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The molecular electrometer at 40 分子静电计在40。

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-09-08 DOI: 10.1016/j.bbamem.2025.184453

Peter M. Macdonald

In 1987 Seelig and colleagues proposed that the phosphocholine headgroup of phosphatidylcholine behaved as a universal sensor of surface electrostatic charge, both cationic and anionic, in lipid bilayers (J. Seelig, P.M. Macdonald, P.G. Scherer, Phospholipid Head Groups as Sensors of Electric Charge in Membranes. Biochemistry, 26 (1987) 7535–7541.) Changes in the deuterium NMR quadrupolar splitting measured with specifically-deuterated positions within the choline headgroup in response to surface charges were attributed to a conformational change within the phosphocholine group corresponding to a “tilt” of the choline group towards or away from the direction of the bilayer normal as the PN dipole sought to align with the surface electrostatic field. In the ensuing nearly 4 decades this so-called “Molecular Electrometer” concept has become accepted doctrine in membrane science and has been employed to examine lipid bilayer surface electrostatics in a host of situations involving multiple membrane- associating biologically significant factors from ions, to anesthetics, to peptides and proteins. In this review, I describe the history of the science behind the Molecular Electrometer, the evolution of methods for examining the Molecular Electrometer response and provide a survey of its application in the myriad instances of membrane-associating molecules affecting and being affected by surface electrostatics. Lastly, I include an overview of the efforts of molecular dynamics simulations to be guided by and to account for the Molecular Electrometer effect in simulations of lipid bilayers.

1987年，Seelig和同事提出磷脂酰胆碱的磷脂胆碱头基团在脂质双分子层中充当表面静电电荷的通用传感器，包括阳离子和阴离子(J. Seelig， P.M.Macdonald， P.G. Scherer，磷脂头基团作为膜中电荷的传感器。生物化学，26(1987)7535-7541。在胆碱头基团内特定氘化位置测量的氘核磁共振四极分裂的变化，响应于表面电荷，归因于胆碱基团内部的构象变化，对应于胆碱基团向双分子层正态线方向“倾斜”，因为PN偶极子试图与表面静电场对齐。在随后的近40年里，这种所谓的“分子静电计”概念已成为膜科学中公认的原则，并已被用于检查脂质双分子层表面静电，涉及多种与膜相关的生物重要因素，从离子到麻醉剂，到肽和蛋白质。在这篇综述中，我描述了分子静电计背后的科学历史，检查分子静电计响应的方法的演变，并提供了它在无数膜相关分子影响和被表面静电影响的实例中的应用调查。最后，我概述了分子动力学模拟的努力，以指导和解释分子静电计效应在脂质双层模拟中的作用。

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引用次数: 0

Order parameters in membranes: Following Joachim Seelig's path 膜中的有序参数：遵循Joachim Seelig的路径。

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-31 DOI: 10.1016/j.bbamem.2025.184449

Estelle Morvan , Axelle Grélard , Erick J. Dufourc

Following the publication of biological membrane models in the 1970s, Joachim Seelig was the first to experimentally demonstrate the dynamic nature of these membranes. He conducted the first ssNMR experiments to measure the order parameters of the CD (²H) bond of lipids deuterium-labelled, showing a fairly fluid membrane interior. Since then, the order parameters of the CD, CH and CC bonds have been measured. They can be used to describe the dynamics of membranes on several space and time scales: intramolecular (Å/ns-ps), molecular (nm/ns–100 ns) and collective (membrane deformations, μm/μs). The profile of CD, CH, CC order parameters across the membrane bilayer allows us to describe the lipid membrane as being very rigid at the glycerol and chain levels and very fluid at its center and surface. This is true for lipid chains carrying double bonds, rings or branched methyl groups. Bipolar lipids that span the entire membrane do not have a very fluid membrane interior. Sterols modulate membrane dynamics, increasing order parameters in the fluid phase and decreasing them in the gel phases. They can be described as regulators of membrane dynamics, as they maintain the membrane in a dynamic state that varies very little when environmental factors change (temperature, pH, etc.). The description of order parameters by statistical mechanics allows the length of the chains, the thickness of the bilayer and the membrane elastic constants to be calculated accurately. The surface area of each lipid in the membrane can also be calculated from the plateau of order parameters (positions C3-C10):

(A_{L}) = 83 (1 - (S_{CD}^{plat}))

.

