Interactions of alpha- and beta-N-acetyl-D-glucosamines with hen and turkey lysozymes.

The binding constants of alpha- and beta-GlcNAc to hen and turkey lysozymes [EC 3.2.1.17] were determined at various pH's using the method proposed by Ikeda and Hamaguchi (1975) J. Biochem. 77, 1-16). The pH dependence of the binding of beta-GlcNAc to hen lysozyme was essentially the same as that for turkey lysozyme. The pH dependence curves of the binding constants of beta-GlcNAc to hen and turkey lysozymes were interpreted in terms of the participation of Glu 35 (pK 6.0), Asp 52 (pK 3.5), Asp 48 (pK 4.5), and Asp 66 (pK 1.5). The binding constants of alpha-GlcNAc to hen and turkey lysozymes were the same below pH 3.5 but were different above this pH. The main participant residues in the binding of alpha-GlcNAc were Glu 35, Asp 48, and Asp 66 for hen lysozyme and Glu 35 and Asp 66 for turkey lysozyme. The results obtained here were well explained by the following assumptions: (1) above about pH 4, alpha-GlcNAc binds to hen lysozyme in both alpha- and beta-modes, which correspond to the binding orientation of alpha-GlcNAc and that of beta-GlcNAc, respectively, as determined by X-ray crystallographic studies, but it binds predominantly in the beta-mode below about pH 4, (2) beta-GlcNAc binds to hen and turkey lysozymes predominantly in the beta-mode above about pH 4 and in both alpha- and beta-modes below pH 4, and (3) alpha-GlcNAc binds to turkey lysozyme predominantly in the beta-mode over the whole pH range studied.     

Inhibition of bull and rabbit sperm enzymes by alpha-chlorohydrin.

High concentrations of alpha-chlorohydrin were found to inhibit hyaluronidase, beta-glucuronidase, and aryl sulphatases in bull and rabbit spermatozoa, but not acrosin and neuraminidase. Preincubation of the enzyme and alpha-chlorohydrin was essential to achieve the maximum inhibition which was irreversible.     

Inhibition of in Vivo Conversion of Methionine to Ethylene by l-Canaline and 2,4-Dinitrophenol

l-Canaline, a potent inhibitor of pyridoxal phosphate-mediated reactions, markedly inhibited the conversion of methionine to ethylene and carbon dioxide by apple tissue. A 50% inhibition of methionine conversion into ethylene was obtained with 50 mum canaline and almost complete inhibition with 300 mum canaline. When 2,4-dinitrophenol, an oxidative phosphorylation uncoupler, was fed to apple tissue, it inhibited the conversion of radioactive methionine to ethylene by 50% at a concentration of 60 mum and by 90% at a concentration of 100 mum. Production of labeled carbon dioxide from acetate-1-(14)C was increased by 2,4-dinitrophenol, indicating that the inhibition of ethylene production was due to uncoupling of phosphorylation. Auxin-induced ethylene production by mungbean (Phaseolus mungo L.) hypocotyl sections was similarly inhibited by these inhibitors.These results support the proposal that pyridoxal phosphate is involved in the formation of ethylene from methionine, substantiate the requirement for ATP in ethylene production, and suggest that this ATP requirement occurs in the step (s) between methionine and ethylene. The biosynthetic mechanism probably involves activation of methionine by ATP followed by a pyridoxal phosphate-mediated gamma-elimination.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

极端暴雨下山地丘陵区小流域洪水淹没强度对景观特征的响应——以河南“7·20”暴雨为例

通过分析郑州7·20暴雨事件中贾峪河山地丘陵区小流域的洪水过程,探究景观特征对洪水淹没强度影响的时空分布规律,并提出增强流域洪水韧性的规划建议,以缓解河南省山区所面临的社会经济发展、生态环境改善等问题。基于高分6号遥感数据、先进陆地观测卫星(ALOS,Advance Land Observing Satellite)相控阵L频段合成孔径雷达(PALSAR)的地表高程数据和小时降雨量数据,利用MIKE 21水动力模型构建贾峪河流域二维水文模型,分析其2021年7月20日0-24时期上、中、下游的洪水淹没深度和面积,并结合双变量空间自相关模型方法,探究洪水淹没强度与各景观组成和地形因素在时间和空间上的相关性的差异以及其空间聚类类型。研究表明:(1)贾峪河流域淹没面积在0-6时快速增长,于18时达到最大9.59km²,此时各区域淹没面积占比从大到小依次为下游18.88%、上游8.25%、中游12.03%,淹没深度在3m以上的面积占36.11%。(2)地形因素(平均Moran''s I=0.159)对洪水强度的影响大于土地类型(平均Moran''s I=0.096),主要影响因子相对高程、地形湿度指数、矿坑面积百分比、水体面积百分比、建设用地面积百分比、耕地面积百分比以及林地和草地面积百分比与洪水淹没强度之间的相关性随时间变化呈增大趋势,均在暴雨中后期18-24时达到最强。(3)交互探测结果表明,多因子叠加会增强各景观特征对洪水淹没强度的影响。上游影响洪水淹没强度的主要驱动力为矿坑和相对高程,中游和下游的主要影响为水体和地形湿度指数。(4)洪水淹没强度24时的平均值与景观特征指数之间的"高-高"和"高-低"地区的面积占比约0.47%-9.85%,主要分布在上游的中部山区和北部河道周围、中游的河道两侧和下游的河道以及常庄水库周边地区。研究结论建议在上游露天矿坑就地改造为蓄水池并恢复植被,中游和下游应提升河岸带绿地质量,增加下游城区绿色基础设施,减轻城市洪水风险。     

