 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS., Conclusions: ProTα via its C-terminal peptide (aa 91 - 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

Objectives: The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies., Methods: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides., Results: In addition to thymosin β4 (Tβ4) and β10 (Tβ10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ4 and Tβ10 together with other proteolytic fragments. The sulfoxide derivative of both Tβ4 and Tβ10 and the acetylated derivatives at lysine residues of Tβ4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too., Conclusion: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

Unlabelled: Thymosin β4 (Tβ4) and its amino-terminal fragment comprising N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) have been reported to act as anti-inflammatory and anti-fibrotic agents in vitro and in vivo. In recent papers, we have shown that Tβ4 exerts a widely protective role in mice treated with bleomycin, and in particular, we have demonstrated its inhibitory effects on both inflammation and early fibrosis., Objectives: In this study, the putative anti-proliferative and anti-fibrogenic effects of Tβ4 and Ac-SDKP were evaluated in vitro. In addition, the effects of Tβ4 up to 21 days were evaluated in the bleomycin mouse model of lung fibrosis., Methods: We utilized both control and TGF-β-stimulated primary human lung fibroblasts isolated from both idiopathic pulmonary fibrosis (IPF) and control tissues. The in vivo effects of Tβ4 were assessed in CD1 mice treated with bleomycin., Results: In the in vitro experiments, we observed significant anti-proliferative effects of Ac-SDKP in IPF fibroblasts. In those cells, Ac-SDKP significantly inhibited TGF-β-induced α-SMA and collagen expression, hallmarks of fibroblast differentiation into myofibroblasts triggered by TGF-β. In vivo, despite its previously described protective role in mice treated with bleomycin at 7 days, Tβ4 failed to prevent fibrosis induced by the drug at 14 and 21 days., Conclusion: We conclude that, compared to Tβ4, Ac-SDKP may have greater potential as an anti-fibrotic agent in the lung. Further in vivo experiments are warranted.

Thymosin-β4 (Tβ4) and profilin are the two major sequestering proteins that maintain the pool of monomeric actin (G-actin) within cells of higher eukaryotes. Tβ4 prevents G-actin from joining a filament, whereas profilin:actin only supports barbed-end elongation. Here, we report two Tβ4:actin structures. The first structure shows that Tβ4 has two helices that bind at the barbed and pointed faces of G-actin, preventing the incorporation of the bound G-actin into a filament. The second structure displays a more open nucleotide binding cleft on G-actin, which is typical of profilin:actin structures, with a concomitant disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, show that the exchange of bound actin between Tβ4 and profilin involves both steric and allosteric components. The sensitivity of profilin to the conformational state of actin indicates a similar allosteric mechanism for the dissociation of profilin during filament elongation.

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides., (Copyright © 2014 Elsevier Inc. All rights reserved.)

After a cardiac event, proper treatment and care of the damaged tissue is crucial in restoring optimal cardiac function and preventing future cardiac events. Recently, thymosin β4 has been found to play a vital role in cardiac cell health and development by regulating angiogenesis, inflammatory responses, and wound healing. We proposed that defined poly(ϵ-caprolactone) (PCL) nanoscaffolds coated with thymosin β4 could efficiently differentiate murine-derived cardiomyocytes into functioning cardiac tissue. PCL nanoscaffolds were developed through electrospinning technology, and subsequently coated with a thymosin β4 solution. Cardiomyocytes were seeded on coated and uncoated nanoscaffolds and observed for six days via fluorescent and electron microscopy. Our results demonstrated a robust growth and differentiation of cardiomyocytes on coated nanoscaffolds compared with uncoated, showing potential for nanoscaffold-mediated cardiac cell replacement in vivo after an MI or other cardiac event.

