Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system

Background  

Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.     

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.     

Non‐housekeeping,non‐essential GroEL (chaperonin) has acquired novel structure and function beneficial under stress in cyanobacteria

GroELs which are prokaryotic members of the chaperonin (Cpn)/Hsp60 family are molecular chaperones of which Escherichia coli GroEL is a model for subsequent research. The majority of bacterial species including E. coli and Bacillus subtilis have only one essential groEL gene that forms an operon with the co‐chaperone groES gene. In contrast to these model bacteria, two or three groEL genes exist in cyanobacterial genomes. One of them, groEL2, does not form an operon with the groES gene, whereas the other(s) does. In the case of cyanobacteria containing two GroEL homologs, one of the GroELs, GroEL1, substitutes for the native GroEL in an E. coli cell, but GroEL2 does not. Unlike the E. coli GroEL, GroEL2 is not essential, but it plays an important role which is not substitutable by GroEL1 under stress. Regulation of expression and biochemical properties of GroEL2 are different/diversified from GroEL1 and E. coli GroEL in many aspects. We postulate that the groEL2 gene has acquired a novel, beneficial function especially under stresses and become preserved by natural selection, with the groEL1 gene retaining the original, house‐keeping function. In this review, we will focus on difference between the two GroELs in cyanobacteria, and divergence of GroEL2 from the E. coli GroEL. We will also compare cyanobacterial GroELs with the chloroplast Cpns (60α and 60β) which are thought to be evolved from the cyanobacterial GroEL1. Chloroplast Cpns appear to follow the different path from cyanobacterial GroELs in the evolution after gene duplication of the corresponding ancestral groEL gene.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.     

Double-stranded DNA binding, an unusual property of DNA polymerase epsilon, promotes epigenetic silencing in Saccharomyces cerevisiae

We have previously shown that DNA polymerase epsilon (Pol epsilon)of Saccharomyces cerevisiae binds stably to double-stranded DNA (dsDNA), a property not generally associated with DNA polymerases. Here, by reconstituting Pol epsilon activity from Pol2p-Dpb2p and Dpb3p-Dpb4p, its two component subassemblies, we report that Dpb3p-Dpb4p, a heterodimer of histone-fold motif-containing subunits, is responsible for the dsDNA binding. Substitution of specific lysine residues in Dpb3p, highlighted by homology modeling of Dpb3p-Dpb4p based on the structure of the histone H2A-H2B dimer, indicated that they play roles in binding of dsDNA by Dpb3p-Dpb4p, in a manner similar to the histone-DNA interaction. The lysine-substituted dpb3 mutants also displayed reduced telomeric silencing, whose degree paralleled that of the dsDNA-binding activity of Pol epsilon in the corresponding dpb3 mutants. Furthermore, additional amino acid substitutions to lysines in Dpb4p, to compensate for the loss of positive charges in the Dpb3p mutants, resulted in simultaneous restoration of dsDNA-binding activity by Pol epsilon and telomeric silencing. We conclude that the dsDNA-binding property of Pol epsilon is required for epigenetic silencing at telomeres.     

Arabidopsis lonely guy (LOG) multiple mutants reveal a central role of the LOG-dependent pathway in cytokinin activation

Cytokinins are phytohormones that play key roles in the maintenance of stem cell activity in plants. Although alternative single-step and two-step activation pathways for cytokinin have been proposed, the significance of the single-step pathway which is catalyzed by LONELY GUY (LOG), is not fully understood. We analyzed the metabolic flow of cytokinin activation in Arabidopsis log multiple mutants using stable isotope-labeled tracers and characterized the mutants' morphological and developmental phenotypes. In tracer experiments, cytokinin activation was inhibited most pronouncedly by log7, while the other log mutations had cumulative effects. Although sextuple or lower-order mutants did not show drastic phenotypes in vegetative growth, the log1log2log3log4log5log7log8 septuple T-DNA insertion mutant in which the LOG-dependent pathway is impaired, displayed severe retardation of shoot and root growth with defects in the maintenance of the apical meristems. Detailed observation of the mutants showed that LOG7 was required for the maintenance of shoot apical meristem size. LOG7 was also suggested to play a role for normal primary root growth together with LOG3 and LOG4. These results suggest a dominant role of the single-step activation pathway mediated by LOGs for cytokinin production, and overlapping but differentiated functions of the members of the LOG gene family in growth and development.     

江西鄱阳湖湖口水域船舶通行对长江江豚发声行为的影响

江西鄱阳湖是长江江豚(Neophocaena phocaenoides)的重要栖息地, 湖中栖息着约400 头江豚。多年的观察表明, 船舶交通是鄱阳湖中江豚面临的重要威胁之一。为了评估船舶通行对长江江豚发声行为的影响,尤其是了解船舶通行期间及其前后江豚的发声和行为特征, 作者于2007 年6 月27 日—7 月1 日在江西鄱阳湖湖口水域采用固定被动声学系统, 即安装在监测点(29°42′38″ N, 116°11′11″ E)的一套水下声学数据记录系统, 对周边通行船舶的水下噪声及江豚声纳信号脉冲事件进行了定点监测和记录, 并对所记录的数据进行了定量和统计分析。在整个监测的109h 中, 声学记录仪共记录到船舶494 艘, 江豚声纳脉冲串信号13413个。船舶出现与江豚出现存在弱的负相关关系(r = -0.029, N = 6550, P Z = -0.370, P> 0.05); 当有船舶经过时, 江豚的发声频次显著降低(Z = -10.050, P Z = -0.275, P> 0.05; Z = -0.119, P> 0.05); 船舶通行之前和之后, 江豚的发声频次、脉冲串持续时间、脉冲间间隔的差异性均不显著(χ2= 5.255, P> 0.05; χ2= 3.511, P> 0.05; χ2= 5.155, P>0.05); 在船舶经过时, 江豚对游动方向没有明显的选择性(χ2= 0.861, P> 0.05)。基于分析结果推测, 在狭窄水域中江豚躲避船舶干扰通常采取“临时性”策略, 而非长距离逃避。由于鄱阳湖湖口水域水道相对狭窄, 尽管研究的结果表明江豚对船舶有一定的敏感反应, 但是在相对狭窄的水域中, 江豚躲避船舶的行为难以充分表现。另外, 江豚对该水域中高密度航行船舶的噪声可能存在一定的“适应性”, 导致当遭遇船舶时, 江豚的声行为反应不十分强烈。因此, 建议有必要在不同尺度的水体中采用声学数据记录仪继续开展类似的观察,以进一步了解江豚对船舶的行为响应, 尤其是观察江豚躲避船舶的行为及发声特征