Plasma Membrane Protein Clusters Appear in CFTR-Expressing Xenopus Laevis Oocytes after cAMP Stimulation

Membrane trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is supposed to be an important mechanism controlled by the intracellular messenger cAMP. This has been shown with fluorescence techniques, electron microscopy and membrane capacitance measurements. In order to visualize protein insertion we applied atomic force microscopy (AFM) to inside-out oriented plasma membrane patches of CFTR-expressing Xenopus laevis oocytes before and after cAMP-stimulation. In a first step, oocytes injected with CFTR-cRNA were voltage-clamped, verifying successful CFTR expression. Water-injected oocytes served as controls. Then, plasma membrane patches were excised, placed (inside out) on glass and scanned by AFM. Before cAMP-stimulation plasma membranes of both water-injected and CFTR-expressing oocytes contained about 200 proteins per μm². Molecular protein masses were estimated from molecular volumes measured by AFM. Before cAMP-stimulation, protein distribution showed a peak value of 11 nm protein height corresponding to 475 kDa. During cAMP-stimulation with 1 mm isobutylmethylxanthine (IBMX) plasma membrane protein density increased in water-injected oocytes to 700 proteins per μm² while the peak value shifted to 7 nm protein height corresponding to 95 kDa. In contrast, CFTR-expressing oocytes showed after cAMP-stimulation about 400 proteins per μm² while protein distribution exhibited two peak values, one peak at 10 nm protein height corresponding to 275 kDa and another one at 14 nm corresponding to 750 kDa. They could represent heteromeric protein clusters associated with CFTR. In conclusion, we visualized plasma membrane protein insertion upon cAMP-stimulation and quantified protein distribution with AFM at molecular level. We propose that CFTR causes clustering of plasma membrane proteins. Received: 11 September 2000/Revised: 13 December 2000     

A biomechanical model to simulate the effect of a high vertical loading on trunk flexural stiffness

A human trunk model was developed to simulate the effect of a high vertical loading on trunk flexural stiffness. A force–length relationship is attributed to each muscle of the multi-body model. Trunk stiffness and muscle forces were evaluated experimentally and numerically for various applied loads. Experimental evaluation of trunk stiffness was carried out by measuring changes in reaction force following a sudden horizontal displacement at the T10 level prior to paraspinal reflexes induction. Results showed that the trunk stiffness increases under small applied loads, peaks when the loads were further increased and decreases when higher loads are applied. A sensitivity analysis to muscle force–length relationship is provided to determine the model's limitations. This model pointed out the importance of taking into account the changes in muscle length to evaluate the effect of spinal loads beyond the safe limit that cannot be evaluated experimentally and to predict the trunk instability under vertical load.     

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell–cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm² areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding.     

Structural Identification of Individual Helical Amyloid Filaments by Integration of Cryo-Electron Microscopy-Derived Maps in Comparative Morphometric Atomic Force Microscopy Image Analysis

The presence of amyloid fibrils is a hallmark of more than 50 human disorders, including neurodegenerative diseases and systemic amyloidoses. A key unresolved challenge in understanding the involvement of amyloid in disease is to explain the relationship between individual structural polymorphs of amyloid fibrils, in potentially mixed populations, and the specific pathologies with which they are associated. Although cryo-electron microscopy (cryo-EM) and solid-state nuclear magnetic resonance (ssNMR) spectroscopy methods have been successfully employed in recent years to determine the structures of amyloid fibrils with high resolution detail, they rely on ensemble averaging of fibril structures in the entire sample or significant subpopulations. Here, we report a method for structural identification of individual fibril structures imaged by atomic force microscopy (AFM) by integration of high-resolution maps of amyloid fibrils determined by cryo-EM in comparative AFM image analysis. This approach was demonstrated using the hitherto structurally unresolved amyloid fibrils formed in vitro from a fragment of tau (297–391), termed ‘dGAE’. Our approach established unequivocally that dGAE amyloid fibrils bear no structural relationship to heparin-induced tau fibrils formed in vitro. Furthermore, our comparative analysis resulted in the prediction that dGAE fibrils are structurally closely related to the paired helical filaments (PHFs) isolated from Alzheimer’s disease (AD) brain tissue characterised by cryo-EM. These results show the utility of individual particle structural analysis using AFM, provide a workflow of how cryo-EM data can be incorporated into AFM image analysis and facilitate an integrated structural analysis of amyloid polymorphism.     

An assessment of protein-ligand binding site polarizability

Electronic polarizability, an important physical property of biomolecules, is currently ignored in most biomolecular calculations. Yet, it is widely believed that polarization could account for a substantial fraction of the total nonbonded energy of a system. This belief is supported by studies of small complexes in vacuum. This perception is driving the development of a new class of polarizable force fields for biomolecular calculations. However, the quantification of this term for protein-ligand complexes has never been attempted. Here we explore the polarizable nature of protein-ligand complexes in order to evaluate the importance of this effect. We introduce two indexes describing the polarizability of protein binding sites. These we apply to a large range of pharmaceutically relevant complexes. We offer a recommendation of particular complexes as test systems with which to determine the effects of polarizability and as test cases with which to test the new generation of force fields. Additionally, we provide a tabulation of the amino acid composition of these binding sites and show that composition can be specific for certain classes of proteins. We also show that the relative abundance of some amino acids is different in binding sites than elsewhere in a protein's structure.     