Objective: Thymosin beta 4 (Tβ4) is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin, eye, heart, and neural system following injury. To fully realize its utility as a treatment for disease caused by injury, the authors constructed a cost-effective novel Tβ4 dimer and demonstrated that it was better able to accelerate tissue repair than native Tβ4., Methods: A prokaryotic vector harboring two complete Tβ4 genes with a short linker was constructed and expressed in Escherichia coli. A pilot-scale fermentation (10 L) was performed to produce engineered bacteria and the Tβ4 dimer was purified by one-step hydrophobic interaction chromatography. The activities of the Tβ4 dimer to promote endothelial cell proliferation, migration, and sprouting were assessed by tetramethylbenzidine (methylthiazol tetrazolium), trans-well, scratch, and tube formation assays. The ability to accelerate dermal healing was assessed on rats., Results: After fermentation, the Tβ4 dimer accounted for about 30% of all the bacteria proteins. The purity of the Tβ4 dimer reached 98% after hydrophobic interaction chromatography purification. An average of 562.4 mg/L Tβ4 dimer was acquired using a 10 L fermenter. In each assay, the dimeric Tβ4 exhibited enhanced activities compared with native Tβ4. Notably, the ability of the dimeric Tβ4 to promote cell migration was almost two times higher than that of Tβ4. The rate of dermal healing in the dimeric Tβ4-treated rats was approximately 1 day faster than with native Tβ4-treated rats., Conclusion: The dimeric Tβ4 exhibited enhanced activity on wound healing than native Tβ4, and the purification process was simple and cost-effective. This data could be of significant benefit for the high pain and morbidity associated with chronic wounds disease. A better strategy to develop Tβ4 as a treatment for other diseases caused by injuries such as heart attack, neurotrophic keratitis, and multiple sclerosis was also described.

Background: Active cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100-109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100-109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents., Results: Monocyte-derived DCs matured in vitro with proTα or proTα(100-109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100-109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100-109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF., Conclusions: Our results suggest that proTα and proTα(100-109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100-109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as "alarmins", provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.

Repertoires of proteins and small peptides play numerous physiological roles as hormones, antimicrobial peptides, and cellular signaling factors. The beta-thymosins are a group of small acidic peptides involved in processes such as actin sequestration, neuronal development, wound healing, tissue repair, and angiogenesis. Recent characterization of the beta thymosins as immunological regulators in invertebrates led to our identification and characterization of a beta-thymosin homologue (Tβ) from Haliotis discus discus. The cDNA possessed an ORF of 132 bp encoding a protein of 44 amino acids with a molecular mass of 4977 Da. The amino acid sequence shows high identity with another molluskan beta-thymosin and has a characteristic actin binding motif (LKKTET) and glutamyl donors. Phylogenetic analysis showed a close relationship with molluskan homologues, as well as its distinct identity and common ancestral origin. Genomic analysis revealed a 3 exon-2 intron structure similar to the other homologues. In silico promoter analysis also revealed significant transcription factor binding sites, providing evidence for the expression of this gene under different cellular conditions, including stress or pathogenic attack. Tissue distribution profiling revealed a ubiquitous presence in all the examined tissues, but with the highest expression in mantle and hemocyte. Immune challenge with lipopolysaccharide, poly I:C and Vibrio parahemolyticus induced beta-thymosin expression in gill and hemocytes, affirming an immune-related role in invertebrates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Thymosin β4 sequesters actin by formation of a 1:1 complex. This transient binding in the complex was stabilized by formation of covalent bonds using the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and a microbial transglutaminase. The localization of cross-linking sites was determined after separating the products using SDS-PAGE by tryptic in-gel digestion and high-resolution HPLC-ESI-MS. Three cross-linked fragments were identified after chemical cross-linking, indicating three contact sites. Because the cross-linked fragments were detected simultaneously with the corresponding non-cross-linked fragments, the three contact sites were not formed in parallel. K3 of thymosin β4 was cross-linked to E167 of actin, K18 or K19 of thymosin β4 to one of the first three amino acids of actin (DDE), and S43 of thymosin β4 to H40 of actin. The imidazole ring of histidine was proven to be an acyl acceptor for carbodiimide-mediated cross-linking. Molecular modeling proved an extended conformation of thymosin β4 along the subdomains 1 to 3 of actin. The enzymatic cross-linking using a microbial transglutaminase led to the formation of three cross-linking sites. Q41 of actin was cross-linked to K19 of thymosin β4, and K61 of actin to Q39 of thymosin β4. The third cross-linking site was identified between Q41 of actin and Q39 of thymosin β4, which are simultaneously cross-linked to K16, K18, or K19 of thymosin β4. When both cross-linking reactions are taken together, the complex formation of actin by thymosin β4 is more likely to be flexible than rigid and is localized along the subdomains 1 to 3 of actin.

Thymosin β4 is the prototype of β-thymosins and is present in almost every mammalian cell. It is regarded to be the main intracellular G-actin sequestering peptide. Thymosin β4 serves as a specific glutaminyl substrate for guinea pig transglutaminase. In the absence of an appropriate additional aminyl donor an ε-amino group of thymosin β4 serves also as an aminyl substrate and an intramolecular bond is formed concomitantly NH3 (17 Da) is lost. The molecular mass of the product is 4,949.6 Da. This is 16.3 Da less than the molecular mass of thymosin β4 (4,965.9 Da). Digestion with endopeptidases and Edman degradation of the fragments identified the exact position of the ring forming isopeptide bond. In spite of 3 glutaminyl and 9 lysyl residues of thymosin β4 only one isopeptide bond between Lys16 and Gln36 was formed (cyclic thymosin β4). These two amino acid residues are conserved in all β-thymosins. Cyclic thymosin β4 still forms a complex with G-actin albeit the stability of the complex is about one fiftieth of the stability of the thymosin β4 × G-actin complex.

