The CRTS I double-block ballastless track under the coupling effect of temperature and falling-shaft impact is more adaptable to actual operation. However, domestic and foreign research has only ...focused on either temperature or train loadings. In this study, the finite-element method was used to analyze the dynamic response of the double-block ballastless track structure. The results suggests that the displacement and acceleration characteristics of the ballastless track structure under different working conditions change significantly than no temperature gradient. Moreover, the vibration responses at the different working conditions under the impact excitation of a dropping wheelset show that. The findings of this study revealed that temperature has significant impact on falling-shaft which cannot be ignored, consequently it, paper provides referential information for the design, construction, operation and maintenance of the CRTS I double-block ballastless track structure under with large temperature difference.
This paper analyzes the influence of foundation deformation and the variation coefficient of vehicle parameters on the reliability of a vehicle vertical safety. Based on the theory of stochastic ...analysis of nonlinear vehicle–track coupled systems, combining the generalized probability density evolution theory, this paper takes the reduction rate of wheel load as the measurement index, considering the combined effects of stochasticity of track irregularity, stochasticity of vehicle parameters and foundation deformation, and studies the reliability of vehicle vertical safety under different working conditions. The results showed that (1) compared with the up-arch deformation, the settlement deformation has a greater impact on the operation safety; (2) with the increase of the variation coefficient of the vehicle parameters, the reliability of the vehicle vertical safety gradually decreases, so it should be combined with vehicle maintenance when setting the settlement limits; (3) when the vehicle operation speed is lower than 375 km/h, the stochasticity of the vehicle parameters has a more significant impact on the vehicle vertical safety, while when the speed is higher than 375 km/h, the foundation deformation amplitude has a more significant influence; (4) when the running speed is higher than 350 km/h, there may be a better set of vehicle parameters to ensure driving safety. It can be seen that in the determination of the high-speed railway foundation deformation limit value, the influence of deformation direction, vehicle parameters stochasticity, and operation speed should be considered.
amylose extender (ae−
) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by ...the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae−
maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae−
mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with γ-32PATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn2+-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.
Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes ...(SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with γ-³²P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis.
The amylose extender (ae⁻) mutant of maize lacks starch branching enzyme IIb (SBEIIb) activity, resulting in amylopectin with reduced branch point frequency, and longer glucan chains. Recent studies ...indicate isozymes of soluble starch synthases form high molecular weight complexes with SBEII isoforms. This study investigated the effect of the loss of SBEIIb activity on interactions between starch biosynthetic enzymes in maize endosperm amyloplasts. Results show distinct patterns of protein-protein interactions in amyloplasts of ae⁻ mutants compared with the wild type, suggesting functional complementation for loss of SBEIIb by SBEI, SBEIIa, and SP. Coimmunoprecipitation experiments and affinity chromatography using recombinant proteins showed that, in amyloplasts from normal endosperm, protein-protein interactions involving starch synthase I (SSI), SSIIa, and SBEIIb could be detected. By contrast, in ae⁻ amyloplasts, SSI and SSIIa interacted with SBEI, SBEIIa, and SP. All interactions in the wild-type were strongly enhanced by ATP, and broken by alkaline phosphatase, indicating a role for protein phosphorylation in their assembly. Whilst ATP and alkaline phosphatase had no effect on the stability of the protein complexes from ae⁻ endosperm, radiolabelling experiments showed SP and SBEI were both phosphorylated within the mutant protein complex. It is proposed that, during amylopectin biosynthesis, SSI and SSIIa form the core of a phosphorylation-dependent glucan-synthesizing protein complex which, in normal endosperm, recruits SBEIIb, but when SBEIIb is absent (ae⁻), recruits SBEI, SBEIIa, and SP. Differences in stromal protein complexes are mirrored in the complement of the starch synthesizing enzymes detected in the starch granules of each genotype, reinforcing the hypothesis that the complexes play a functional role in starch biosynthesis.
The sugary-2 mutation in maize (Zea mays L.) is a result of the loss of catalytic activity of the endosperm-specific SS (starch synthase) IIa isoform causing major alterations to amylopectin ...architecture. The present study reports a biochemical and molecular analysis of an allelic variant of the sugary-2 mutation expressing a catalytically inactive form of SSIIa and sheds new light on its central role in protein-protein interactions and determination of the starch granule proteome. The mutant SSIIa revealed two amino acid substitutions, one being a highly conserved residue (Gly522→Arg) responsible for the loss of catalytic activity and the inability of the mutant SSIIa to bind to starch. Analysis of protein-protein interactions in sugary-2 amyloplasts revealed the same trimeric assembly of soluble SSI, SSIIa and SBE (starch-branching enzyme) IIb found in wild-type amyloplasts, but with greatly reduced activities of SSI and SBEIIb. Chemical cross-linking studies demonstrated that SSIIa is at the core of the complex, interacting with SSI and SBEIIb, which do not interact directly with each other. The sugary-2 mutant starch granules were devoid of amylopectin-synthesizing enzymes, despite the fact that the respective affinities of SSI and SBEIIb from sugary-2 for amylopectin were the same as observed in wild-type. The data support a model whereby granule-bound proteins involved in amylopectin synthesis are partitioned into the starch granule as a result of their association within protein complexes, and that SSIIa plays a crucial role in trafficking SSI and SBEIIb into the granule matrix.
