具备定点偶联功能的HBc-VLPs的制备与性质鉴定

乙型肝炎病毒核心蛋白HBc,可在体外自组装形成二十面体对称结构的病毒样微粒VLPs。VLPs可将外源序列重复且高密度地展示在表面,VLPs进入机体后能够快速诱导机体产生针对外源性抗原的特异性体液免疫及细胞免疫应答,具有极强的免疫原性与生物活性。因此,HBc-VLPs可以作为一种安全、有效的疫苗载体。文中设计了一种能够实现与抗原定点偶联的HBc-VLPs,并开发了一套高效制备HBc-VLPs的方法。通过定点突变技术,使翻译后的多肽序列第80位氨基酸由Ala变为Cys,在HBc-VLPs的主要免疫显性区域引入一个定点交联位点,构建了原核表达载体pET28a(+)-hbc,表达、纯化获得了高纯度的HBc(A80C) 单体蛋白;在PB缓冲体系中,HBc(A80C) 蛋白自组装形成HBc-VLPs纳米粒子。粒度仪的测定结果表明,HBc-VLPs纳米微粒的平均粒径为29.8 nm,透射电子显微镜观察到HBc-VLPs形成粒径约为30 nm的球形微粒,其形态与天然的HBV微粒相似。以流感病毒M2e抗原肽为模式抗原,通过Sulfo-SMCC氨基-巯基双功能交联剂,将M2e定点连接于HBc-VLPs通过突变引入的Cys残基处,制备了M2e-HBc-VLPs模式疫苗,通过细胞荧光示踪,验证了HBc-VLPs结构的完整性与M2e的正确交联。动物免疫实验表明该疫苗能够有效刺激小鼠产生抗原特异性的IgG抗体,验证了疫苗载体HBc-VLPs的有效性。研究结果为HBc-VLPs作为疫苗载体的研究奠定了基础,能够促进HBc-VLPs载体疫苗的研发以及HBc-VLPs在其他领域的应用。     

Antibacterial Diphenyl Ether,Benzophenone and Xanthone Derivatives from Aspergillus flavipes

The extract of the strain Aspergillus flavipes DL‐11 exerted antibacterial activities against six Gram‐positive bacteria. During the following bioassay‐guided separation, ten diphenyl ethers ( 1 – 10 ), two benzophenones ( 11 – 12 ), together with two xanthones ( 13 – 14 ) were isolated. Among them, 4′‐chloroasterric acid ( 1 ) was a new chlorinated diphenyl ether. Their structures were elucidated by extensive spectroscopic data analysis, including IR, HR‐ESI‐MS, NMR experiments, and by comparison with the literature data. All compounds showed moderate to strong antibacterial effects on different Gram‐positive bacteria with MIC values that ranged from 3.13 to 50 μg/mL, but none of the compounds exhibited activity against Gram‐negative bacteria Vibrio parahaemolyticus ATCC17802 (MIC>100 μg/mL). In particular, the MICs of some compounds are at the level of positive control.     

Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti‐tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti‐tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME—such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell‐based microenvironment therapies—to provide novel ideas for producing breakthroughs in tumour therapy strategies.     

Comparative proteomic analysis of apomictic monosomic addition line of <Emphasis Type="Italic">Beta corolliflora</Emphasis> and <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. in sugar beet

Apomixis refers to a process in which plants produce seed without fertilization through female syngamy that produces embryos genetically identical to the maternal parent. In sugar beet, interspecific hybrids between diploid Beta vulgaris and tetraploid Beta corolliflora were established and monosomic addition line M14 was selected because of the apomictic phenotype. By using two-dimensional electrophoresis gels we identified the proteins which were differently expressed between the M14 and B. vulgaris. A total of 27 protein spots which varied expressed between lines were isolated and successfully identified with MALDI-TOF MS. Among them five protein spots were found to be only presented in M14 and two protein spots only expressed in Beta. According to their functional annotations described in Swissprot database, these proteins were, respectively, involved in important biological pathways, such as cell division, functionally classified using the KEGG functional classification system. The result may be useful for us to better understand the genetic mechanism of apomixes.     

生长期对不同品种(系)木薯品质特性的影响

本文研究了不同生长期对不同品种(系)木薯含粉率、氢氰酸和单宁含量等品质特性的影响.结果表明,随着生长期的延长,木薯含粉率呈现先增加后下降的趋势,氢氰酸和单宁含量的变化趋势因品种(系)而异.品种南植在生长7个月收获时,木薯淀粉含量高,氢氰酸和单宁含量较低,品种D、SC和ZM在生长9个月收获时,木薯淀粉含量高,氢氰酸和单宁含量较低,因此适合食品用淀粉的深加工;品种C和E在生长8个月收获时,淀粉含量高,但氢氰酸和单宁含量也较高.本研究探明了不同生长期和品种对鲜木薯含粉率、单宁和氢氰酸含量的影响,为合理选择生长期和木薯品种提供理论支撑.     