Thymosin β4 (Tβ4) is a small peptide composed of 43 amino acids. It has many important biological functions, such as promoting cardiac repair and wound healing, and therefore has great potential in clinical applications. In this report, we describe a novel and efficient way to produce highly purified and active Tβ4. It was expressed in a soluble form using a DsbA and hexahistindine tag in Escherichia coli (E. coli). Using high cell density cultivation, the final biomass concentration was about 50 g L(-1) dry cell weight with the expression level of the fusion protein being 40%. To obtain highly purified protein, a purification process involving a five-step column procedure was implemented. The purity of Tβ4 was above 98% and all the host cell related impurities, such as endotoxin, host cell protein and residual DNA levels, were within the permissible range listed in the Chinese Pharmacopoeia. The E-rosette test demonstrated that the bioactivity of purified Tβ4 was consistent with other published work. This is the first report producing highly purified Tβ4 from genetically engineered sources., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We sought to identify serological markers capable of diagnosing preeclampsia (PE). We performed serum peptide analysis (liquid chromatography mass spectrometry) of 62 unique samples from 31 PE patients and 31 healthy pregnant controls, with two-thirds used as a training set and the other third as a testing set. Differential serum peptide profiling identified 52 significant serum peptides, and a 19-peptide panel collectively discriminating PE in training sets (n = 21 PE, n = 21 control; specificity = 85.7% and sensitivity = 100%) and testing sets (n = 10 PE, n = 10 control; specificity = 80% and sensitivity = 100%). The panel peptides were derived from 6 different protein precursors: 13 from fibrinogen alpha (FGA), 1 from alpha-1-antitrypsin (A1AT), 1 from apolipoprotein L1 (APO-L1), 1 from inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), 2 from kininogen-1 (KNG1), and 1 from thymosin beta-4 (TMSB4). We concluded that serum peptides can accurately discriminate active PE. Measurement of a 19-peptide panel could be performed quickly and in a quantitative mass spectrometric platform available in clinical laboratories. This serum peptide panel quantification could provide clinical utility in predicting PE or differential diagnosis of PE from confounding chronic hypertension.

Prothymosin alpha (ProTα), a nuclear protein, plays multiple functions including cell survival. Most recently, we demonstrated that the active 30-amino acid peptide sequence/P30 (amino acids 49-78) in ProTα retains its substantial activity in neuroprotection in vitro and in vivo as well as in the inhibition of cerebral blood vessel damages by the ischemic stress in retina and brain. But, it has remained to identify the minimum peptide sequence in ProTα that retains neuroprotective activity. The present study using the experiments of alanine scanning suggested that any amino acid in 9-amino acid peptide sequence/P9 (amino acids 52-60) of P30 peptide is necessary for its survival activity of cultured rat cortical neurons against the ischemic stress. In the retinal ischemia-perfusion model, intravitreous injection of P9 24h after ischemia significantly inhibited the cellular and functional damages at day 7. On the other hand, 2,3,5-triphenyltetrazolium chloride (TTC) staining and electroretinogram assessment showed that systemic delivery with P9 1h after the cerebral ischemia (1h tMCAO) significantly blocks the ischemia-induced brain damages. In addition, systemic P9 delivery markedly inhibited the cerebral ischemia (tMCAO)-induced disruption of blood vessels in brain. Taken together, the present study provides a therapeutic importance of 9-amino acid peptide sequence against ischemic damages., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Tumour-associated lymphocytes (TALs) present in effusions of ovarian cancer patients exhibit impaired activities, due to the immunosuppressive environment of the ascites. Means to enhance their cytotoxicity against autologous tumour cells are of clinical importance. The immunomodulator prothymosin alpha (proTα) increases the specific lysis of tumour cells by activated CD8(+) T-lymphocytes and its immunoreactivity is exerted by the carboxy-terminal decapeptide, proTα(100-109). These two molecules were studied on TALs in vitro, and in SCID mice bearing human ovarian tumours., Methods: TALs and tumour cells were isolated from 41 ovarian cancer patients and co-cultured in the presence of proTα or proTα(100-109). The cytotoxicity of peptide-stimulated TALs was tested against autologous tumour cells and K562. Ex vivo peptide-stimulated TALs from three patients were adoptively transferred intraperitoneally in SCID mice, previously inoculated with each patient's autologous tumour cells., Results: ProTα and its immunoreactive peptide proTα(100-109), enhanced the cytotoxic activity of TALs against autologous tumour cells in vitro, but marginally affected the lysis of K562. The effect of proTα and proTα(100-109) was higher after 7-14 days of stimulation, whereas TAL cytotoxicity was significantly decreased after 21 days. Mice administered TALs, ex vivo activated with proTα or proTα(100-109) for 7 days, showed a relatively lower tumour increase rate and a prolongation of their survival, compared to controls., Conclusion: Our data demonstrate that, in the presence of tumour antigens, proTα and proTα(100-109) enhance the depressed cytotoxicity of TALs against autologous tumour cells in vitro and retard tumour growth in vivo., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor for cytoprotective gene activation in the oxidative stress response. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses the transcriptional activity of Nrf2. During oxidative stress, Nrf2 is released from Keap1 and is translocated into the nucleus, where it interacts with the small Maf protein to initiate gene transcription. Prothymosin α (ProTα), an intrinsically disordered protein, also interacts with the Kelch domain of Keap1 and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, we have characterized the interaction between ProTα and the Kelch domain of Keap1 by using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, peptide array analysis, site-directed mutagenesis, and molecular dynamic simulations. The results of nuclear magnetic resonance chemical shift mapping, amide hydrogen exchange, and spin relaxation measurements revealed that ProTα retains a high level of flexibility, even in the bound state with Kelch. This finding is in agreement with the observations from the molecular dynamic simulations of the ProTα-Kelch complex. Mutational analysis of ProTα, guided by peptide array data and isothermal titration calorimetry, further pinpointed that the region (38)NANEENGE(45) of ProTα is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

