Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

The relation between connective tissue cells and intercellular substances, including basement membranes.

Basement membranes are distributed widely in the body forming an extracellular matrix for epithelial and endothelial cells. The collagenous and glycoprotein constituents of basement membranes are synthesized by these two cell types. Disturbance of the interactions between basement membranes and their associated epithelial and endothelial cells can lead to the pathological changes seen in diseases involving basement membranes. These changes are illustrated here by reference to glomerulonephritis induced by the deposition of immune complexes in the glomerulus of the kidney, and chronic inflammatory changes occurring in the lung after inhalation of asbestos. In these diseases basement membrane changes can occur in several ways. Hydrolytic enzymes released from inflammatory cells degrade basement membranes while other constituents by epithelial and endothelial cells. Alternatively the physical separation of epithelial and endothelial cells from their basement membrances by space-occupying substances such as immune complexes can interfere with feedback mechanisms leading to synthesis of basement membrane constituents and cell proliferation. Studies of these pathological changes at a cellular level should shed new light on the ways in which cells interact with their pericellular environment.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.     

Dynamics of Membrane Tethers Reveal Novel Aspects of Cytoskeleton-Membrane Interactions in Axons

Mechanical properties of cell membranes are known to be significantly influenced by the underlying cortical cytoskeleton. The technique of pulling membrane tethers from cells is one of the most effective ways of studying the membrane mechanics and the membrane-cortex interaction. In this article, we show that axon membranes make an interesting system to explore as they exhibit both free membrane-like behavior where the tether-membrane junction is movable on the surface of the axons (unlike many other cell membranes) as well as cell-like behavior where there are transient and spontaneous eruptions in the tether force that vanish when F-actin is depolymerized. We analyze the passive and spontaneous responses of axonal membrane tethers and propose theoretical models to explain the observed behavior.     

The significance of nasal carriage of Staphylococcus aureus as risk factor for human skin infections

The effects of 2,2',5,5'-tetrachlorobiphenyl (TeCB), a PCB congener, and biphenyl on the cytoplasmic membranes of Ralstonia eutropha H850 were investigated by measuring fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe, and determining the cellular fatty acid compositions. TeCB significantly affected the membrane of R. eutropha H850 cells grown on fructose by decreasing DPH fluorescence polarization. In contrast, the membrane of cells grown on biphenyl showed a considerably less significant effect of TeCB on membrane polarization than in fructose-grown cells. An increase in the ratio of total saturated to unsaturated fatty acids in cells grown on biphenyl suggested less of a fluidizing effect of TeCB on membranes in those cells. When biphenyl-grown cells were transferred back to a fructose medium, they required 25 generations for the membrane polarization and fatty acid compositions of these cells to revert back to those of the initial fructose-grown cells. The re-adaptation to a change in temperature required only five generations to return to normal. These results show that biphenyl affects cells in more ways than simply fluidizing the cytoplasmic membrane.     

The role of the plasma membrane in the development of Dictyostelium discoideum. II. Developmental and topographic analysis of polypeptide and glycoprotein composition

Previous workers have shown in a variety of ways that cell contact is required for the differentiation of Dictyostelium discoideum. Because interactions between cells are probably mediated by molecules on their plasma membranes, we have characterized the polypeptide composition of the membrane of cells at different stages of development. At least 55 polypeptides are found in the plasma membrane of vegetative cells. The polypeptide composition of the plasma membranes changes considerably during development. Treatment of intact cells with pronase indicated that many of the altered components appear to be located on the external surface of the plasma membrane where they could participate in interactions between cells. Similar digestion of the isolated membranes destroys most of their polypeptides, indicating that the bulk of the proteins of the plasma membrane are not completely embedded in the membrane. Several polypeptides appear to change in sensitivity to pronase during development. There are several changes in glycoprotein composition which occur between log phase and aggregation phase. An almost complete change in glycoprotein species occurs between aggregation and pre-culmination. Unlike the polypeptides, the glycoproteins are very resistant to pronase treatment in intact cells. However, some are pronase sensitive in isolated membranes.     

