Verifiable Hypotheses for Thymosin β4-Dependent and -Independent Angiogenic Induction of Trichinella spiralis-Triggered Nurse Cell Formation

Trichinella spiralis has been reported to induce angiogenesis for nutrient supply and waste disposal by the induction of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. However, the action mechanism to induce VEGF in nurse cells by T. spiralis is not known. Hypoxia in nurse cells was suggested as a possible mechanism; however, the presence of hypoxic conditions in infected muscle or nurse cells and whether hypoxia indeed induces the expression of VEGF and subsequent angiogenesis in the infected muscle are both a matter of debate. Our recent studies have shown that thymosin β4, a potent VEGF inducing protein, is expressed in the very early stages of T. spiralis muscle infection suggesting the induction of VEGF in early stage nurse cells. Nevertheless, we now show that hypoxic conditions were not detected in any nurse cell stage but were detected only in the accumulated inflammatory cells. These studies propose that induction of angiogenesis by VEGF in T. spiralis-infected nurse cells was mediated by thymosin β4 and is unrelated to hypoxic conditions.     

固相片段缩合法制备胸腺素α_1的工艺

以固相多肽合成(SPPS)为基础,应用固相片段合成方法制备胸腺素α1。系统考察了不同分段方法、缩合时间、反应溶剂、缩合剂及乙酰化的不同顺序对片段缩合反应的影响。通过试验确定了最佳合成工艺条件:采用三段(1~10、11~18和19~28)分别合成方式,以V(DMF)/V(DCM)等于3.0的混合溶液为反应溶剂,HATU为缩合剂,N末端氨基先乙酰化后进行片段间缩合的途径进行合成,多肽片段间缩合反应时间采用20.0 h。该条件下合成的胸腺素α1,粗品收率达64.3%,纯度达61.1%,与逐步合成法相比较,产物纯度提高了29.9%。     

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.     

高纯胸腺肽α1的制备

为了获得高纯产品,在制备胸腺肽α1(Tα1)工艺中分别对合成、纯化两步骤进行控制。在固相多肽合成过程中,以TBTU/HOBt/DIEA体系作为缩合试剂,在易形成α螺旋和β转角的肽序位置,通过采用HOBt/DIC补投的方法促使偶联反应完全。在分离纯化阶段,结合反相色谱和离子色谱的优势,以反相离子对液相色谱法为主要分离手段,通过多步分离实现纯化。获得的粗肽纯度为67.2%,胸腺肽α1产品纯度达99.5%以上,总收率为17.2%。此法可作为制备高纯多肽药物的有效手段。     

Potential Role of Thymosin Beta 4 in Liver Fibrosis

Liver fibrosis, the main characteristic of chronic liver diseases, is strongly associated with the activation of hepatic stellate cells (HSCs), which are responsible for extracellular matrix production. As such, investigating the effective regulators controlling HSC activation provides important clues for developing therapeutics to inhibit liver fibrosis. Thymosin beta 4 (Tβ4), a major actin-sequestering protein, is known to be involved in various cellular responses. A growing body of evidence suggests that Tβ4 has a potential role in the pathogenesis of liver fibrosis and that it is especially associated with the activation of HSCs. However, it remains unclear whether Tβ4 promotes or suppresses the activation of HSCs. Herein, we review the potential role of Tβ4 in liver fibrosis by describing the effects of exogenous and endogenous Tβ4, and we discuss the possible signaling pathway regulated by Tβ4. Exogenous Tβ4 reduces liver fibrosis by inhibiting the proliferation and migration of HSCs. Tβ4 is expressed endogenously in the activated HSCs, but this endogenous Tβ4 displays opposite effects in HSC activation, either as an activator or an inhibitor. Although the role of Tβ4 has not been established, it is apparent that Tβ4 influences HSC activation, suggesting that Tβ4 is a potential therapeutic target for treating liver diseases.     