β-thymosins, including thymosin β4 (Tβ4), Tβ10, and Tβ15, are a family of highly conserved 5 kDa peptides. They are involved not only in normal cell migration, but also in tumor metastasis. However, the molecular mechanisms of β-thymosins to regulate cell migration and other functions are not fully understood. Recently, this important area is under active investigation worldwide. Many new discoveries have been made from molecular biology and cell culture models as well as animal models and human diseases. This timely review provides the most updated information about functional roles and molecular mechanisms of β-thymosins in normal tissues and disease conditions.

Thymosin β4 (Tβ4) is a low molecular weight peptide found in several mammalian tissues and is known mainly by its ability to bind cytoskeletal actin, influencing cell migration and differentiation, and promoting tissue repair. Considering the functional role of this peptide, the main goal of this work was to characterize Tβ4 in mammals' saliva by using evolutionary and proteomic tools. For this, mammalian Tβ4 sequences were retrieved from NCBI, SwissProt and Ensembl databases. The alignment of Tβ4 amino acid sequences showed a high degree of conservation between species. The gene seems to be evolving under negative selection as indicated by a dN/dS ratio of 0.05. Whole saliva was collected from dog, human, rabbit, cow, horse and sheep and the salivary peptides were isolated through filtration and analyzed by LC-MS/MS. Spectra was processed against the database constructed with the retrieved Tβ4 sequences. For the first time, the identification of this peptide was achieved in rat, dog, horse and bovine saliva. Detection in these mammal species and its amino acid conservation suggest an important role of Tβ4 in the homeostasis of the mammalian oral cavity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that thymosin β4-sulfoxide lies downstream of hydrogen peroxide in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T-cell/CD14+ monocyte co-cultures, thymosin β4-sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, thymosin β4-sulfoxide is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

Alpha thymosins are a family of immunostimulating peptides first isolated from thymus gland. In the present work, the structure of the ReO(V)³⁺ complex of an alpha 1 thymosin derivative containing the metal-chelating N,N-dimethylglycyl-L-seryl-L-cysteinyl group was studied with NMR, CD, and ESI. The analysis indicated the existence of two interconverting diastereomers depending on the orientation of the side chain of the chelated Ser syn- or anti- to the oxygen of the ReO(V)³⁺ core. The two diastereomers could be separated on HPLC under a slow gradient showing the ratio of syn/anti to be 3:2, in agreement with the NMR data. The conversion process was shown to involve the coordination of a water molecule to the ReO(V)³⁺ core through the incubation of the complex in ¹⁸O-enriched water and subsequent ESI analysis. HPLC analysis of the analogous radioactive (⁹⁹m)TcO(V)³⁺ complex showed the formation of two isomers in the same syn/anti 3:2 ratio. Biodistribution studies of the (⁹⁹m)TcO(V)³⁺ complex in Swiss albino mice with experimentally induced inflammation showed higher accumulated radioactivity in inflamed tissue compared to normal (ratio of inflamed/control tissue 3.9). (⁹⁹m)Tc-labeled complexes of alpha thymosin derivatives are expected to facilitate research on alpha thymosins and accelerate exploitation of these peptides in immunotherapy protocols., (© 2012 John Wiley & Sons A/S.)

