The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.     

Functional analysis of Arg-308 mutants of Flp recombinase. Possible role of Arg-308 in coupling substrate binding to catalysis

The arginine residue at position 308 in the Flp recombinase corresponds to the only invariant arginine within the Int family of recombinases. Alterations of this residue result in Flp variants that retain substrate recognition, but form weaker protein-DNA complexes than wild type Flp. Furthermore, their DNA cleavage activity is significantly diminished. A conservative change of R308K results in a functional Flp variant; however, this protein has a lowered temperature optimum for recombination. The Arg-308 mutants can be stabilized on the DNA substrate through cooperativity with a partner Flp mutant that is tight binding. Thus, interactions between Flp monomers must be a relevant feature of the normal recombination reaction.     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

空间组织模式：微生物群落组装的“游戏”规则

在生物膜形成过程中,微生物种群之间通过主动或者被动的生物过程所形成的独特空间结构被称为空间组织模式。微生物空间组织模式广泛存在于自然和人工环境中,比如医疗、工业和生态系统等,是微生物形成和维持特定群落结构并发挥功能的主要方式,也是形成和维持微生物群落多样性的关键机制。然而,由于微生物群落的复杂性及相关研究方法的局限性,微生物空间组织模式方面的研究目前仍然处于起始阶段。本文梳理了微生物空间组织模式领域的研究进展,系统总结了空间组织模式初始阶段（微生物界面附着）和成熟阶段（空间自组织）的形成过程与协同机制,以及其对微生物养分利用和元素循环、微生物多样性维持和种群进化及功能的影响和调控机制,并分析了影响微生物空间组织模式的关键环境因素。     

织金洞钟乳石沉积物表面可培养细菌多样性和网络分析

【背景】洞穴环境中蕴含丰富而独特的细菌资源,对洞穴环境中细菌的分离培养有助于了解洞穴细菌多样性及细菌资源的挖掘和利用。【目的】利用不同培养基分离细菌,探究钟乳石表面可培养细菌的多样性及其种间互作关系。【方法】通过11种培养基对织金洞内钟乳石沉积物表面的细菌进行分离纯化,并利用16S rRNA基因序列分析初步确定分离菌株的分类地位。在R软件下,通过Bipartite包分析可培养细菌属间的相互作用。【结果】从钟乳石沉积物表面共分离出206株细菌,它们隶属于4门25属45种,香农(Shannon)指数为4.78,辛普森(Simpson)指数为0.95。变形菌门(Proteobacteria)和芽孢杆菌属(Bacillus)分别为样品中可培养细菌的优势门(47.09%)和优势属(29.61%)。无机寡营养培养基有助于洞穴钟乳石沉积物细菌的分离。属水平-采样点网络分析表明,可培养细菌分布具有非随机、显著的嵌套性。芽孢杆菌属(Bacillus)、德沃斯氏菌属(Devosia)、产碱杆菌属(Alcaligenes)、节杆菌属(Arthrobacter)和短杆菌属(Brevibacterium)在细菌群落中存在较多的有效合作值(Effective Partners)和亲密度(Closeness),被其他细菌所依赖程度(Species Strength)较高,是该群落中的重要组成类群。【结论】织金洞内钟乳石沉积物表面存在丰富的细菌资源,分析细菌类群在群落中的作用应结合细菌相对丰度和网络分析。     

乙酸钠调控小球藻生长及代谢产物

【背景】小球藻是一种单细胞绿藻,在不同培养条件下可积累高附加值的代谢产物,这些产物可用于生产生物燃料、食品、保健品、药品等。然而这些代谢产物在藻细胞中的生产率较低且很难通过经济可行的方法将其分离,这使其工业化规模生产受到限制。【目的】研究乙酸钠对小球藻生物量的影响,并分析其对小球藻代谢产物的调控作用。【方法】通过在小球藻培养液中添加不同浓度的乙酸钠(1.0、2.0、3.0、4.0、5.0 g/L),研究其调控小球藻生长和代谢的作用机理。【结果】在添加3.0 g/L乙酸钠的培养液中,小球藻的生物量是对照组的5.2倍,尽管藻细胞中蛋白质含量无明显变化,但油脂和类胡萝卜素含量是对照组的2.4倍和1.2倍,多糖和叶绿素a含量却仅为对照组的54.6%和54.4%。【结论】乙酸钠不仅会影响藻细胞的生长,还会调控其代谢过程,这为深入探索乙酸钠在调控小球藻生长及代谢过程的作用机制提供了理论基础和技术资料。     

Nontraditional translation is the key to UFMylation and beyond

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.