随着20世纪70年代生物膜模型的发表，Joachim Seelig是第一个通过实验证明这些膜的动态特性的人。他进行了第一次ssNMR实验，测量了氘标记的脂质CD （2H）键的有序参数，显示出相当流体的膜内部。从那时起，CD， CH和CC键的有序参数被测量。它们可以用来描述膜在几个空间和时间尺度上的动力学：分子内（Å/ns-ps），分子（nm/1-100 ns）和集体（膜变形，μm/μs）。CD， CH， CC在膜双分子层上的顺序参数的轮廓使我们能够将脂质膜描述为在甘油和链水平上非常坚硬，在其中心和表面非常流动。这对于带有双键、环或支链甲基的脂链来说是正确的。跨越整个膜的双极脂质没有一个非常流体的膜内部。甾醇调节膜动力学，增加流体相的序参量，减少凝胶相的序参量。它们可以被描述为膜动力学的调节器，因为当环境因素（温度、pH等）发生变化时，它们使膜保持在动态状态，变化很小。用统计力学方法描述的有序参数可以精确计算链的长度、双层膜的厚度和膜的弹性常数。膜中各脂类的表面积也可由序参数平台（位置C3-C10）计算得到：AL=831-SCDplat。

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引用次数: 0

Biophysical investigations of the membrane interactions of collagen VI-derived host defense peptides vi型胶原衍生宿主防御肽膜相互作用的生物物理研究。

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-28 DOI: 10.1016/j.bbamem.2025.184448

Kathakali De , Karin Bryder , Christopher Aisenbrey , Matthias Mörgelin , Burkhard Bechinger

Collagen VI is an extracellular matrix protein forming complex microfibrillar networks in connective tissues. Specifically, we focused on its role in innate immunity, in particular on cationic sequence motifs from the α3(VI)-chain, which exhibit strong antibacterial properties against both Gram-positive and Gram-negative bacteria in vitro and in vivo. Cytotoxicity assays revealed minimal to no adverse effects, even at concentrations effective against bacterial pathogens. This favorable safety profile suggests that these antimicrobial peptides selectively target bacterial membranes while sparing host cells, making them promising candidates for therapeutic development.

The membrane structure and interactions of two antimicrobial peptides were investigated in quantitative detail using solid-state NMR, CD and fluorescence spectroscopies. Whereas calcein release was somewhat more pronounced from POPE/POPG 3/1 vesicles when compared to POPC/30 % cholesterol, this activity is about two orders of magnitude increased when POPC/POPG 3/1 liposomes are investigated. This pronounced lipid dependence was reproduced with magainin 2, a well-known linear cationic AMP. In lipid titration experiments both collagen-derived peptides showed a transition from predominantly random coil to helical conformations. Quantitative evaluation of membrane association required the presence of PEG-lipids which are known to prevent the agglutination of POPE/POPG 3/1 liposomes. A dissociation constant in the 260 μM range was observed for GVR28 while the binding isotherms reveal an intermediate state when SFV33 associates with bacterial membranes. ²H solid-state NMR reveals considerable membrane disorder of the deuterated PG palmitoyl chain in POPE/POPG membranes. The ensemble of biophysical data suggests two distinct modes of action for the collagen derived peptides.