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., (© 2012 New York Academy of Sciences.)

A human serum albumin and Thymosin α1 (HSA-Tα1) fusion protein was designed and over-expressed in Pichia pastoris. To purify the fusion protein, a new native preparative electrophoresis system that involved a modified device with a sample receiving chamber, and an assay method with Coomassie Blue G-250 tracing the collection of the protein of interest. In this device, two gels were run in parallel: native vertical collecting polyacrylamide gel electrophoresis (PAGE) and native vertical tracing PAGE. Samples mixed with or without Coomassie Blue G-250 loading buffer were separately loaded to the two aforementioned gels, and the fractions were collected until the tracing protein band combined with dye reached 1 cm from the sample-receiving chamber at the bottom of the gel. Approximately nine fractions were collected at regular intervals of 15 min. HSA-Tα1 fusion protein with 95% relative homogeneity was harvested and manifested similar immunological activities as synthetic Tα1 after a single-step purification of this preparative PAGE. As a result, this system offers a new, rapid and simple method for the purification of the protein of interest.

A sustained release formulation for the therapeutic peptide thymosin β4 (Tβ4) that can be localized to the heart and reduce the concentration and frequency of dose is being explored as a means to improve its delivery in humans. This review contains concepts involved in the delivery of peptides to the heart and the synthesis of polymer microspheres for the sustained release of peptides, including Tβ4. Initial results of poly(lactic-co-glycolic acid) microspheres synthesized with specific tolerances for intramyocardial injection that demonstrate the encapsulation and release of Tβ4 from double-emulsion microspheres are also presented., (© 2012 New York Academy of Sciences.)

Thymosin β4 (Tβ4) is a peptide with multiple biological functions. Here, we focus on the role of Tβ4 in vascularization, and review our studies of the controlled delivery of Tβ4 through its incorporation in biomaterials. Tβ4 promotes vascularization through VEGF induction and AcSDKP-induced migration and differentiation of endothelial cells. We developed a collagen-chitosan hydrogel for the controlled release of Tβ4 over 28 days. In vitro, the Tβ4-encapsulated hydrogel increased migration of endothelial cells and tube formation from epicardial explants that were cultivated on top of the hydrogel, compared to Tβ4-free hydrogel and soluble Tβ4 in the culture medium. In vivo, subcutaneously injected Tβ4-containing collagen-chitosan hydrogel in rats led to enhanced vascularization compared to Tβ4-free hydrogel and collagen hydrogel with Tβ4. Furthermore, the injection of the Tβ4-encapsulated hydrogel in the infarct region improved angiogenesis, reduced tissue loss, and retained left ventricular wall thickness after myocardial infarction in rats., (© 2012 New York Academy of Sciences.)

Evolution probably invented the β-thymosin domain in a single-celled close relative of multicellular animals. Expansion from single genes to the small family of monomeric β-thymosins of present-day vertebrates may have started with a very ancient duplication, before the rounds of whole-genome duplications. In land vertebrates and fish, this family consists of the descendants of five genes of their jawed vertebrate common ancestor. Identifying this common ancestry depends on the genes possessing conserved sets of flanking sequences, as the relationships are not recognizable from amino-acid sequences. One of these genes has given rise both to a group of fish β-thymosins and to a hitherto unrecognized group of β-thymosins of birds and reptiles. The resulting classification may prove useful in relation to the β-thymosins of model organisms, such as the zebrafish, and for identifying important noncoding sequence elements, exemplified here by a conserved sequence in the 3'untranslated region of transcripts from the β4 subfamily., (© 2012 New York Academy of Sciences.)

The formulation TB-500 is suspected to be used as doping agent in sport. This work describes the detection and the identification of the N-terminal acetylated 17-23 fragment of human thymosin beta 4 (Ac-LKKTETQ) in TB-500 by means of high-performance liquid chromatography/high resolution mass spectrometry using an Orbitrap Exactive benchtop mass spectrometer. Ac-LKKTETQ was also synthesized by solid-phase peptide synthesis, and an analytical strategy for detection in plasma and urine by high-performance liquid chromatography/low resolution triple-quadrupole mass spectrometry was suggested.