猪心中提取胸腺素的研究

实验选取猪心为原料,探讨提取胸腺素活性物质的可能性及方法,从而找出一种廉价的原料来源,并提高猪心的利用价值.试验表明采用有机溶剂萃取法可以从中提出胸腺素物质,综合考虑相关的因素和条件的影响,通过做正交实验,确定出较佳的胸腺素提取工艺条件为：pH值为3.0,水浴加热温度为85℃,水浴加热时间为10min.     

转基因聚球藻7942培养中光传递及其对细胞生长的影响

对转人源胸腺素a1基因聚球藻7942光生物反应器分批培养过程中光的分布、平均光强变化及其对藻细胞生长的影响进行了分析. 结果表明，Hyperbolic光衰减模型较之Lambert-Beer光衰减模型能更好地描述转基因聚球藻7942培养液中的光衰减；随着藻细胞密度的增加，光生物反应器内平均光强不断减小，藻细胞的光能比吸收速率也不断降低，反应器中“暗区”体积不断加大；培养时间0~1.5 d，藻细胞能获得充足光能，因而呈指数生长；1.5 d后藻细胞处于光限制，比生长速率不断下降，但体积光能吸收速率达到最大并保持恒定，藻细胞进入线性生长期；5 d后光生物反应器“暗区”体积超过了反应器总体积的50%，藻细胞吸收的光能用于维持的比例不断增加，从而导致生长速率下降；培养8 d，转基因聚球藻7942中人源胸腺素a1表达量为4.53 mg/L.     

Recombinant Human Thymosin β4 (rhTβ4) Modulates the Anti-Inflammatory Responses to Alleviate Benzalkonium Chloride (BAC)-Induced Dry Eye Disease

Dry eye disease (DED) is a multifactorial ocular disorder that interferes with daily living and reduces quality of life. However, there is no most ideal therapeutic treatment to address all the deleterious defects of DED. The purpose of this study was to investigate the ability of recombinant human thymosin β4 (rhTβ4) to promote healing in a benzalkonium chloride (BAC)-induced mice DED model and the anti-inflammatory effects involved in that process. Eye drops consisting of 0.05% and 0.1% rhTβ4 were used for treatment of DED. Tear volume and corneal staining scores were measured after 7 days. Periodic acid-Schiff staining for gobleT cells in conjunctiva, immunohistochemical staining for CD4⁺ T cells, TUNEL assay for apoptotic positive cells in cornea and conjunctiva, qRT-PCR and ELISA assays for multiple cytokines were performed. All clinical parameters showed improvement in both the 0.05% and 0.1% rhTβ4 groups. Specifically, topical application of rhTβ4 significantly increased conjunctival gobleT cells and reduced apoptotic cells in conjunctiva. Mechanically, the rhTβ4 groups showed significantly reduced inflammatory cytokine levels and CD4⁺ T cells in conjunctiva by blocking NF-κB (nuclear factor kappa B) activation, suggesting that 0.05–0.1% rhTβ4 eye drops may be used as a potential therapeutic treatment for DED.     

Synthesis of thymosin β4Xen and its comparison with the natural tritetraconpeptide

Thymosin β₄^Xen, a 43 residue peptide recently isolated from Xenopus laevis, was synthesized by automatic solid phase procedure and compared with the natural product, isolated from the ovaries of Xenopus laevis For the synthesis N-methylpyrrolidone was chosen as solvent instead of the commonly used dimethylformamide because this solvent seems to be superior for solid phase peptide synthesis due to the favorable swelling properties of the polystyrene resin in this solvent and its dissolving power against the resin-bound peptide which reduces intermolecular aggregation. With acetic anhydride/pyridine and hydroxysuccinimide acetate two different acetylation reagents were tested for the final acetylation step, which gave both comparable results as shown by analytical HPLC investigations. The crude synthetic product was purified by HPLC, confirmed by ASA and LD-MS and was identical compared with the natural thymosin β₄^Xen