胶原VI是一种细胞外基质蛋白，在结缔组织中形成复杂的微纤维网络。具体来说，我们关注的是它在先天免疫中的作用，特别是α3（VI）链上的阳离子序列基序，它在体外和体内对革兰氏阳性和革兰氏阴性细菌都表现出很强的抗菌特性。细胞毒性试验显示，即使在对细菌病原体有效的浓度下，也几乎没有副作用。这种良好的安全性表明，这些抗菌肽选择性地靶向细菌膜，同时保留宿主细胞，使其成为治疗开发的有希望的候选者。利用固体核磁共振、CD和荧光光谱对两种抗菌肽的膜结构和相互作用进行了定量详细研究。与POPC/30 %胆固醇相比，POPE/POPG 3/1囊泡释放的钙黄蛋白更为明显，而当研究POPC/POPG 3/1脂质体时，这种活性增加了大约两个数量级。这种明显的脂质依赖性在magainin 2（一种众所周知的线性阳离子AMP）中得到了再现。在脂质滴定实验中，两种胶原来源的肽都表现出从主要随机线圈到螺旋构象的转变。膜关联的定量评估需要peg -脂质的存在，已知peg -脂质可以防止POPE/POPG 3/1脂质体的凝集。GVR28的解离常数在260 μM范围内，而结合等温线显示SFV33与细菌膜结合时处于中间状态。2H固态核磁共振显示，在POPE/POPG膜中氘化PG棕榈酰链存在明显的膜无序性。综合生物物理数据表明胶原衍生肽有两种不同的作用模式。

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引用次数: 0

Modulation of TNAP activity and apatite formation in biomimetic matrix vesicles studied by 31P solid-state NMR 31P固体核磁共振研究仿生基质囊泡中TNAP活性和磷灰石形成的调节。

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-27 DOI: 10.1016/j.bbamem.2025.184446

B.Z. Favarin , N. Nassif , T. Azaïs , J. Guignier , S. Mebarek , R. Buchet , J.L. Millán , A.P. Ramos , A.J. Costa-Filho , P. Ciancaglini

Skeletal and dental mineralization relies on a precisely regulated sequence of events culminating in apatite deposition onto collagen fibrils. Matrix vesicles (MVs), extracellular vesicles released by mineralization-competent cells, play a pivotal role in this process through the catalytic activity of alkaline phosphatase (TNAP). The lipid composition of MVs, particularly phosphatidylserine (PS)-calcium complexes, facilitates the nucleation of amorphous calcium phosphate and apatite formation. However, the interplay between the TNAP structure, the lipid membrane environment, and its enzymatic activity remains incompletely understood.

Biomimetic models of MVs, as proteoliposomes made with dipalmitoylphosphatidylcholine (DPPC) and various TNAP mutants, were used to investigate the TNAP's activity and mineralization potential. Molecular docking and site-directed mutagenesis revealed that specific cysteine substitutions near TNAP's catalytic and anchoring sites influence structural stability, enzymatic activity, and incorporation into lipid bilayers. Notably, TNAP mutants S221C and P307C exhibited enhanced catalytic efficiency in DPPC liposomes, while A420C showed reduced activity due to steric hindrance near the catalytic site. Solid-state NMR and cryo-TEM analyses confirmed hydroxyapatite formation, with significant contributions from lipid-anchored TNAP to the mineralization process.

These findings highlight the critical influence of the lipid environment on TNAP's functional properties and provide insights into the mechanisms governing biomineralization and related pathologies, including hypophosphatasia associated with various TNAP mutations. The study underscores the importance of ATP and pyrophosphate hydrolysis by TNAP in modulating apatite formation and reveals the role of specific TNAP mutations in regulating enzymatic activity, stability, and mineral propagation. Understanding these interactions could lead to alternate therapeutic strategies in treatment and regenerative medicine.

