Phospholipid base exchange enzyme activity in sarcolemmal membranes from the heart of cardiomyopathic hamsters

The activity of phospholipid base exchange enzymes has been evaluated in cardiac sarcolemmal membranes from Syrian Golden hamsters and from a hamster strain (UM-X7.1) characterized by a genetic form of hypertrophic cardiomyopathy. No choline base exchange activity and only a little serine base exchange activity were detected, whereas the ethanolamine base exchange enzyme was found highly active in membranes from both strains. For this reason, the present study is focussed on the ethanolamine base exchange enzyme. The apparent Km for ethanolamine of ethanolamine base exchange enzyme from Syrian Golden membranes and from UM-X7.1 strain membranes are 18 and 32 μM, respectively. The specific activity of the sarcolemmal ethanolamine base exchange enzyme is lower in the UM-X7.1 strain than in Syrian Golden hamsters. The calcium-dependence of the enzyme appears different when the membranes from the two strains are compared. Indeed, after removal of the membrane-bound divalent cations, comparable activities are found in both membrane preparations, whereas, upon addition of Ca²⁺ to the incubation mixtures, the activity of the enzyme is enhanced in the membranes from Syrian Golden strain more than in those from UM-X7.1 strain. The cholesterol content of sarcolemmal membranes is higher in the cardiomyopathic strain than in the Syrian Golden hamsters. A possible relation between changes of the membrane lipid composition and of the ethanolamine base exchange activity is discussed.     

Preparation and Evaluation of Electrospun Zein/HA Fibers Based on Two Methods of Adding HA Nanoparticles

Zein/HA fibrous membranes were successfully prepared by electrospinning the zein/HA solution mixed by magnetic stirrer （Method I） or ultrasonic power （Method Ⅱ）. The morphology of zeirdHA nanocomposite fibers and the distribution of HA within the fibers electrospun by two methods were researched by Scanning Electron Microscopy （SEM） and Energy-dispersive X-ray spectroscopy （EDX）. In Method I, the distribution of HA nanoparticles is not homogeneous and HA particles tend to agglom- erate. The relatively homogeneous HA distribution can be observed in the membranes electrospun by Method Ⅱ. Using mag- netic stirrer to prepare the electrospinning solution improves the wettability of zein/HA membranes. From the viewpoint of application, electrospun zein/HA membranes fabricated by the solution mixed via Methods I and II both possessed reasonable tensile strength and elongation at break for both handling and sterilization. Considering two aspects of strength and elongation, electrospun zein/HA membranes fabricated by Method I are more balanced than those fabricated by Method Ⅱ. Biological performances of the control zein and zein/HA membranes were assessed by in vitro culture of hMSCs. Results show that both types of the membranes can support cell proliferation. The cells cultured on the zein/HA membranes electrospun by Method I with 5 wt% HA （on weight ofzein） show significantly higher proliferation than those cultured on the control zein membranes on the seventh day. The electrospun zein/HA fibrous membranes show promises for bone tissue engineering applications.     

Structural and Functional Damage Caused by Boron Deficiency in Sunflower Leaves

Boron deficiency induced a dramatic inhibition in sunflower plant growth, shown by a reduction in dry mass of roots and shoots of plants grown for 10 d in nutrient solution supplied with 0.02 μM B. This low B supply facilitated the appearance of brown purple pigmentation on the plant leaves over the entire growth period. Compared to B-sufficient (BS) leaves, leakage from B-deficient (BD) leaves was 20 fold higher for potassium, 38 fold for sucrose, and 6 fold for phenolic compounds. High level of membrane peroxidation was detected by measuring peroxidase activities as well as peroxidative products in BD sunflower plants. Soluble and bound peroxidase activities measured in BD thylakoid membranes were accelerated two fold compared to those detected in BS-membranes. No detectable change in soluble peroxidase activity in roots whereas a 4 fold stimulation in bound peroxidase activity was detected. Thylakoid membranes subjected to low B supply showed enhancement in lipoxygenase activity and malondialdehyde (MDA) content in parallel with 40 and 30 % decrease of linoleic and linolenic acid contents (related to total unsaturated fatty acids). A slower rate of Hill reaction activity (40 %) and a suppressed flow of electron transfer of the whole chain (30 %) were detected in BD thylakoid membranes. This reduction was accompanied with a decline in the activity of photosystem 2 shown by a diminished rate of oxygen evolution (42 %) coupled with a quenching (27.5 %) in chlorophyll a fluorescence emission spectra at 685 nm (F₆₈₅). Thus B is an important element for membrane maintenance, protection, and function by minimizing or limiting production of free oxygen radicals in thylakoid membranes of sunflower leaves. This revised version was published online in June 2006 with corrections to the Cover Date.     