Membrane lipids and transporter function

Transport proteins are essential for cells in allowing the exchange of substances between cells and their environment across the lipid bilayer forming a tight barrier. Membrane lipids modulate the function of transmembrane proteins such as transporters in two ways: Lipids are tightly and specifically bound to transport proteins and in addition they modulate from the bulk of the lipid bilayer the function of transport proteins. This overview summarizes currently available information at the ultrastructural level on lipids tightly bound to transport proteins and the impact of altered bulk membrane lipid composition. Human diseases leading to altered lipid homeostasis will lead to altered membrane lipid composition, which in turn affect the function of transporter proteins.     

Structural lability of membranes]

The present-day state of the problem of membrane structural lability is reviewed. The ways of initiation and the nature of structural rearrangements, the mechanisms of generalization of local structural perturbations in membranes and the effects of rearrangements on the functional activity of organoids and cells are discussed.     

Effect of barium and tetraethylammonium on membrane circulation in frog retinal photoreceptors

We studied the influence of altered ionic conditions on the recycling of synaptic vesicle membrane in frog retinal photoreceptors using horseradish peroxidase to monitor synaptic activity and trace the fate of internalized membrane. The addition of 1.2 mM barium or 20 mM tetraethylammonium to isolated retinas maintained in Ringer's solution, changes the usual balance of membrane circulation in the rod cells; the cone cells are much less affected. Retrieval of synaptic vesicle membrane in the rods, which normally regenerates small vesicles, becomes mediated predominantly by large sacs and vacuoles ("cisternae"). Because these cisternae can be labeled with peroxidase, they appear to arise from endocytized membrane. Morphometric analysis suggests strongly that the cisternae are formed of circulating synaptic vesicle membrane. The effects of barium and tetraethylammonium can be inhibited by high extracellular potassium, by high intensity light, and by 5 mM cobalt. They seem likely to depend on potassium channels, though additional more complex mediation may also be involved. The alterations in membrane retrieval that we find are of interest in terms of the multiple pathways of membrane cycling now being uncovered. They open potential experimental approaches to the controls of this circulation. In addition, the findings extend our previous ones demonstrating that rod cells and cone cells differ in their responses to divalent cations in ways that seem likely to be of physiological importance.     

The role of microvesicles and its active molecules in regulating cellular biology

Cell‐derived microvesicles are membrane vesicles produced by the outward budding of the plasma membrane and released by almost all types of cells. These have been considered as another mechanism of intercellular communication, because they carry active molecules, such as proteins, lipids and nucleic acids. Furthermore, these are present in circulating fluids, such as blood and urine, and are closely correlated to the progression of pathophysiological conditions in many diseases. Recent studies have revealed that microvesicles have a dual effect of damage and protection of receptor cells. However, the nature of the active molecules involved in this effect remains unclear. The present study mainly emphasized the mechanism of microvesicles and the active molecules mediating the different biological effects of receptor cells by affecting autophagy, apoptosis and inflammation pathways. The effective ways of blocking microvesicles and its active molecules in mediating cell damage when microvesicles exert harmful effects were also discussed.     

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. II. The responses to annular illumination