骨骼和牙齿矿化依赖于一个精确调控的事件序列，最终导致磷灰石沉积到胶原原纤维上。基质囊泡（Matrix vesicles, MVs）是矿化能力细胞释放的细胞外囊泡，通过碱性磷酸酶（TNAP）的催化活性在这一过程中发挥关键作用。mv的脂质组成，特别是磷脂酰丝氨酸(PS)-钙复合物，促进了无定形磷酸钙的成核和磷灰石的形成。然而，TNAP结构、脂膜环境及其酶活性之间的相互作用仍不完全清楚。利用双棕榈酰磷脂酰胆碱（DPPC）和各种TNAP突变体构建的MVs蛋白脂质体的仿生模型，研究了TNAP的活性和矿化潜力。分子对接和定点诱变表明，在TNAP的催化位点和锚定位点附近的特定半胱氨酸取代会影响结构稳定性、酶活性和脂质双分子层的掺入。值得注意的是，TNAP突变体S221C和P307C在DPPC脂质体中表现出增强的催化效率，而A420C由于催化位点附近的位阻而表现出活性降低。固体核磁共振和低温透射电镜分析证实了羟基磷灰石的形成，脂质锚定的TNAP对矿化过程有重要贡献。这些发现强调了脂质环境对TNAP功能特性的关键影响，并提供了对生物矿化和相关病理（包括与各种TNAP突变相关的低磷酸症）的机制的见解。该研究强调了ATP和TNAP水解焦磷酸盐在调节磷灰石形成中的重要性，并揭示了特定TNAP突变在调节酶活性、稳定性和矿物繁殖中的作用。了解这些相互作用可能会导致治疗和再生医学的替代治疗策略。

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引用次数: 0

Complexes of star-shaped block copolymers of poly(2-alkyl-2-oxazine)s and curcumin can affect lipid bilayers mimicking biomembranes 聚（2-烷基-2-氧嗪）s和姜黄素的星形嵌段共聚物配合物可以影响模拟生物膜的脂质双层

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-22 DOI: 10.1016/j.bbamem.2025.184443

Svetlana S. Efimova , Vera A. Martynyuk , Nina D. Kozina , Tatyana Y. Kirila , Alexander P. Filippov , Olga S. Ostroumova

Poly(2-alkyl-2-oxazine)s (PAlOz) are promising tools for developing delivery systems due to their high biocompatibility, resistance to enzyme hydrolysis, and ability to degrade in biological environments. Here, we investigated the effects of hexaaza[2₆]cyclophane (CPh6), poly(2-methyl-2-oxazine) (PMedOz), a block copolymer of poly(2-ethyl-2-oxazine) (PEtOz) and poly(2-isopropyl-2-oxazine) (PiPrOz), star-shaped block copolymers with six PAlOz arms and a CPh6 branching center (CPh6-PAlOz), and the complexes of all these macromolecules with curcumin on lipid bilayers mimicking the membranes of normal and cancer cells. Curcumin alone demonstrated a pronounced ability to reduce the boundary potential of lipid bilayers composed of phosphatidylcholine or phosphatidylserine, while PMedOz and PEtOz-b-PiPrOz copolymers exhibited either no or weak effects on the electrical properties of biomimetic model membranes. CPh6-PAlOz star-shaped block copolymers were able to interact with K⁺-nonactin. Differential scanning microcalorimetry of the gel-to-liquid crystalline phase transition of membrane lipids indicated that curcumin and all tested macromolecules had more pronounced effects on phosphatidylserine melting than on the phase behavior of phosphatidylcholine. A star-shaped block copolymer with a [PEtOz]/[PiPrOz] ration of 0.8 significantly decreased the melting point of phosphatidylserine. The disordering effects of complexes of curcumin with CPh6, PEtOz-b-PiPrOz copolymers, or the CPh6-PAlOz star-shaped block copolymer with a [PEtOz]/[PiPrOz] ratio of 5 on phosphatidylserine bilayers were less than the algebraic sum of the effects of the polymers and curcumin separately. These data indicate that the carrier cannot be considered an inert matrix that does not affect the biological activity of the transferred active compound, and this should be taken into account when assessing the biological consequences.