Thymosin β4 (Tβ4) is a small, low-molecular-weight peptide ubiquitously expressed in all cells and extracellular fluids. It is a major actin sequestering protein present in the cells. In addition to this, Tβ4 has also been shown to be involved in endothelial cell migration, angiogenesis, corneal wound healing, and stem cell differentiation. It is also released by platelets after activation. The amount of Tβ4 increases at sites of injury and thus suggests an important role of this biopeptide in wound healing. Herein, we provide an overview of the role of Tβ4 in thrombosis and platelet aggregation.

Thymosin α1-thymopentin (Tα1-TP5) fusion peptide has been proved to be an immune regulator based on its higher immunoregulatory activity than Tα1 and TP5. To obtain Tα1-TP5 more effectively and economically, Tα1-TP5 was genetically fused to a self-cleaving intein-chitin binding domain tag for purification via chitin beads in Escherichia coli. After affinity purification, the target peptide was released from the chitin beads via self-cleaving intein ((INTervening protEIN) induced by dithiothreitol. Further, Tα1-TP5 was purified by Superdex 30 and identified by Tricine-SDS-PAGE and electrospray ionization-mass spectrometry. Finally, about 7.6 mg Tα1-TP5 purified from the soluble fraction and inclusion bodies was obtained from 1 L culture media. The purity was 95% after a series of chromatographic purification steps. In vitro, the purified Tα1-TP5 could stimulate the proliferation of mouse splenic lymphocytes. Overall, this work demonstrated that Tα1-TP5 was purified with low cost and high efficiency, greatly expanding its potential use as an immune regulator., (Copyright © 2012 Elsevier Inc. All rights reserved.)

β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone., (Copyright © 2011 Elsevier Inc. All rights reserved.)

800 MHz NMR structure of the 28-residue peptide thymosin alpha-1 in 40% TFE/60% water (v/v) has been determined. Restrained molecular dynamic simulations with an explicit solvent box containing 40% TFE/60% TIP3P water (v/v) were used, in order to get the 3D model of the NMR structure. We found that the peptide adopts a structured conformation having two stable regions: an alpha-helix region from residues 14 to 26 and two double β-turns in the N-terminal twelve residues which form a distorted helical structure., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rapid vascularization at the infarcted site is crucial for cardiac repair following myocardial infarction. Thymosin β4 (Tβ4), a 43-amino acid peptide, is both angiogenic and cardioprotective. Tβ4 in soluble form was previously shown to promote cell migration from quiescent adult cardiac explants. Here we developed a collagen-chitosan hydrogel for the encapsulation of Tβ4, which allowed its controlled release over 28days to elicit localized and prolonged effects. Contrastingly, Tβ4 was fully released over 3days when encapsulated in collagen-only hydrogels due to charge repulsion and lack of interconnected pores as shown by SEM. The charge of encapsulated molecules affected their release from collagen-chitosan hydrogels. While the release of neutral polyalanine was size-controlled diffusion, that of negatively-charged Tβ4 and positively-charged polylysine was affected by electrostatic interactions of peptides with collagen/chitosan molecules. Hydrogels with encapsulated Tβ4 significantly increased cell migration and outgrowth of CD31-positive capillaries from mouse and rat epicardial explants in vitro, compared to Tβ4-free and soluble controls. Potential advantage of Tβ4 over commonly-used angiogenic growth factors is that it can induce recruitment and differentiation of both endothelial and smooth muscle cells necessary for vascular stability. Importantly, Tβ4-encapsulated collagen-chitosan hydrogels promoted angiogenesis in vivo upon subcutaneous injection, compared to collagen-only hydrogels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

By an unknown mechanism, β-thymosins are extracellular modulators of angiogenesis, inflammation, wound healing, and development. We were interested in identifying β-thymosin interactors and determining their importance in β-thymosins signaling in human vein endothelial cells (HUVECs). We performed pulldown experiments with biotinylated thymosin β-4 (Tβ4) in comparison to neutravidin beads alone and used mass spectrometric analysis to identify differentially interacting proteins. By this method, we identified F1-F0 ATP synthase, a known target of antiangiogenic angiostatin. By surface plasmon resonance, we determined for Tβ4 binding to the β subunit of ATP synthase a K(D) of 12 nM. Blocking antibodies and antagonists (oligomycin, IC(50) ∼1.8 μM; piceatannol, IC(50) ∼1.05 μM; and angiostatin, IC(50) ∼2.9 μg/ml) of ATP synthase inhibited the Tβ4-induced increase in cell surface ATP levels, as measured by luciferase assay, and the Tβ4-induced increase in HUVEC migration, as measured by transwell migration assay. Silencing of the ATP-responsive purinergic receptor P2X4 with siRNA also blocked Tβ4-induced HUVEC migration in a transwell assay. Furthermore, in silico we identified common amphiphilic α-helical structural similarities between β-thymosins and the inhibitory factor 1 (IF1), an inhibitor of ATP synthase hydrolysis. In summary, we have identified an extracellular signaling pathway where Tβ4 increases cell surface ATP levels via ATP synthase and have shown further that ATP-responsive P2X4 receptor is required for Tβ4-induced HUVEC migration.