Resolving the molecular structure of microtubules under physiological conditions with scanning force microscopy

We have imaged microtubules, essential structural elements of the cytoskeleton in eukaryotic cells, in physiological conditions by scanning force microscopy. We have achieved molecular resolution without the use of cross-linking and chemical fixation methods. With tip forces below 0.3 nN, protofilaments with ~6 nm separation could be clearly distinguished. Lattice defects in the microtubule wall were directly visible, including point defects and protofilament separations. Higher tip forces destroyed the top half of the microtubules, revealing the inner surface of the substrate-attached protofilaments. Monomers could be resolved on these inner surfaces.Abbreviations APTS (3-aminopropyl)triethoxysilane - DETA N¹-[3-(trimethoxysilyl)propyl]diethylenetriamine - EM electron microscopy - MT microtubule - SFM scanning force microscopy     

The harsh life on the 15th century Croatia‐Ottoman Empire military border: Analyzing and identifying the reasons for the massacre in Čepin

Excavation of the historic period cemetery in ?epin, Croatia revealed the presence of a large number of perimortem injuries distributed among males, females, and subadults. Archaeological and historical data suggest these individuals were victims of a raid carried out by Turkish akinji light cavalry in 1441. Comparisons with the frequencies of perimortem trauma in 12 other, temporally congruent skeletal series from the Balkans (n = 2,123 skeletons) support this assumption. The role of the akinji in the Ottoman army was twofold: to supply war captives, and to terrorize and disperse local populations before the advance of regular troops. This article tests the hypothesis that the purpose of the 1441 raid was the latter. To accomplish this, perimortem trauma in the series were analyzed by sex, age, location, and depth of the injury. A total of 82 perimortem injuries were recorded in 12 males, 7 females, and 3 subadults. The demographic profile of the victims suggests that young adults were specifically targeted in the attack. Significant sex differences are noted in the number, distribution, and pattern of perimortem trauma. Females exhibit significantly more perimortem injuries per individual, and per bone affected, than males. The morphology and pattern of perimortem trauma in females is suggestive of gratuitous violence. Cumulatively, analysis of the osteological data suggest that the objective of the 1441 akinji raid was to spread terror and panic in the ?epin area, either as revenge for recent military setbacks, or as part of a long‐term strategy intended to depopulate the area around Osijek. Am J Phys Anthropol, 2010. © 2009 Wiley‐Liss, Inc.     

Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts

Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-β-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed.     

杭州西溪国家湿地公园1993年以来景观演变及其驱动力分析

在遥感和GIS技术支持下,以杭州西溪国家湿地公园的TM影像为主要数据源,研究了西溪湿地近10年的景观空间格局特征和演变情况,探讨了景观演变的驱动因素.结果表明,景观多样性指数由1993年的1.7854上升到2001年的1.8438和2003年的2.2096, 景观多样性指数在1993年以后持续上升.景观的破碎化指数从1993年的0.0036增加到2001年的0.0042和2003年的0.0047,表明西溪整个景观的破碎化程度随时间而加深,各类景观受人类活动的干扰在增强.人为活动成为西溪湿地格局演变的主要驱动因素.房地产开发是地处城市边缘的西溪湿地景观演变的主要内在动力,景观整体多样性的演变受到景区社会经济发展水平和各种政策的强烈影响.     

Activation-induced force enhancement in human adductor pollicis

It has been known for a long time that the steady-state isometric force after muscle stretch is bigger than the corresponding force obtained in a purely isometric contraction for electrically stimulated and maximal voluntary contractions (MVC). Recent studies using sub-maximal voluntary contractions showed that force enhancement only occurred in a sub-group of subjects suggesting that force enhancement for sub-maximal voluntary contractions has properties different from those of electrically-induced and maximal voluntary contractions. Specifically, force enhancement for sub-maximal voluntary contractions may contain an activation-dependent component that is independent of muscle stretching. To address this hypothesis, we tested for force enhancement using (i) sub-maximal electrically-induced contractions and stretch and (ii) using various activation levels preceding an isometric reference contraction at 30% of MVC (no stretch). All tests were performed on human adductor pollicis muscles. Force enhancement following stretching was found for all subjects (n = 10) and all activation levels (10%, 30%, and 60% of MVC) for electrically-induced contractions. In contrast, force enhancement at 30% of MVC, preceded by 6 s of 10%, 60%, and 100% of MVC was only found in a sub-set of the subjects and only for the 60% and 100% conditions. This result suggests that there is an activation-dependent force enhancement for some subjects for sub-maximal voluntary contractions. This activation-dependent force enhancement was always smaller than the stretch-induced force enhancement obtained at the corresponding activation levels. Active muscle stretching increased the force enhancement in all subjects, independent whether they showed activation dependence or not. It appears that post-activation potentiation, and the associated phosphorylation of the myosin light chains, might account for the stretch-independent force enhancement observed here.