放牧对祁连山高寒典型草原土壤质量的影响

土壤质量是维持陆地生态系统稳定性与功能多样性的基础。放牧作为草地资源最广泛的利用方式之一,其对草地土壤质量的影响却缺乏量化标准,且两者之间的作用机理尚不明确。以祁连山高寒草原两个季节性牧场为研究对象,结合生态系统耦合与生态系统多功能性,探究了放牧对高寒草原土壤质量的影响与潜在机制。试验结果表明：基于最小数据集,不同放牧率下土壤质量指数差异显著(P<0.05),冬季牧场和春秋季牧场放牧率分别在2.45头月^-1 hm^-2和0.80头月^-1 hm^-2时土壤质量指数最高。土壤速效磷、有机碳、氮磷比和土壤pH是决定冬季牧场土壤质量的关键因子,而春秋季牧场中则是土壤有机碳、碳氮比和土壤pH;两个季节性牧场土壤质量指数与物种丰富度指数(P<0.05)和香浓维纳多样性指数(P<0.0001)呈显著正相关。高寒草原季节性牧场放牧地植物群落物种多样性与土壤因子耦合度在0.67—0.81之间,平均耦合度为0.74,属于中度协调;随着放牧率的增加,生态系统多功能性指数逐渐减低且与土壤质量指数变化趋势相似,...     

Long noncoding RNA lncMREF promotes myogenic differentiation and muscle regeneration by interacting with the Smarca5/p300 complex

Long noncoding RNAs (lncRNAs) play important roles in the spatial and temporal regulation of muscle development and regeneration. Nevertheless, the determination of their biological functions and mechanisms underlying muscle regeneration remains challenging. Here, we identified a lncRNA named lncMREF (lncRNA muscle regeneration enhancement factor) as a conserved positive regulator of muscle regeneration among mice, pigs and humans. Functional studies demonstrated that lncMREF, which is mainly expressed in differentiated muscle satellite cells, promotes myogenic differentiation and muscle regeneration. Mechanistically, lncMREF interacts with Smarca5 to promote chromatin accessibility when muscle satellite cells are activated and start to differentiate, thereby facilitating genomic binding of p300/CBP/H3K27ac to upregulate the expression of myogenic regulators, such as MyoD and cell differentiation. Our results unravel a novel temporal-specific epigenetic regulation during muscle regeneration and reveal that lncMREF/Smarca5-mediated epigenetic programming is responsible for muscle cell differentiation, which provides new insights into the regulatory mechanism of muscle regeneration.     

CRISPR/Cas9-induced structural variations expand in T lymphocytes in vivo

CRISPR/Cas9 has been adapted to disrupt endogenous genes in adoptive T-lymphocyte therapy to prevent graft-versus-host disease. However, genome editing also generates prevalent deleterious structural variations (SVs), including chromosomal translocations and large deletions, raising safety concerns about reinfused T cells. Here, we dynamically monitored the progression of SVs in a mouse model of T-cell receptor (TCR)-transgenic T-cell adoptive transfer, mimicking TCR T therapeutics. Remarkably, CRISPR/Cas9-induced SVs persist and undergo clonal expansion in vivo after three weeks or even two months, evidenced by high enrichment and low junctional diversity of identified SVs post infusion. Specifically, we detected 128 expanded translocations, with 20 615 as the highest number of amplicons. The identified SVs are stochastically selected among different individuals and show an inconspicuous locus preference. Similar to SVs, viral DNA integrations are routinely detected in edited T cells and also undergo clonal expansion. The persistent SVs and viral DNA integrations in the infused T cells may constantly threaten genome integrity, drawing immediate attention to the safety of CRISPR/Cas9-engineered T cells mediated immunotherapy.     

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase

Twinkle is a mitochondrial replicative helicase which can self-load onto and unwind mitochondrial DNA. Nearly 60 mutations on Twinkle have been linked to human mitochondrial diseases. Using cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM), we obtained the atomic-resolution structure of a vertebrate Twinkle homolog with DNA and captured in real-time how Twinkle is self-loaded onto DNA. Our data highlight the important role of the non-catalytic N-terminal domain of Twinkle. The N-terminal domain directly contacts the C-terminal helicase domain, and the contact interface is a hotspot for disease-related mutations. Mutations at the interface destabilize Twinkle hexamer and reduce helicase activity. With HS-AFM, we observed that a highly dynamic Twinkle domain, which is likely to be the N-terminal domain, can protrude ∼5 nm to transiently capture nearby DNA and initialize Twinkle loading onto DNA. Moreover, structural analysis and subunit doping experiments suggest that Twinkle hydrolyzes ATP stochastically, which is distinct from related helicases from bacteriophages.