Wheat germ poly(A)-binding protein increases the ATPase and the RNA helicase activity of translation initiation factors eIF4A, eIF4B, and eIF-iso4F

Recent studies demonstrated that wheat germ poly(A)-binding protein (PABP) interacted with translation eukaryotic initiation factor (eIF)-iso4G and eIF4B, and these interactions increased the poly(A) binding activity of PABP (Le, H., Tanguay, R. L., Balasta, M. L., Wei, C. C., Browning, K. S., Metz, A. M., Goss, D. J., and Gallie, D. R. (1997) J. Biol. Chem. 272, 16247-16255) and the cap binding activity of eIF-iso4F (Wei, C. C., Balasta, M. L., Ren, J., and Goss, D. J. (1998) Biochemistry 37, 1910-1916). We report here that the interaction between PABP and eIF-iso4G has a substantial effect on the ATPase activity and RNA helicase activity of (eIF4A + eIF4B + eIF-iso4F) complex. ATPase kinetic assays show, in the presence of poly(U), PABP can increase the parameter (k(cat)/K(m)) by 3.5-fold with a 2-fold decrease of K(m) for the (eIF4A + eIF-iso4F) complex. In the presence of globin messenger RNA, the ATPase activity of the complex (eIF4A + eIF-iso4F) was increased 2-fold by the presence of PABP. RNA helicase assays demonstrated that the presence of PABP enhanced the RNA duplex unwinding activity of the initiation factor complex. These results suggest that, in terms of the scanning model of translation initiation, PABP may enhance the mRNA scanning rate of the complex formed by eIF4A, eIF4B, and eIF4F or eIF-(iso)4F and increase the rate of translation.     

XBP1 depletion precedes ubiquitin aggregation and Golgi fragmentation in TDP‐43 transgenic rats

Protein inclusion is a prominent feature of neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) that is characterized by the presence of ubiquitinated TDP‐43 inclusion. Presence of protein inclusions indicates an interruption to protein degradation machinery or the overload of misfolded proteins. In response to the increase in misfolded proteins, cells usually initiate a mechanism called unfolded protein response (UPR) to reduce misfolded proteins in the lumen of endoplasmic reticules. Here, we examined the effects of mutant TDP‐43 on the UPR in transgenic rats that express mutant human TDP‐43 restrictedly in the neurons of the forebrain. Over‐expression of mutant TDP‐43 in rats caused prominent aggregation of ubiquitin and remarkable fragmentation of Golgi complexes prior to neuronal loss. While ubiquitin aggregates and Golgi fragments were accumulating, neurons expressing mutant TDP‐43 failed to up‐regulate chaperones residing in the endoplasmic reticules and failed to initiate the UPR. Prior to ubiquitin aggregation and Golgi fragmentation, neurons were depleted of X‐box‐binding protein 1 (XBP1), a key player of UPR machinery. Although it remains to determine how mutation of TDP‐43 leads to the failure of the UPR, our data demonstrate that failure of the UPR is implicated in TDP‐43 pathogenesis.     

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells

Background

In previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified.

Methods

A novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro.

Results

LIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane.

Conclusions

These observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.

     

A novel ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

A novel β-glucosidase (G-II) was purified to homogeneity from a culture filtrate of the phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-II specifically cleaved the β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb₁ to produce ginsenoside Rd, but did not hydrolyze the other β-d-glucosidic linkages in protopanaxadiol-type ginsenosides. In specificity tests, G-II was active against pNPG and disaccharides such as cellobiose and gentiobiose, but exhibited very low activities against other aryl-glycosides and methyl-α-glycosides. G-II consisted of two identical subunits with a native molecular mass of 180 kDa and a pI of 4.4. The optimal pH of G-II was pH 5.5, and the enzyme was highly stable over a range of pH 5.0–11.0. The optimal temperature was 45 °C, and the enzyme became unstable at temperatures above 40 °C. The K_m and V_max values against pNPG were 0.19 mM and 57.7 μmol/(min mg), respectively. The enzyme was inhibited by Zn²⁺, Cu²⁺ (over 50 mM) and SDS (250 mM). However, the inhibition by SDS was partially reversed by 10 mM dithiothreitol. Three oligopeptide fragments obtained after enzymatic digestion of G-II were sequenced by nanoESI-MS/MS. The amino acid sequence homology analysis showed that G-II possessed significant homology with the family 3 β-glucosidases.