Protein–protein interactions between starch phosphorylase (SP) and other starch biosynthetic enzymes were investigated using isolated maize endosperm amyloplasts and a recombinant maize enzyme. ...Plastidial SP is a stromal enzyme existing as a multimeric protein in amyloplasts. Biochemical analysis of the recombinant maize SP indicated that the tetrameric form was catalytically active in both glucan-synthetic and phosphorolytic directions. Protein–protein interaction experiments employing the recombinant SP as an affinity ligand with amyloplast extracts showed that the multimeric state of SP determined interactions with other enzymes of the starch biosynthetic pathway. The monomeric form of SP interacts with starch branching enzyme I (SBEI) and SBEIIb, whereas only SBEI interacts with the tetrameric form of SP. In all cases, protein–protein interactions were broken when amyloplast lysates were dephosphorylated in vitro, and enhanced following pre-treatment with ATP, suggesting a mechanism of protein complex formation regulated by protein phosphorylation. In vitro protein phosphorylation experiments with γ-32P-ATP show that SP is phosphorylated by a plastidial protein kinase. Evidence is presented which suggests SBEIIb modulates the catalytic activity of SP through the formation of a heteromeric protein complex.
•New information on regulation of storage starch biosynthesis in crop plants.•Starch phosphorylase regulated by protein phosphorylation and protein interactions.•Multimeric states of starch phosphorylase determine protein interaction partners.•Starch phosphorylase and branching enzyme regulate each other's activities.
Mutations affecting specific starch biosynthetic enzymes commonly have pleiotropic effects on other enzymes in the same metabolic pathway. Such genetic evidence indicates functional relationships ...between components of the starch biosynthetic system, including starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes; however, the molecular explanation for these functional interactions is not known. One possibility is that specific SSs, BEs, and/or starch debranching enzymes associate physically with each other in multisubunit complexes. To test this hypothesis, this study sought to identify stable associations between three separate SS polypeptides (SSI, SSIIa, and SSIII) and three separate BE polypeptides (BEI, BEIIa, and BEIIb) from maize (Zea mays) amyloplasts. Detection methods included in vivo protein-protein interaction tests in yeast (Saccharomyces cerevisiae) nuclei, immunoprecipitation, and affinity purification using recombinant proteins as the solid phase ligand. Eight different instances were detected of specific pairs of proteins associating either directly or indirectly in the same multisubunit complex, and direct, pairwise interactions were indicated by the in vivo test in yeast. In addition, SSIIa, SSIII, BEIIa, and BEIIb all comigrated in gel permeation chromatography in a high molecular mass form of approximately 600 kD, and SSIIa, BEIIa, and BEIIb also migrated in a second high molecular form, lacking SSIII, of approximately 300 kD. Monomer forms of all four proteins were also detected by gel permeation chromatography. The 600- and 300-kD complexes were stable at high salt concentration, suggesting that hydrophobic effects are involved in the association between subunits.
Under long-term temperature loading, long bridges stretch and deform, causing the beam and rail to move relative to each other. This phenomenon could cause a gradual slide of the rail relative to the ...fastener composite pad and ultimately results in the slipping out of the fastener pad at the end of the ballastless track bridge changes. The slipping out of the fastener composite pad changes the longitudinal resistance of the fastener and causes shock of the train on the rail structure due to direct contact between the rail and the tie, thus inhibiting the normal use of the line. In this paper, a reciprocating tensile test was carried out on a WJ-8 small resistance fastener to explore the breaking process of the fastener composite pad, identify the interaction mechanism of the fastener composite pad under the rail, and ultimately determine the influence of the pad on the longitudinal resistance of the fastener. The results show that the two factors that affect the longitudinal resistance of the fastener under reciprocating loading are the surface roughness and displacement of the fastener composite pad. While the surface roughness of the fastener composite pad is the main influencing factor in the early stage, the displacement of the composite pad has a greater impact on the later stage. The research results of this paper can provide an important reference for the maintenance and repair of ballastless track fasteners on bridges.
•Furfuryl alcohol is employed as the anodic substrate to construct the hybrid water splitting system.•The Cu2Co(OH)x/NF is utilized as the integrated anodic electrode, and it is facilely prepared via ...microwave-assisted heating method.•Fabricated catalyst depicts 219 mV lower onset potential for furfuryl alcohol oxidation than OER at 10 mA cm–2.•Hydrogen generation at low electric energy consumption coupled with value-added anodic product generation can be achieved with this hybrid water splitting system.•The reasons for selection of furfuryl alcohol oxidation as the substitute half-reaction of OER are explained in detail.
Both anode feedstock and integrated electrodes are highly significant for the development of hybrid water electrolysis technology. Herein, biomass-derived furfuryl alcohol is selected as the anode feedstock, mainly because it contains no base-labile group (e.g., -CHO) and does not spontaneously undergo a Cannizzaro-type transformation under alkaline conditions. Bimetallic hydroxide nanowire arrays with nickel foam as the substrate (denoted Cu2Co(OH)x/NF) are designed and readily prepared via a microwave-assisted heating method. Then, Cu2Co(OH)x/NF is used as an integrated electrode for electrocatalytic furfuryl alcohol oxidation to value-added furoic acid with enhanced hydrogen production in 1.0 M NaOH. The input potential is reduced by approximately 219 mV when the oxygen evolution reaction is replaced by furfuryl alcohol oxidation to drive a current density of 10 mA cm−2. Moreover, the as-obtained electrode can be recycled several times without an obvious decline in carboxylate yield or Faradaic efficiency.
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