聚（2-烷基-2-恶嗪）s （PAlOz）由于其高生物相容性、抗酶水解和在生物环境中降解的能力而成为开发递送系统的有前途的工具。在这里，我们研究了六氮杂环烷（CPh6），聚（2-甲基-2-恶嗪）（PMedOz），聚（2-乙基-2-恶嗪）（PEtOz）和聚（2-异丙基-2-恶嗪）（PiPrOz）的嵌段共聚物，具有六个PAlOz臂和一个CPh6分支中心的星形嵌段共聚物（CPh6-PAlOz），以及所有这些大分子与curcumin的配合物对模拟正常和癌细胞膜的脂质双层的影响。姜黄素可以明显降低磷脂酰胆碱或磷脂酰丝氨酸组成的脂质双分子层的边界电位，而PMedOz和PEtOz-b-PiPrOz共聚物对仿生模型膜的电学性能没有或只有微弱的影响。CPh6-PAlOz星形嵌段共聚物能够与K+-非肌动蛋白相互作用。膜脂凝胶-液晶相变的差示扫描微热法表明，姜黄素和所有被测大分子对磷脂酰丝氨酸熔融的影响比对磷脂酰胆碱相行为的影响更为显著。[PEtOz]/[PiPrOz]比为0.8的星形嵌段共聚物显著降低了磷脂酰丝氨酸的熔点。姜黄素与CPh6、PEtOz-b-PiPrOz共聚物或[PEtOz]/[PiPrOz]比为5的CPh6- paloz星形嵌段共聚物的配合物对磷脂酰丝氨酸双分子层的无序作用小于聚合物与姜黄素各自作用的代数和。这些数据表明，载体不能被认为是不影响转移活性化合物生物活性的惰性基质，在评估生物后果时应考虑到这一点。

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引用次数: 0

Assessing the anticancer potential of spider venom peptide Latarcin Ltc2a against triple negative breast cancer 评估蜘蛛毒液肽Latarcin Ltc2a对三阴性乳腺癌的抗癌潜力

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-20 DOI: 10.1016/j.bbamem.2025.184442

Prasanjeet Kaur , Srabaita Roy , Shilpi Minocha , Archana Chugh

Cancer remains one of the most formidable challenges to human health, necessitating constant exploration of innovative therapeutic strategies. Among the myriad potential candidates, peptides from venom have emerged as potent sources of bioactive molecules possessing diverse pharmacological properties. In this study, we repurposed a spider venom-derived antimicrobial peptide, Ltc2a, into a selective anticancer agent, bridging microbial defense with cancer therapeutics. Our findings reveal that Ltc2a exhibits selective cytotoxicity towards cancer cells compared to normal cells at just 2 μM of the peptide concentration. Ltc2a induced rapid cytotoxicity within 1 h in breast cancer cells and it was accompanied by membrane disruption as shown by propidium iodide (PI) positive staining and visible damage to cancer cell membranes under field emission scanning electron microscopy (FESEM). In vivo studies using a zebrafish model indicated favorable uptake and a lack of acute toxicity, depicting 80 % survival rate up to 4 μM of tested peptide concentration. Interestingly, the truncated variants of Ltc2a retained their alpha helical structure and demonstrated preferential uptake in MDA-MB-231 cells over HEK293T cells. These findings highlight the therapeutic potential of Ltc2a as selective anticancer peptide with minimal toxicity, paving the way for further preclinical development.

癌症仍然是人类健康面临的最严峻挑战之一，需要不断探索创新的治疗策略。在众多潜在的候选者中，来自毒液的肽已成为具有多种药理特性的生物活性分子的有力来源。在这项研究中，我们将蜘蛛毒液衍生的抗菌肽Ltc2a改造成一种选择性抗癌剂，将微生物防御与癌症治疗联系起来。我们的研究结果表明，与正常细胞相比，Ltc2a在2 μM的肽浓度下对癌细胞表现出选择性的细胞毒性。Ltc2a在1 h内对乳腺癌细胞产生快速的细胞毒性，并伴有膜破坏，如碘化丙啶（PI）阳性染色和场发射扫描电镜（FESEM）下可见的癌细胞膜损伤。使用斑马鱼模型进行的体内研究表明，在4 μM的测试肽浓度下，其具有良好的吸收性和无急性毒性，存活率为80%。有趣的是，Ltc2a的截断变体保留了其α螺旋结构，并且在MDA-MB-231细胞中表现出比HEK293T细胞更优先的摄取。这些发现突出了Ltc2a作为毒性最小的选择性抗癌肽的治疗潜力，为进一步的临床前开发铺平了道路。