EFFECTS OF VASOACTIVE INTESTINAL PEPTIDE ON ACTIVITIES OF SUCCINIC DEHYDROGENASE AND ALKALINE PHOSPHATASE IN RAT HEPATOMA CELLS

Using cytochemical method,microspectrophotometry and image analysis,effects of va-soactive intestinal peptide(VIP)on activities of succinic dehydrogenase(SDH)and alkalinephosphatase(ALP)in rat hepatoma cells were studied in vitro.The results showed that thehepatoma cell expressed potent positive reactions of SDH and ALP,the positive positionswere located at the cell membranes and/or cytoplasm.Having been treated with VIP,ALPdecreased obviously in activity(P<0. 01,compared with hepatoma cells untreated by VIP).The sites of ALP activty were chiefly located at the cell membranes,particularly at the cell-cell contacts.Cultured rat hepatoma cells had intensive SDH activity in their cytoplasm.Compared with untreated eclls,there was no marked difference in the intensity of SDH activ-ity in VIP-treated hepatoma cells(P>0.05).     

Investigating design principles of micropatterned encapsulation systems containing high-density microtissue arrays

Immunoisolation is an important strategy to protect transplanted cells from rejection by the host immune system.Recently,microfabrication techniques have been used to create hydrogel membranes to encapsulate microtissue in an arrayed organization.The method illustrates a new macroencapsulation paradigm that may allow transplantation of a large number of cells with microscale spatial control,while maintaining an encapsulation device that is easily maneuverable and remaining integrated following transplantation.This study aims to investigate the design principles that relate to the translational application of micropatterned encapsulation membranes,namely,the control over the transplantation density/quantity of arrayed microtissues and the fidelity of pre-formed microtissues to micropatterns.Agarose hydrogel membranes with microwell patterns were used as a model encapsulation system to exemplify these principles.Our results show that high-density micropatterns can be generated in hydrogel membranes,which can potentially maximize the percentage volume of cellular content and thereby the transplantation efficiency of the encapsulation device.Direct seeding of microtissues demonstrates that microwell structures can efficiently position and organize pre-formed microtissues,suggesting the capability of micropatterned devices for manipulation of cellular transplants at multicellular or tissue levels.Detailed theoretical analysis was performed to provide insights into the relationship between micropatterns and the transplantation capacity of membrane-based encapsulation.Our study lays the ground for developing new macroencapsulation systems with microscale cellular/tissue patterns for regenerative transplantation.     

rSac3, a new member of Sac domain phosphoinositide phosphatases family

Inositol phospholipids are concentrated in the cytosolic surface of membranes. Phosphatidylinositol （PtdIns）, the precursor of phosphoinositides, is synthesized primarily in the endoplasmic reticulum. Reversible phosphorylation in the inositol ring of PtdIns at positions 3, 4 and 5 results in the generation of seven phosphoinositide species： PtdIns（3）P, PtdIns（4）P, PtdIns（5）P, PtdIns（3,4）P2, PtdIns（3,5）P2, PtdIns（4,5）P2, PtdIns（3,4,5）P3. Phos- phoinositides can be rapidly interconverted from one species to another by strategically localized kinases and phosphatases [1]. Phosphoinositides play a fundamental role in controlling membrane-cytosol interfaces [2]. They are essential regulators of many cellular functions, including classical signal transduction, membrane trafficking, cytoskeletal organization, nuclear events, cell survival versus apoptosis, motility and the permeability and transport of membranes [1, 3].[第一段]     

Understanding protein translocation across chloroplast membranes: Translocons and motor proteins

Subcellular organelles in eukaryotes are surrounded by lipid membranes.In an endomembrane system,vesicle trafficking is the primary mechanism for the delivery of organellar proteins to specific organelles.However,organellar proteins for chloroplasts,mitochondria,the nucleus,and peroxisomes that are translated in the cytosol are directly imported into their target organelles.Chloroplasts are a plant-specific organelle with outer and inner envelope membranes,a dual-membrane structure that is simil...     