Prothymosin α (ProTα) is a multifunctional protein that, in mammalian cells, is involved in nuclear metabolism through its interaction with histones and that also has a cytosolic role as an apoptotic inhibitor. ProTα is phosphorylated by a protein kinase (ProTαK), the activity of which is dependent on phosphorylation. ProTα phosphorylation also correlates with cell proliferation. Mass spectrometric analysis of ProTαK purified from human tumor lymphocytes (NC37 cells) enabled us to identify this enzyme as the M2-type isoenzyme of pyruvate kinase. A study on the relationship between ProTαK activity and pyruvate kinase isoforms in NC37 cells and in other cell types confirmed that the M2 isoform is the enzyme responsible for ProTαK activity in proliferating cells. Yet, about 10% of the cellular pool of the M2 isoform shows specific affinity for ProTα and is responsible for ProTαK activity. This pool of M2 protein possesses no observable pyruvate kinase activity and changes its responses to various effectors of pyruvate kinase activity; however, these responses to PK effectors are maintained by the main cellular fraction containing the M2 isoform. Acquisition of ProTαK activity by M2 seems to be due to the phosphorylation of serine and threonine residues, which, besides being essential for its catalytic activity, induces a trimeric association of ProTαK. This association can be shifted to a tetrameric form by fructose 1, 6-bisphosphate, which results in a decrease in ProTαK activity., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProTα) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to neurodegenerative diseases, premature aging and cancer. In order to understand the mechanisms these two disordered proteins employ to bind to Keap1, we performed extensive 0.5-1.0 microsecond atomistic molecular dynamics (MD) simulations and isothermal titration calorimetry experiments to investigate the structure/dynamics of free-state ProTα and Neh2 and their thermodynamics of bindings. The results show that in their free states, both ProTα and Neh2 have propensities to form bound-state-like β-turn structures but to different extents. We also found that, for both proteins, residues outside the Keap1-binding motifs may play important roles in stabilizing the bound-state-like structures. Based on our findings, we propose that the binding of disordered ProTα and Neh2 to Keap1 occurs synergistically via preformed structural elements (PSEs) and coupled folding and binding, with a heavy bias towards PSEs, particularly for Neh2. Our results provide insights into the molecular mechanisms Neh2 and ProTα bind to Keap1, information that is useful for developing therapeutics to enhance the oxidative stress response.

Thymosin-β4 (Tβ4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tβ4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tβ4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tβ4., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The nuclear protein prothymosin-α (ProTα), which lacks a signal peptide sequence, is released from neurons and astrocytes on ischemic stress and exerts a unique form of neuroprotection through an anti-necrotic mechanism. Ischemic stress-induced ProTα release is initiated by a nuclear release, followed by extracellular release in a non-vesicular manner, in C6 glioma cells. These processes are caused by ATP loss and elevated Ca²(+), respectively. S100A13, a Ca²(+)-binding protein, was identified to be a major protein co-released with ProTα in an immunoprecipitation assay. The Ca²(+)-dependent interaction between ProTα and S100A13 was found to require the C-terminal peptide sequences of both proteins. In C6 glioma cells expressing a Δ88-98 mutant of S100A13, serum deprivation caused the release of S100A13 mutant, but not of ProTα. When cells were administered apoptogenic compounds, ProTα was cleaved by caspase-3 to generate a C-terminal peptide-deficient fragment, which lacks the nuclear localization signal (NLS). However, there was no extracellular release of ProTα. All these results suggest that necrosis-inducing stress induces an extacellular release of ProTα in a non-vesicular manner, whereas apoptosis-inducing stress does not, owing to the loss of its interaction with S100A13, a cargo molecule for extracellular release.

