Novel aminopeptidase N inhibitors derived from antineoplaston AS2–5 (Part I)

Overexpression of zinc-dependent metalloproteinase, aminopeptidase N (APN/CD13), is considered to be involved in the process of tumor invasion and metastasis. Herein we describe the synthesis and in vitro enzymatic inhibition assay of antineoplaston AS2–5 scaffold peptidomimetic compounds. The results demonstrated that most of these l-iso-glutamine derivatives displayed selective inhibitory activity against APN as compared with MMP-2, with IC₅₀ values in the micromole range. The structure–activity relationships were also briefly discussed.     

GTPase Activity of MxB Contributes to Its Nuclear Location,Interaction with Nucleoporins and Anti-HIV-1 Activity

The human myxovirus resistance 2(Mx2/Mx B) protein, a member of interferon(IFN)-inducible dynamin-like large GTPases, restricts a number of virus infections. Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization. However, the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1. To understand the role of Mx B GTPase activity, including GTP binding and GTP hydrolysis, in restriction of HIV-1 infection, we genetically separated these two functions and evaluated their contributions to restriction. We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication. The GTPase activity of Mx B contributed to its nuclear location, interaction with nucleoporins(NUPs) and HIV-1 capsids. Furthermore, Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity. The function of GTPase activity was therefore multi-faceted, led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.     

Frequency Analysis and Anti-Shock Mechanism of Woodpecker＇s Head Structure

The mechanical properties of the skull and the anti-shock characteristics of woodpecker＇s head were investigated by ex- periment and numerical simulation. We measured the micro-Young＇s modulus of the skull by nano-indentation method and calculated the macro-equivalent Young＇s modulus of the skull at different positions using homogenization theory. Based on the Computerized Tomography （CT） images of woodpecker head, we then built complete and symmetric finite element models of woodpecker＇s skull and its internal structure and performed modal analysis and stress spectrum analysis. The numerical results show that the application of pre-tension force to the hyoid bone can increase the natural frequency of woodpecker＇s head. The first natural frequency under the pre-tension force of 25 N reaches 57 Hz, which is increased by 21.3% from the non-pre-tension state and is more than twice the working frequency of woodpecker （20 Hz 25 Hz）. On the application of impact force to the tip of beak for 0.6 ms, high magnitudes of stress component occur at around 100 Hz and 8,000 Hz, far away from both the working frequencies and the natural frequencies of woodpecker head. The large gaps among the natural, working and stress response frequencies enable the woodpecker to effectively protect its brain from the resonance injury.     

Morusin induces apoptosis and suppresses NF-kappaB activity in human colorectal cancer HT-29 cells

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G₁ phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB.     

白介素-1β对高糖刺激的人肾小管上皮细胞转分化的影响

目的探讨炎症细胞因子白介素-1β（interleukin-1βIL-1β）对高糖刺激的人肾小管上皮细胞转分化的影响。-方法体外培养人肾近曲小管上皮细胞株（HKCs）,随机分为正常对照组（5.5 mmol/L normal glucose）;高糖组（30 mmol/L high glucose）;高糖＋IL-1β（5ng/ml）组。分别于处理后24h、48h、72h收集细胞,采用免疫细胞化学染色和Western蛋白印迹法检测细胞角蛋白-18（cytokeratin-18 CK-18）、α-平滑肌肌动蛋白（α-smooth muscle actinα-SMA）水平。结果高糖能够诱导肾小管上皮细胞α-SMA蛋白的合成增加,而肾小管上皮细胞的标志物CK-18的表达逐渐减少;IL-1β与高糖同时刺激可使肾小管上皮细胞α-SMA蛋白表达进一步增多,而其自身标志物CK-18的表达则明显下降。结论炎症因子IL-1β能增强高糖对肾小管上皮细胞转分化的作用。     