Evolutionary conservative phenomena in eukaryotic cells and evolutionary cell biology

Ⅰ.The significance of evolutionary viewpoint in cell research and the evolutionary conserva-tive phenomena at cellular and subcellular levelsEvolutionary theory is one of the fundamental principles of modern biology,and there-fore,the evolutionary viewpoint is one of the basic viewpoints in the research of biologi-cal problems. In living organisms,all the organic structures at various levels,from macro-molecules(nucleic acids,proteins and polysaccharides),the complexes of macro-molecules(nucleosomes,ribsomes,membranes),organelles,cells,tissues, organs,systems,up to organisms,are all the results of evolution.The mode and the mechanism     

Cloning of the promoter region of plasma membrane aquaporin BnPIP1 from Brassica napus and its functional analysis

Aquaporins make water-selective channels in plants, facilitating the permeation of water through membranes and adjusting water fast transport during seed germination, cell elongation, stoma movement, fertilization and responses to environmental stresses. They belong to the MIP (major intrinsic protein) family with molecular weight of 2629 kD and are characterized by six membrane-spanning a-helixes connected by five loops and short N-terminal and C-terminal domains in the cytoplasm[13]. The p…     

Effect of ConA—receptor interaction on the structure of cell membrane

Recently,the effect of ligand receptor interaction on the membrane structure of liposomes has been studied extensively,However,little is known about how it exists on biological membranes,In this paper,the effect of Concanavalin A(ConA) receptorinteratcion on the structure of cell membranes was studied by Circular DIchrosim(CD) and 31P Nuclear Magnetic Resonance(NMR).CD results of both the purified macrophage membranes and human erythrocyte hgosts(EG) showed that the conformation of membrane proteins changed after ConA binding.For further research,31P-NMR was used to detect the orgainzation of phosp[holipid molecules on macrophage membranes.After ConA binding,the tendercy to form non bilayer structure increased with the amount of ConA.The changes of 31P-NMR spectra of living macrophages might be partly due to the above stated reason too.In addition,ConA-receptor interaction also induced similar results of 31P-NMR spectra in EG.In contrast,wheat germ agglutinin (WGA),another kind of lectin,rarely showed the same influence.     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

In vitro reassembly of nuclear envelopes and organelles in Xenopus egg extracts

We reconstituted bilayer nuclear membranes, multilayer membranes, and organelles from mixtures ofXenopus laevis egg extracts and demembranatedXenopus sperm nuclei. Varying proportions of the cytosolic and vesicular fractions from the eggs were used in the reconstitution mixtures. A cytosol：vesicle ratio of 10：1 promoted reassembly of the normal bilayer nuclear membrane with inserted nuclear pore complexes around the decondensed Xenopus sperm chromatin. A cytosol： vesicle ratio of 5：1 caused decondensed and dispersed sperm chromatin to be either surrounded by or divided by unusual multilayer membrane structures with inlaid pore complexes. A cytosol：vesicle ratio of 2.5：1 promoted reconstitution of mitochondria, endoplasmic reticulum networks, and Golgi apparatus. During reassembly of the endoplasmic reticulum and Golgi apparatus, vesicular fragments of the corresponding organelles fused together and changed their shape to form flattened cistemae, which were then stacked one on top of another.     

Ultracytochemical localization of H＋—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

H^-adenosine triphosphatase (H^ -ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H^ -ATPase,N-ethylmaleimide (NEM) or 4,4‘-diisothiocyanostilbene-2,2‘disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H^ -ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H^ -ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H^ -ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H^ -ATPase which may be derived from lysosomal membranes.     

The Basal Level Ethylene Response is Important to the Wall and Endomembrane Structure in the Hypocotyl Cells of Etiolated Arabidopsis Seedlings

The sub-cellular events that occur during the ethylene-modulated cell elongation were characterized by examining the ultra-structure of etiolated Arabidopsis seedling hypocotyl cells. Preventing the basal level ethylene response facilitated cell elongation, and the cells exhibited wall loosening and separation phenotype. Nearby the wall separation sites were frequently associated with an increase in the cortical rough endoplasmic reticulum (rER) membranes, the presence of paramural bodies, and the circular Golgi formation. The cortical rER proliferation and circular Golgi phenotype were reverted by the protein biosynthesis inhibitor cycloheximide. The cortical rER membranes were longer when the ethylene response was prevented and shortened with elevated ethylene responses. Proteomic changes between wild type and the ethylene-insensitive mutant ethylene insensitive2 (ein2) seedling hypocotyls indicated that distinct subsets of proteins involving endomembrane trafficking, remodeling, and wall modifications were differentially expressed. FM4-64 staining supported the proteomic changes, which indicated reduced endocytosis activity with alleviation of the ethylene response. The basal level ethylene response has an important role in endomembrane trafficking, biological materials transport and maintenance of the endomembrane organization. It is possible that endomembrane alterations may partly associate with the wall modifications, though the biological significance of the alterations should be addressed in future studies.     