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引用次数: 0

The coronavirus spike HR2 domain: An obscure player entering the limelight during membrane fusion? 冠状病毒刺突HR2结构域：膜融合过程中一个不起眼的参与者？

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-19 DOI: 10.1016/j.bbamem.2025.184445

Elena T. Aliper , Roman G. Efremov

The coronavirus spike protein, the key entity effectuating membrane fusion, cannot exist without membrane-active fragments. In addition to fusion peptides, among such domains are HR1 and HR2. Crucial to the spike's refolding and membrane fusion, they are believed to both interact with each other and bind to the membranes that are merged. To elucidate HR2's precise role in this process, an understanding of its structure and behaviour is required. Here, we used various computational approaches to study SARS-CoV-2 spike HR2's (1163-1211) interaction with membranes in the context within which it operates in live spike. During simulations with model bilayers, HR2 remained hugely unresponsive to the presence of a membrane, however, when extended to include the transmembrane domain (TMD) (1212-1234) and/or membrane-active preHR2 fragment (1147-1161), HR2’s binding to model bilayers was markedly enhanced. The trimeric coiled-coil of HR2 does not dissociate either on its own or with added TMD and/or preHR2. Molecular hydrophobicity potential (MHP) mapping showed that HR2's central part possesses a tilted oblique-oriented motif characteristic of “textbook” membrane-active peptides, albeit flanked by highly hydrophilic fragments. A truncated HR2 only encompassing this motif had a greater affinity for membranes, suggesting HR2 has a modular structure with a membrane-active segment masked by flanking regions and might be potentiated by HR2-adjacent domains and other factors coming into play after the spike gets enzymatically cleaved. Such a modular structure may have evolved for HR2's membrane activity to be regulated very subtly and “switched on” at precisely the right moment during viral fusion.

冠状病毒刺突蛋白是实现膜融合的关键实体，它的存在离不开膜活性片段。除了融合肽外，这些结构域还包括HR1和HR2。它们对突刺的再折叠和膜融合至关重要，据信它们相互作用并结合在合并的膜上。为了阐明HR2在这一过程中的确切作用，需要了解其结构和行为。在这里，我们使用了各种计算方法来研究SARS-CoV-2刺突HR2（1163-1211）在活刺突中与膜的相互作用。在模型双分子层的模拟中，HR2对膜的存在仍然没有反应，然而，当扩展到包括跨膜结构域（TMD）（1212-1234）和/或膜活性preHR2片段（1147-1161）时，HR2与模型双分子层的结合明显增强。HR2的三聚体卷曲线圈既不能单独解离，也不能与添加的TMD和/或preHR2分离。分子疏水性电位（MHP）图谱显示，HR2的中心部分具有“教科书”膜活性肽的倾斜斜基序特征，尽管两侧有高度亲水的片段。仅包含该基序的截断的HR2对膜具有更大的亲和力，这表明HR2具有模块化结构，具有被侧翼区域掩盖的膜活性片段，并且可能被HR2邻近结构域和其他因子在刺突被酶切后起作用。这种模块化结构可能已经进化为HR2的膜活性被非常微妙地调节，并在病毒融合的正确时刻精确地“开启”。

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引用次数: 0

Study on the correlation between proton transfer of phenolic hydroxyl groups of 6 flavonoid compounds and their antioxidant activities 6种黄酮类化合物酚羟基质子转移与抗氧化活性的相关性研究

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-19 DOI: 10.1016/j.bbamem.2025.184444