Osmolyte-induced protein folding free energy changes

Upon addition of protecting osmolyte to an aqueous solution of an intrinsically unstructured protein, spectral observables are often seen to change in a sigmoid fashion as a function of increasing osmolyte concentration. Commonly, such data are analyzed using the linear extrapolation model (LEM), a method that defines a scale from 0%-100% folded species at each osmolyte concentration by means of extending pre- and post-folding baselines into the transition region. Defining the 0%-100% folding scale correctly for each osmolyte is an important part of the analysis, leading to evaluation of the fraction of folded protein existing in the absence of osmolytes. In this study, we used reduced and carboxyamidated RNase T1 (RCAM-T1) as an intrinsically unstructured protein, and determined the thermodynamic stability of RCAM-T1 induced by naturally occurring osmolytes. Because the folded fraction of the protein population determined by experiments of thermal and urea-induced denaturation is nonzero in the absence of osmolytes at 15 degrees C, the commonly used LEM can lead to false values of DeltaG[stackD-->N0] for protein folding due to the arbitrary assumption that the protein is 100% unfolded in the presence of buffer alone. To correct this problem, titration of the protein solution with urea and extrapolating back to zero urea concentration gives the spectral value for 100% denatured protein. With fluorescence as the observable we redefine F/F0 to F/F0extrap = 1.0 and require that the denatured-state baseline have this value as its intercept. By so doing, the 0%-100% scale-corrected DeltaG[D-->N0] values of RCAM-T1 folding in the presence of various osmolytes are then found to be identical, with small error, demonstrating that DeltaG[D-->N0] is independent of the osmolytes used. Such a finding is an important step in validating this quantity derived from the LEM as having the properties expected of an authentic thermodynamic parameter. The rank order of osmolyte efficacies in stabilizing RCAM-T1 is sarcosine > sucrose > sorbitol > proline > betaine > glycerol.     

基于细菌群感效应人工构建分子开关

对生物体内已有的或者人工组装的生物合成途径进行优化操作涉及两个重要问题:代谢途径中关键酶的活性及蛋白表达水平。对于酶表达水平的研究,传统的做法是采用强启动子控制下的靶蛋白过量表达策略。靶蛋白的过量表达通常会导致细胞内积累大量的无活性包涵体,从而严重影响细胞的生理状态和相关生物途径的有效运转。针对这一问题,设计一种分子开关来精确调控生物合成过程中关键酶的表达水平,对于研究生物合成途径的代谢节律以及促进生物合成途径高效运转都具有重要的实用价值。基于细菌群落中普遍存在群感效应的基本原理并结合酶促催化的动力学特征,首先在大肠杆菌群落中建立信号分子高丝氨酸内酯(AHL)介导的细胞–细胞交流机制,将靶基因egfp置入到启动子PluxI的控制之下。在细胞生长过程中,产生的AHL累积到一定浓度启动靶基因表达。通过在细胞生长的不同阶段启动AHL降解酶AiiA的表达控制环境中信号分子AHL的浓度水平,从而控制靶基因egfp的转录效率,最终实现对靶蛋白EGFP表达水平的精确控制。通过检测细胞的生长状态、靶基因在mRNA水平、蛋白质水平的表达情况证明人工设计的分子开关可以便捷高效地控制靶基因表达水平,具有时空调节的严谨性。该分子开关有望广泛应用于代谢工程和合成生物学等研究领域中。     

Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa

Chronic lung infection by mucoid Pseudomonas aeruginosa is one of the major pathologic features in patients with cystic fibrosis. Mucoid P.?aeruginosa is notorious for its biofilm forming capability and resistance to immune attacks. In this study, the roles of extracellular polymeric substances from biofilms formed by mucoid P.?aeruginosa were investigated. Alginate is not an essential structure component for mucoid P.?aeruginosa biofilms. Genetic studies revealed that Pel and Psl polysaccharides serve as essential scaffold and mediate macrocolony formation in mucoid P.?aeruginosa biofilms. The Psl polysaccharide is more important than Pel polysaccharide in mucoid P.?aeruginosa biofilm structure maintenance and phagocytosis resistance. The polysaccharides were further found to protect mucoid P.?aeruginosa strain from host immune clearance in a mouse model of acute lung infection.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

不同型别的基因工程干扰素抗病毒活性的比较

对大肠杆菌生产的不同型别的基因工程干扰素rIFN-α1(α1)、rIFN-αA(αA)、rIFN-β17ser(β17ser)和rIFN-γ(γ),以及自然人白细胞干扰素nIFN-αco,在不同细胞上对不同病毒的抗病毒活性做了比较研究。证明:①α1抗病毒作用的细胞谱较广,尤其在牛肾MDBK细胞和猪肾PK细胞上有很高的活性,分别为在人细胞上的29倍和7倍。β17ser和γ在异种细胞上活性极低,在鼠、猪和牛肾细胞上的活性为人细胞上的1～2％以下。②5种干扰素对麻疹、CoxB1、Sindbis、腺病毒7型和Ⅰ、Ⅱ型单纯疱疹病毒的抗病毒活性无明显差异。但不同病毒对干扰素的敏感性有明显差别,以Sindbis病毒为最敏感,7型腺病毒最不敏感。③5种干扰素对流行性出血热病毒均有明显的抗病毒作用,尤以人α1型和β干扰素作用最强,α1对出血热病毒的抗病毒活性是对滤泡性口膜炎病毒(VSV)的1/2.85,人β干扰素是对VSV的1/4.1。上述结果为人基因工程干扰素的临床应用提供了实验依据。