An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

Two polypeptide chain constituents of the major protein of the cornified layer of newborn rat epidermis.

The insoluble component of stratum corneum of rat epidermis yields two major bands after extraction with 8 M urea-mercaptoethanol-dithiothreitol. The ratio of these two bands is about 1:1 in terms of protein stain intensity and S-[14C]carboxymethyl label. Both polypeptides were purified to homogeneity by DE-52-cellulose, sodium dodecyl sulfate hydroxylapatite C column chromatography, and preparative DodSO4-polyacrylamide gel electrophoresis. The heavier polypeptide contains 30% alpha helix and the lighter contains 27% alpha helix as determined by circular dichroism studies. Both are sensitive to Pronase and resistant to trypsin, collagenase, and elastase. The lighter chain is stable to pepsin but the heavier can be partially degraded to a smaller polypeptide with a molecular weight similar to that of light chain. Amino acid analysis shows that the light chain contains 12 more tyrosine residues than does the heavy chain, suggesting that the light chain is not generated from the heavy chain. However, the two chains may have a common peptide region. Antiserum prepared against the heavier polypeptide can be completely absorbed by purified lighter polypeptide and vice versa indicating that both chains have some common antigenic determinants. Antibody against either chain can cross-react with the stratum corneum and keratohyalin granules in the epidermis of newborn rat as indicated by fluorescent microscopic observation. Similarly, this antibody also cross-reacts with the cell surface or the contents of spinous and granular cells, and very weakly with basal cells, indicating that the two proteins may be present in the lower strata as well as the stratum corneum.     

Synthesis of myosin heavy and light chains in muscle cultures

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.     

Isolement du vernodalin et du vernolepin a partir de Vernonia guineensis: Authenticité du squelette elemane

The isolation of vernoladin and vernolepin is reported. The authenticity of the occurrence of the elemane skeleton in nature is discussed, and a biosynthetic scheme concerning the genus Vernonia is proposed.     

蚯蚓作用下污泥重金属形态变化及其与化学生物学性质变化的关系

城市污泥处理是一项世界性难题,污泥农业利用是其最简单有效的资源化利用方式之一,但污泥中较高的重金属含量限制了其实际推广应用,利用蚯蚓-超富集植物联合修复污泥重金属的方法已引起国内外研究者的关注。以新鲜城市脱水污泥为研究对象,接种赤子爱胜蚓(Eisenia fetida)进行室内培养试验,系统研究蚯蚓作用下污泥重金属形态的变化,及其与污泥氧化还原条件、化学和微生物性质变化的关系,以期为蚯蚓-超富集植物联合修复技术在污泥重金属处理中的应用提供理论依据。结果表明,试验前期蚯蚓在污泥中能正常生长和存活,前20 d总生物量增加了52%。蚯蚓可以显著促进污泥中的Cu、Zn、Cd、Ni等重金属从残渣态和铁锰态等稳定形态向交换态和水溶态等有效形态转化。还可以显著降低污泥中还原性物质的含量,减缓p H值下降速度,降低总有机碳含量,促进铵态氮向硝态氮转化,减少污泥微生物的数量并增加其种群活性。蚯蚓作用下,污泥中重金属的活化程度与还原性物质的含量呈显著负相关,而与微生物种群的活性呈显著正相关(P0.05)。综上所述,蚯蚓可以促进污泥重金属的活化,并改善污泥的肥力条件,为修复植物在污泥中的正常生长和对重金属离子的快速吸收提供有利条件。     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

Host cell RecA activates a mobile element-encoded mutagenic DNA polymerase

Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA′₂B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA′₂B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.     

LGR5 regulates osteogenic differentiation of human thoracic ligamentum flavum cells by Wnt signalling pathway

Thoracic ossification of the ligamentum flavum (TOLF) is ectopic ossification of the spinal ligaments. Histologically, the development of TOLF can be described as the process of endochondral ossification. However, the underlying aetiology has not been completely clarified. In this investigation, the gene expression profile associated with leucine‐rich repeat‐containing G‐protein‐coupled receptors (LGR) and Wnt signalling pathway in the thoracic ligamentum flavum cells (TLFCs) of different ossification stages was analysed via RNA sequencing. We further confirmed the significant differences in the related gene expression profile by Gene Ontology (GO) enrichment analysis. LGR5 was first identified in primary human TLFCs during osteogenic differentiation. To evaluate the effect of LGR5 on osteogenic differentiation, LGR5 has been knocked down and overexpressed in human TLFCs. We observed that the knockdown of LGR5 inhibited the activity of Wnt signalling and attenuated the potential osteogenic differentiation of TLFCs, while overexpression of LGR5 activated the Wnt signalling pathway and increased osteogenic differentiation. Our results provide important evidence for the potent positive mediatory effects of LGR5 on osteogenesis by enhancing the Wnt signalling pathway in TOLF.     

A new bacteria-free strategy induced by MaGal2 facilitates pinewood nematode escape immune response from its vector beetle

Symbiotic microbes play a crucial role in regulating parasite–host interactions; however, the role of bacterial associates in parasite–host interactions requires elucidation. In this study, we showed that, instead of introducing numerous symbiotic bacteria, dispersal of 4th-stage juvenile (J_IV) pinewood nematodes (PWNs), Bursaphelenchus xylophilus, only introduced few bacteria to its vector beetle, Monochamus alternatus (Ma). J_IV showed weak binding ability to five dominant bacteria species isolated from the beetles’ pupal chamber. This was especially the case for binding to the opportunistic pathogenic species Serratia marcescens; the nematodes’ bacteria binding ability at this critical stage when it infiltrates Ma for dispersal was much weaker compared with Caenorhabditis elegans, Diplogasteroides asiaticus, and propagative-stage PWN. The associated bacterium S. marcescens, which was isolated from the beetles’ pupal chambers, was unfavorable to Ma, because it caused a higher mortality rate upon injection into tracheae. In addition, S. marcescens in the tracheae caused more immune effector disorders compared with PWN alone. Ma_Galectin2 (MaGal2), a pattern-recognition receptor, was up-regulated following PWN loading. Recombinant MaGal2 protein formed aggregates with five dominant associated bacteria in vitro. Moreover, MaGal2 knockdown beetles had up-regulated prophenoloxidase gene expression, increased phenoloxidase activity, and decreased PWN loading. Our study revealed a previously unknown strategy for immune evasion of this plant pathogen inside its vector, and provides novel insights into the role of bacteria in parasite–host interactions.     

四合木种群的生态适应性

通过四合木异地播种生长量比较、以及四合木对土壤的适应性和对水热综合因子的适应性分析,得到如下结果：（1）四合木生长量高低的顺序为乌海区的东胜土（12．12g）〉乌海区的乌海土（10．62g）〉东胜区的乌海土（3．10g）〉东胜区的东胜土（2．59g）。同一地区不同土壤间四合木生长量之间没有差异（P≥0．05）,不同地区相同土壤间的生长量之间却有十分显著的差异,说明了土壤条件对四合木的生存影响是次要的,气候条件是保证四合木生存的最重要的条件。（2）四合木分布区与异地保护区之间9种土壤微量元素具有差异,但由于四合木植株中的微量元素含量均高于土壤中的有效量,说明四合木对微量元素具有富集作用,土壤微量元素含量不是四合木成活与生长的限制因子。（3）四合木分布区与异地保护区之间各月平均温度差异显著;全年气温日较差以及温暖指数、寒冷指数、湿润指数和水热综合因子指数差异显著;生长季地表地温昼夜温差具有特殊性,这可能是异地保护不易成功的限制因子之一。