REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.     

Chemical modulators of autophagy as biological probes and potential therapeutics

Autophagy is an evolutionarily conserved mechanism for protein degradation that is critical for the maintenance of homeostasis in man. Autophagy has unexpected pleiotropic functions that favor survival of the cell, including nutrient supply under starvation, cleaning of the cellular interior, defense against infection and antigen presentation. Moreover, defective autophagy is associated with a diverse range of disease states, including neurodegeneration, cancer and Crohn's disease. Here we discuss the roles of mammalian autophagy in health and disease and highlight recent advances in pharmacological manipulation of autophagic pathways as a therapeutic strategy for a variety of pathological conditions.     

Distinct responses of cones and melanopsin-expressing retinal ganglion cells in the human electroretinogram

ABSTRACT: BACKGROUND: The discovery of the novel photoreceptor, melanopsin-expressing retinal ganglion cells (mRGCs), has raised researchers' interest in photoreceptive tasks performed by the mRGC, especially in non-image-forming visual functions. In a prior study, we investigated the mRGC response to light stimuli independent of rods and cones with the four-primary illumination system, which modulates stimulus levels to the mRGC and cones independently, and mRGC baseline responses were recorded in the electroretinogram (ERG). METHODS: In the present study, we used the same illumination system to compare independent responses of the mRGC and cones in five subjects (mean +/- SD age, 23.0 +/- 1.7 years). The ERG waveforms were examined as direct measurements of responses of the mRGCs and cones to stimulation (250 msec). Implicit times (the time taken to peaks) and peak values from 30 stimuli given to each subject were analyzed. RESULTS: Two distinct positive peaks appeared in the mRGC response, approximately 80 msec after the onset of the stimuli and 30 msec after their offset, while no such peaks appeared in the cone response. The response to the mRGC stimulus was significantly higher than that to the cone stimulus at ~80 msec (p < 0.05) and tended to be higher than the cone stimulus at ~280 msec (p = 0.08). CONCLUSIONS: Implicit time of the first peak was much longer than that to the b-wave and this delay might reflect mRGC's sluggish responses. This is the first report of amplitudes and implicit time in the ERG from the response of the mRGC that is independent of rods and cones and obtained using the four-primary illumination system.     

Design, synthesis, and evaluation of novel 4-thiazolylimidazoles as inhibitors of transforming growth factor-β type I receptor kinase

A novel series of 4-thiazolylimidazoles was synthesized as transforming growth factor-β (TGF-β) type I receptor (also known as activin receptor-like kinase 5 or ALK5) inhibitors. These compounds were evaluated for their ALK5 inhibitory activity in an enzyme assay and their TGF-β-induced Smad2/3 phosphorylation inhibitory activity in a cell-based assay. N-{[5-(1,3-benzothiazol-6-yl)-4-(4-methyl-1,3-thiazol-2-yl)-1H-imidazol-2-yl]methyl}butanamide 20, a potent and selective ALK5 inhibitor, exhibited good enzyme inhibitory activity (IC(50)=8.2nM) as well as inhibitory activity against TGF-β-induced Smad2/3 phosphorylation at a cellular level (IC(50)=32nM).     

Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors

Shigella possess 220 kb plasmid, and the major virulence determinants, called effectors, and the type III secretion system (TTSS) are exclusively encoded by the plasmid. The genome sequences of S. flexneri strains indicate that several ipaH family genes are located on both the plasmid and the chromosome, but whether their chromosomal IpaH cognates can be secreted from Shigella remains unknown. Here we report that S. flexneri strain, YSH6000 encodes seven ipaH cognate genes on the chromosome and that the IpaH proteins are secreted via the TTSS. The secretion kinetics of IpaH proteins by bacteria, however, showed delay compared with those of IpaB, IpaC and IpaD. Expression of the each mRNA of ipaH in Shigella was increased after bacterial entry into epithelial cells, and the IpaH proteins were secreted by intracellular bacteria. Although individual chromosomal ipaH deletion mutants showed no appreciable changes in the pathogenesis in a mouse pulmonary infection model, the DeltaipaH-null mutant, whose chromosome lacks all ipaH genes, was attenuated to mice lethality. Indeed, the histological examination for mouse lungs infected with the DeltaipaH-null showed a greater inflammatory response than induced by wild-type Shigella, suggesting that the chromosomal IpaH proteins act synergistically as effectors to modulate the host inflammatory responses.     

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.     

记中国首次发现的"真古兽类" (eupantotherian)化石

记述了在中国首次发现的采自辽宁黑山县八道壕矿区早白垩世晚期沙海组的一件“eu- pantotherian”(“真古兽类”)下颌骨化石。标本保存了最后两个前臼齿和4个臼齿,它以抬高的下颌角突,半臼齿化的最后一枚前臼齿,臼齿上面积增大但未发育成完整盆形的跟座,尚未形成的facet-5,及加长的最后臼齿等特点有别于所有已知的“eupantotherian”和具有雏形磨楔式臼齿的Kielantherium,被命名为一新属新种,Mozomus shikamai gen.et sp.nov.(鹿间明镇古兽),并由它而创建了一新科,Mozomuridae fam.nov. “Eupantotherian”是早期哺乳动物演化中的一个重要环节,是从无跟座的对齿兽(sym- metrodont)到具有磨楔式(tribosphenic)臼齿兽类的中间类型。早期兽类进化的成功模式是发育成具有磨楔式的臼齿,即在上臼齿上发育出原尖,而下臼齿的跟座形成由3个齿尖围成的盆状。这种结构扩大了牙齿的面积,使咀嚼切割能力更趋完善,今天的有袋类和真兽类均是如此。但在哺乳动物系统发育史上,“eupantotherian”类的化石发现不多,这给探讨具有磨楔式臼齿构造的两大门类(后兽类和真兽类)的起源带来不少困难和疑惑。而传统上的真古兽类形态分异又很大,并不是一个单系类群。其中有跟座发育较好者,如peramurans有可能更接近具有磨楔式臼齿兽类的基部位置,本文记述的Mozomus shikamai也应属于这一类型。具有雏形的被认为处于基干上的磨楔式臼齿类化石,迄今只有两种,即发现在英国早白垩世地层中的滨齿兽(Aegialodon)和蒙古早白垩世晚期Hoobor层的Kielantherium,前者仅有一颗下臼齿,后者由一枚下臼齿和一具有4颗臼齿的下牙床为代表。两种化石在分类上被归入单一的滨齿兽目(Order Aegialodontia Butler,1978),视为Boreosphenidans的基干(stem)。本文记述的Mozomus,其时代与Kielantherium的大体相当,在大小、齿式及臼齿形态上与后者也多有相近之处,但前者以其臼齿的facet-5尚未出现和跟盆发育不全等特点表明它较Kielantherium更为原始,不具备磨楔式臼齿的模式,因之不能归人Aegialodontia,而只能纳入”eupantotheri- ans”。但在后一类的组合中,Mozomus以它半臼齿化的最后前臼齿和面积增大但未发育成盆形的跟座等特点,又是组合中相当进步的类型。无论如何Mozomus的发现是在为数极少的向磨楔式臼齿模式进化的中间环节上增添了一件重要的化石标本,也增加了不少新的信息。它必会引起学者对这一进化过程的更加深入的反思和新的启示。