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8±0.09 nm in diameter and 1.9±0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40―60 nm, and a range of 1.8―5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12―40 nm for their diameter and 0.7―2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 μm2. According to their size, we classified the invaginated pits into two types―larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead pro-toplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still de-tectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in di-ameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6―9.8 nm, thicker in regions covered with proteins.     

Binding of rhodopsin and rhodopsin analogues to transducin,rhodopsin kinase and arrestin-1

AIM: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1. METHODS: Rod outer segments(ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra. Transducin and arrestin-1 were purified to homogeneity by column chromatography, and an enriched-fraction of rhodopsin kinase was obtainedby extracting freshly prepared ROS in the dark. The guanine nucleotide binding activity of transducin was determined by Millipore filtration using β,γ-imido-(3H)-guanosine 5'-triphosphate. Recognition of the reconstituted pigments by rhodopsin kinase was determined by autoradiography following incubation of ROS membranes containing the various regenerated pigments with partially purified rhodopsin kinase in the presence of(γ-32P) ATP. Binding of arrestin-1 to the various pigments in ROS membranes was determined by a sedimentation assay analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. RESULTS: Reconstituted rhodopsin and rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal rendered an absorption spectrum showing a maximum peak at 498 nm, 486 nm and about 467 nm, respectively, in the dark; which was shifted to 380 nm, 404 nm and about 425 nm, respectively, after illumination. The percentage of reconstitution of rhodopsin and the rhodopsin analogues containing 9-cis-retinal and 13-cis-retinal was estimated to be 88%, 81% and 24%, respectively. Although only residual activation of transducin was observed in the dark when reconstituted rhodopsin and 9-cis-retinal-rhodopsin was used, the rhodopsin analogue containing the 13-cis isomer of retinal was capable of activating transducin independently of light. Moreover, only a basal amount of the reconstituted rhodopsin and 9-cis-retinal-rhodopsin was phosphorylated by rhodopsin kinase in the dark, whereas the pigment containing the 13-cis-retinal was highly phosphorylated by rhodopsin kinase even in the dark. In addition, arrestin-1 was incubated with rhodopsin, 9-cis-retinal-rhodopsin or 13-cis-retinal-rhodopsin. Experiments were performed using both phosphorylated and non-phosphorylated regenerated pigments. Basal amounts of arrestin-1 interacted with rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin under dark and light conditions. Residual arrestin-1 was also recognized by the phosphorylated rhodopsin and phosphorylated 9-cis-retinal-rhodopsin in the dark. However, arrestin-1 was recognized by phosphorylated 13-cis-retinal-rhodopsin in the dark. As expected, all reformed pigments were capable of activating transducin and being phosphorylated by rhodopsin kinase in a lightdependent manner. Additionally, all reconstituted photolyzed and phosphorylated pigments were capable of interacting with arrestin-1. CONCLUSION: In the dark, the rhodopsin analogue containing the 13-cis isomer of retinal appears to fold in a pseudo-active conformation that mimics the active photointermediate of rhodopsin.     

Toll-like receptors are potential therapeutic targets in rheumatoid arthritis

Toll-like receptors (TLRs) are found on the membranes of pattern recognition receptors and not only play important roles in activating immune responses but are also involved in the pathogenesis of inflammatory disease, injury and cancer. Furthermore, TLRs are also able to recognize endogenous alarmins released by damaged tissue and necrosis and/or apoptotic cells and are present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands plays an important role in initiating and driving inflammatory diseases. Increasing data suggest a role for TLR signaling in rheumatoid arthritis, which is an autoimmune disease. Although their involvement is not comprehensively understood, the TLRs signaling transducers may provide potential therapeutic targets.     

Electron Tomography in Plant Cell Biology

This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution （4-7 nm）. Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.