Linker histone H1 binds with high affinity to naked and nucleosomal DNA in vitro but is rapidly exchanged between chromatin sites in vivo suggesting the involvement of one or more linker histone chaperones. Using permeabilized cells, we demonstrate that the small acidic protein prothymosin alpha (ProTalpha) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTalpha levels in vivo by siRNA-mediated mRNA degradation resulted in a decreased rate of exchange of linker histones as assayed by photobleaching techniques. These results indicate that ProTalpha is a component of a linker histone chaperone., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been applied to the analysis of a wide range of biomolecules. To date, there are two specific areas of application where MALDI-TOF-MS is viewed as impractical: analysis of low-mass analytes and relative quantitative applications. However, these limitations can be overcome and quantification can be routine. Increased levels of thymosin beta(4) (TB4) have been recently found in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients. Our objective was to apply a label-free quantitative application of MALDI-TOF-MS to measure TB4 levels in human CSF by adding the oxidized form of TB4 as an internal standard. The relative peak area or peak height ratios of the native TB4 to the added oxidized form were evaluated. Considering the relative peak area ratios, healthy individuals showed a mean value of 40.8+/-21.27 ng/ml, whereas CJD patients showed high values with a mean of 154+/-59.07 ng/ml, in agreement with the previous observation found in CJD patients. Similar results were obtained considering peak height ratios. The proposed method may provide a simple and rapid screening method for quantification on CSF of TB4 levels suitable for diagnostic purposes., (2010 Elsevier Inc. All rights reserved.)

Thymosin beta(4), a small ubiquitous protein containing 43 aa, has structure/function activity via its actin-binding domain and numerous biological affects on cells. Since it is the major actin-sequestering molecule in eukaryotic cells and is found essentially in all cells and body fluids, thymosin beta(4) has the potential for significant roles in tissue development, maintenance, repair, and pathology. Several active sites with unique functions have been identified, including the amino-terminal site containing 4 aa (Ac-SDKP) that generally blocks inflammation and reduces fibrosis. Another active site at the amino terminus contains 15 aa, including Ac-SDKP, and promotes cell survival and blocks apoptosis, while a short sequence containing LKKTETQ, the central actin-binding domain (aa 17-23) plus 1 additional amino acid (Q), promotes angiogenesis, wound healing, and cell migration. Several additional biological activities have been identified but not yet localized in the molecule, including its antimicrobial activity, the induction of various genes (including laminin-5, MMPs, TGF beta, zyxin, terminal deoxynucleotidyl transferase, and angiogenesis-related proteins), and the ability to activate ILK/PINCH/Akt, and other signaling molecules important in both apoptosis and inflammatory pathways. This review details these important physiologically and pathologically active sites and their potential therapeutic uses.

Background: Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail., Results: To examine the HsjCib gene, full-length cDNAs were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) and sequencing. Multiple isoforms were identified, and on the basis of the results of northern and Southern hybridization analyses, these isoforms were considered to be transcriptional variants from a single gene. On the basis of their sequence similarity to homologous genes of other organisms, functions in actin assembly were assumed to be different among isoforms. Expression analysis revealed high expression in the head during soldier differentiation, which was consistent with their allometric growth. Although isoform expression was observed in various tissues, different expression levels were observed among tissues, suggesting the possibility of tissue-specific morphogenetic regulation by HsjCib isoforms., Conclusion: This study revealed the characteristics and dynamics of the HsjCib gene during soldier differentiation as a potential representative of downstream effector genes in caste-specific morphogenesis. From the expression patterns observed, this gene is considered to be involved in cephalic morphogenesis and neural reorganization, resulting in the establishment of caste-specific morphology and behavior.

Human thymosin alpha1 is an effective immune system enhancer for the treatment of cancer and viral diseases. Therefore the development of new methods for its synthesis is an urgent problem. In the present work, we propose an efficient scalable scheme for the production of recombinant thymosin alpha1. We used an expression system based on the pET32b+ plasmid and Escherichia coli strain ER2566 to obtain a fusion protein consisting of thymosin alpha1 and thioredoxin separated by a TEV (tobacco etch virus) protease cleavage site. The fusion protein was overexpressed in soluble form and purified by ion-exchange chromatography. After proteolytic cleavage of the fusion protein with TEV protease, recombinant desacetylthymosin alpha1 was isolated by ultrafiltration. Acetic anhydride was used for selective N-terminal acetylation of the obtained peptide (yield=62%). The resultant thymosin alpha1 was purified by RP-HPLC (reversed-phase HPLC). The distinctive feature of this technology is that it is a combination of different approaches: the biotechnological production of recombinant fusion protein, its enzymatic cleavage, and chemical acetylation of desacetylthymosin alpha1. Each stage of the process was optimized to increase the yield of the target peptide, which averaged 29 mg/litre of bacterial culture. The proposed method is simple and cost-effective and is suitable for large-scale production of recombinant thymosin alpha1.