Shuangmei Gong , Changying Yang , Min Li , Xiaobo Li

This study investigated six flavonoids' photophysical properties in various environments, revealing their pKa variations (e.g., quercetin: 7.364 in water vs 9.329 in vesicles) and demonstrating vesicle/liposome systems' protective effects by slowing proton transfer and oxidation. Metal ion complexation occurred preferentially at specific hydroxyl-keto sites (3-OH-4-keto for quercetin/kaempferol, 5-OH-4-keto for apigenin/baicalein), while structural features like phenolic hydroxyl arrangement and double bonds significantly influenced these interactions. Importantly, the work established that vesicular systems provide superior protection against proton transfer and oxidation compared to liposomes.ANS fluorescence quenching studies further characterized their molecular behaviors, providing key insights into flavonoid stabilization mechanisms and pharmacological activity foundations.

本研究研究了6种黄酮类化合物在不同环境下的光物理性质，揭示了它们的pKa变化（例如，槲皮素在水中：7.364比在囊泡中：9.329），并证明了囊泡/脂质体系统通过减缓质子转移和氧化的保护作用。金属离子络合优先发生在特定的羟基酮位点（槲皮素/山奈酚的3- oh -4-酮，芹菜素/黄芩素的5- oh -4-酮），而酚羟基排列和双键等结构特征显著影响这些相互作用。重要的是，这项工作确定了与脂质体相比，囊泡系统提供了更好的保护，防止质子转移和氧化。ANS荧光猝灭研究进一步表征了它们的分子行为，为黄酮类化合物的稳定机制和药理活性基础提供了重要的见解。

引用次数: 0

Accurate antibiotic accumulation in Enterobacteriaceae isolates expressing efflux pumps 表达外排泵的肠杆菌科分离物中抗生素的准确积累

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-08 DOI: 10.1016/j.bbamem.2025.184441

Jinane Tabcheh , Julia Vergalli , Jean-Marie Pagès , Jean Michel Brunel

In Enterobacteriaceae, antibiotic susceptibility is frequently influenced by mechanisms such as membrane modifications, target site mutations, and enzymatic resistance barriers. Recently, there has been a notable rise in Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae isolates exhibiting antibiotic resistance in hospital settings. Of particular concern, some resistant isolates employ membrane-associated resistance mechanisms that significantly lower intracellular antibiotic concentrations, reducing them below the threshold required for therapeutic efficacy. Advancements in methods for quantifying drug accumulation within bacterial cells have provided critical insights into these resistance mechanisms. A key step in these studies relies on cell lysis to release intracellular contents including antibacterial molecules for precise quantification. However, current lysis methods are often time-consuming, underscoring the need for a robust, efficient approach to accurately measure intracellular antibiotic concentrations in isolates exhibiting various levels of efflux pump activity. In this study, we developed a rapid and reliable lysis protocol that minimizes the risk of drug alteration while enabling precise and reproducible measurement of intracellular antibiotic concentrations allowing an evidence-based study of efflux in resistant clinical strains of Enterobacteriaceae. This approach holds significant promise for enhancing our understanding of membrane-associated resistance mechanisms and for informing the optimization of treatment strategies.

在肠杆菌科中，抗生素敏感性经常受到膜修饰、靶点突变和酶抗性屏障等机制的影响。最近，在医院环境中肺炎克雷伯菌、大肠杆菌和阴沟肠杆菌分离株表现出抗生素耐药性的病例显著增加。特别值得关注的是，一些耐药菌株采用膜相关耐药机制，显著降低细胞内抗生素浓度，使其低于治疗效果所需的阈值。定量细菌细胞内药物积累方法的进步为这些耐药机制提供了重要的见解。这些研究的关键步骤依赖于细胞裂解释放细胞内内容物，包括抗菌分子，以进行精确定量。然而，目前的裂解方法往往是耗时的，强调需要一个强大的，有效的方法来准确测量细胞内抗生素浓度的分离表现出不同水平的外排泵活性。在这项研究中，我们开发了一种快速可靠的裂解方案，最大限度地降低了药物改变的风险，同时能够精确和可重复地测量细胞内抗生素浓度，从而对肠杆菌科耐药临床菌株的外排进行循证研究。这种方法对增强我们对膜相关耐药机制的理解和优化治疗策略具有重要的意义。

