E. coli chaperones DnaK,Hsp33 and Spy inhibit bacterial functional amyloid assembly

Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homolog in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.Key words: chaperone, curli, functional amyloid, CsgA, DnaK, Hsp33, Spy     

核心组蛋白的融合表达和纯化

目的:克隆核心组蛋白H2A、H2B、H3和H4的基因,表达并纯化组蛋白与谷胱甘肽S-转移酶(GST)的融合蛋白。方法:用PCR方法从乳腺文库中扩增核心组蛋白H2A、H2B、H3和H4的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-H2A、pGST-H2B、pGST-H3和pGST-H4,分别转化大肠杆菌BL21,表达融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4;用谷胱甘肽-Sepharose 4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果:分别构建了核心组蛋白H2A、H2B、H3和H4的融合表达载体;Western印迹检测表明,融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4获得表达及纯化。结论:表达并纯化了H2A、H2B、H3和H4的融合蛋白,为进一步研究核心组蛋白的功能奠定了基础。     

Kinetic properties of the binding of alpha-lytic protease to peptide boronic acids

The kinetic parameters for peptide boronic acids in their interaction with alpha-lytic protease were determined and found to be similar to those of other serine proteases [Kettner, C., & Shenvi, A. B. (1984) J. Biol. Chem. 259, 15106-15114]. alpha-Lytic protease hydrolyzes substrates with either alanine or valine in the P1 site and has a preference for substrate with a P1 alanine. The most effective inhibitors are tri- and tetrapeptide analogues that have a -boroVal-OH residue in the P1 site. At pH 7.5, MeOSuc-Ala-Ala-Pro-boroVal-OH has a Ki of 6.4 nM and Boc-Ala-Pro-boroVal-OH has a Ki of 0.35 nM. Ac-boroVal-OH and Ac-Pro-boroVal-OH are 220,000- and 500-fold less effective, respectively, than the tetrapeptide analogue. The kinetic properties of the tri- and tetrapeptide analogues are consistent with the mechanism for slow-binding inhibition, E + I in equilibrium EI in equilibrium EI*, while the less effective inhibitors are simple competitive inhibitors. MeO-Suc-Ala-Ala-Pro-boroAla-OH is a simple competitive inhibitor with a Ki of 67 nM at pH 7.5. Other peptide boronic acids, which are analogues of nonsubstrates, are less effective than substrate analogues but still are effective competitive inhibitors. For example, MeOSuc-Ala-Ala-Pro-boroPhe-OH has a Ki of 0.54 microM although substrates with a phenylalanine in the P1 position are not hydrolyzed. Binding for boronic acid analogues of both substrate and nonsubstrate analogues is pH dependent with higher affinity near pH 7.5. Similar binding properties have been observed for pancreatic elastase. Both enzymes have almost identical requirements for an extended peptide inhibitor sequence in order to exhibit highly effective binding and slow-binding characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tremacron hongheense,a New Species of Gesneriaceaefrom Southeastern Yunnan,China

A new species of Gesneriaceae from Honghe County, Southeastern Yunnan, China, Tremacron hongheense WH. Chen & YM. Shui, is described and illustrated. The new species is similar to Tremacron forrestii Craib, but differs by its leaf blade adaxially sparsely long setose (vs. densely white appressed pubescent and sparsely rusty brown villous), corolla tube outside short white glandular (vs. nearly glabrous), corolla lobes red and thickening at apex, especially adaxial lip (vs. yellow and not thickening), stamens 16-18cm long (vs. 04-12cm long), staminode 05-14cm long (vs. 02-04cm long).     

Effects of grazing on plant species diversity and carbon partitioning in semiarid rangelands of northeastern China

Grasslands are one of the most widespread landscapes worldwide, covering approximately one-fifth of the world’s land surface, where grazing is a common practice. How carbon storage responds to grazing in steppes remains poorly understood. We quantified the effects of grazing on community composition and species diversity, and carbon storage in two typical grasslands of northeastern China, one in Horqin and the other one in Hulunbeier. In both grasslands, grazing did not influence plant species diversity. However, it substantially decreased aboveground carbon by 31% and 54% in Horqin and Hulunbeier, respectively. Fenced and grazing treatments showed a similar belowground carbon at both locations. The predominant carbon pool in the study grassland ecosystem was found in the upper 100 cm soil depth, from 98.2 to 99.1% of the total carbon storage. There were no significant effects of grazing on soil carbon neither in the whole profile nor in the uppermost 20 cm soil depth in the two study grasslands. Studies on the effects of varying rangeland management, such as region disparity and grazing systems, may have important consequences on species diversity and carbon partitioning, and thus on rangeland stability and ecosystem functioning.     

Peak plasma interleukin-6 and other peripheral markers of inflammation in the first week of ischaemic stroke correlate with brain infarct volume, stroke severity and long-term outcome

Background

Cerebral ischaemia initiates an inflammatory response in the brain and periphery. We assessed the relationship between peak values of plasma interleukin-6 (IL-6) in the first week after ischaemic stroke, with measures of stroke severity and outcome.

Methods

Thirty-seven patients with ischaemic stroke were prospectively recruited. Plasma IL-6, and other markers of peripheral inflammation, were measured at pre-determined timepoints in the first week after stroke onset. Primary analyses were the association between peak plasma IL-6 concentration with both modified Rankin score (mRS) at 3 months and computed tomography (CT) brain infarct volume.

Results

Peak plasma IL-6 concentration correlated significantly (p < 0.001) with CT brain infarct volume (r = 0.75) and mRS at 3 months (r = 0.72). It correlated similarly with clinical outcome at 12 months or stroke severity. Strong associations were also noted between either peak plasma C-reactive protein (CRP) concentration or white blood cell (WBC) count, and all outcome measures.

Conclusions

These data provide evidence that the magnitude of the peripheral inflammatory response is related to the severity of acute ischaemic stroke, and clinical outcome.     

Identification and characterization of a novel glucose-phosphorylating enzyme in Kluyveromyces lactis

Recent data suggest that hexokinase KlHxk1 (Rag5) represents the only glucose-phosphorylating enzyme of Kluyveromyces lactis, which also is required for glucose signalling. Long-term growth studies of a K. lactis rag5 mutant, however, reveal slow growth on glucose, but no growth on fructose. Isolation of the permissive glucose-phosphorylating enzyme, mass spectrometric tryptic peptide analysis and determination of basic kinetic data identify a novel glucokinase (KlGlk1) encoded by ORF KLLA0C01,155g. In accordance with the growth characteristics of the rag5 mutant, KlGlk1 phosphorylates glucose, but fails to act on fructose as a sugar substrate. Multiple sequence alignment indicates the presence of at least one glucokinase gene in all sequenced yeast genomes.