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引用次数: 0

Rescue of sirtuin inhibitor-dependent decrease in claudin-4 expression and paracellular barrier property in keratinocytes by epigallocatechin gallate 没食子儿茶素没食子酸酯对sirtuin抑制剂依赖性的角化细胞claudin-4表达降低和细胞旁屏障特性的拯救作用

IF 2.5 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochimica et biophysica acta. Biomembranes

Pub Date : 2025-08-06 DOI: 10.1016/j.bbamem.2025.184440

Maika Miwa , Miki Tanabe , Shunsuke Matsuda , Kento Yamakawa , Yuta Yoshino , Norihiro Tada , Akichika Itoh , Akira Ikari

The barrier function of granular layer in the skin is mainly sustained by claudin-1 (CLDN1) and CLDN4, tight junctional components. We recently found that the activity of sirtuin-2 (SIRT2), an anti-aging molecule, is decreased with aging in keratinocytes, leading to the attenuation of CLDN4 expression and paracellular barrier function. SIRT2 may be a novel target for enhancing skin barrier function in elderly people. In vitro SIRT2 activity assay showed that epigallocatechin gallate (EGCG) and green tea extract (GT) have a potent ability to activate SIRT2. Tenovin-1 (Ten-1), a sirtuin-1/2 inhibitor, decreased the SIRT2 activity in human keratinocyte-derived HaCaT cells, which was rescued by EGCG and GT. Ten-1 decreased the protein level of CLDN4, which was rescued by EGCG, whereas CLDN1 expression was changed by neither Ten-1 nor EGCG. Ten-1 decreased the tight junctional localization of CLDN4, transepithelial electrical resistance, and paracellular permeability to FD4, a fluorescence paracellular flux marker, which were rescued by EGCG. Ten-1 increased the acetylation level of CLDN4, which was inhibited by EGCG without affecting NAD⁺ content, a substrate for SIRT2. The protein levels of wild-type and K191A mutant were decreased by Ten-1, whereas that of K196A was not. Furthermore, Ten-1 increased the acetylation levels of WT and K191A mutant. We suggest that Ten-1 decreases CLDN4 expression mediated by the acetylation of K196 of CLDN4 and EGCG is useful to protect from aging-induced dysfunction of paracellular barrier in the keratinocytes.

皮肤颗粒层的屏障功能主要由紧密连接成分CLDN1 （CLDN1）和CLDN4维持。我们最近发现，抗衰老分子sirtuin-2 （SIRT2）的活性随着角质形成细胞的衰老而降低，导致CLDN4表达和细胞旁屏障功能的衰减。SIRT2可能是增强老年人皮肤屏障功能的新靶点。体外SIRT2活性分析表明，表没食子儿茶素没食子酸酯（EGCG）和绿茶提取物（GT）具有有效的SIRT2激活能力。Tenovin-1 （Ten-1）是一种sirtuin-1/2抑制剂，可降低EGCG和GT拯救的人角化细胞来源的HaCaT细胞中SIRT2的活性。Ten-1可降低EGCG拯救的CLDN4的蛋白水平，而CLDN1的表达不受Ten-1和EGCG的影响。10 -1降低了CLDN4的紧密连接定位、经上皮电阻和细胞旁对FD4（一种荧光细胞旁通量标记物）的通透性，这些被EGCG拯救。10 -1增加了CLDN4的乙酰化水平，EGCG抑制了CLDN4的乙酰化，但不影响SIRT2的底物NAD+含量。野生型和K191A突变体的蛋白水平下降了10 -1，而K196A突变体的蛋白水平没有下降。此外，10 -1增加了WT和K191A突变体的乙酰化水平。我们认为，10 -1降低CLDN4和EGCG的K196乙酰化介导的CLDN4表达，有助于保护角质形成细胞免受衰老诱导的细胞旁屏障功能障碍。

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