On-center bipolar cells in the dark-adapted carp retina were divided into four types (A, B, C, and D) on the basis of response wave forms, spectral response properties, and electrical membrane properties. Type A and B cells responded to a spot of light with a transient depolarization followed by a plateau, whereas the response of type C and D cells were approximately rectangular in shape. The center and surround responses of type A cells had maximum spectral response of approximately 525 nm in the lower mesopic range; the polarity of both responses was reversed at positive membrane potentials as the membrane was depolarized by extrinsic current. The center and surround responses of type D cells had a maximum spectral response of approximately 625 nm in the mesopic or photopic range; the polarity of both responses was reversed at membrane potentials that were more negative than those at the dark level. The results suggest that the center and surround responses mediated by rods are generated by changes in sodium conductance, but in opposite ways; whereas those mediated by red cones are generated by changes in potassium and/or chloride conductances. In type B and C cells, which probably receive inputs from both rods and/or green cones as well as red cones, the center responses were composed of the two ionic mechanisms described above. The surround responses of many type B and C cells were dominated by only one ionic mechanism with a negative reversal potential, but in some type B cells the surround responses were resulted from two ionic mechanisms similar to those of the center responses.     

GUV preparation and imaging: Minimizing artifacts

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the “raft model” of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.     

Modes of the formation of elementary bodies and their isolation from the cell in L-form bacteria

Elementary bodies are formed on the cell surface and inside the cell body in all cell types characteristic of L-form cultures, i. e. spherical cells, large bodies and filament structures. The following ways of elementary body formation are described: by budding on the cell surface, appearance immediately in the cytoplasm, in the vacuole, as a result of cytoplasmic fragmentation accompanied by the lysis of the cell, as well as in cases of the separation of cytoplasmic areas surrounded by the membrane or the myelin-like structure. The release of elementary bodies from the cell occurs as a result of the lysis or death of the mother cell, the thinning of the vacuole wall, and possibly due to small transient defects in the membrane, not accompanied by the death of the mother cell. The scheme of the formation and release of elementary bodies from the cell is presented.     

The Croonian Lecture 1999. Intracellular membrane traffic: getting proteins sorted.

The secretory and endocytic pathways within higher cells consist of multiple membrane-bound compartments, each with a characteristic composition, through which proteins move on their way to or from the cell surface. Sorting of proteins within this system is achieved by their selective incorporation into budding vesicles and the specific fusion of these with an appropriate target membrane. Cytosolic coat proteins help to select vesicle contents, while fusion is mediated by membrane proteins termed SNAREs present in both vesicles and target membranes. SNAREs are not the sole determinants of target specificity, but they lie at the heart of the fusion process. The complete set of SNAREs is known in yeast, and analysis of their locations, interactions and functions in vivo gives a comprehensive picture of the traffic routes and the ways in which organelles such as the Golgi apparatus are formed. The principles of protein and lipid sorting revealed by this analysis are likely to apply to a wide variety of eukaryotic cells.     

Integrity under stress: Host membrane remodelling and damage by fungal pathogens

Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore‐forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent “stretching” of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.     

脱水导致种子和花粉细胞膜变化的生物热力学研究进展

膜系统是引起脱水敏感细胞的损伤和死亡的原初位点之一,该文综述了近年来对以下各问题以及它们之间的关系的研究进展:(1)膜相变;(2)膜系统和生物大分子受到的压力;(3)玻璃态的形成及其部位;(4)造成玻璃态形成的溶质分子的大小对膜相变的影响;(5)两性物质在细胞质水相和膜脂脂相间的再分配.旨在为如何有效地监控种子和花粉寿命和最大程度地减少种子和花粉细胞的衰老损伤,预测种质保存的最佳贮藏条件和种质寿命,以便及时更换保存样本等方面的研究提供新的思路.     

The effect of digitonin of the stimulation by insulin of glucose uptake by isolated fat cells.

The effect of digitonin on glucose uptake by isolated fat cells in the presence and absence of insulin has been studied. At low concentrations of digitonin, the stimulation of glucose uptake by insulin was inhibited without severe cell damage as estimated by the leakage of lactate dehydrogenase from the cells. The inhibition of the insulin effect was not reversed by washing the cells or by the addition of cholesterol or lecithin-cholesterol liposomes to the incubation medium of the cells after treatment with digitonin. Cholesterol was shown to be present in the fat cells and it is suggested that the inhibition of the insulin effect is a consequence of the formation of digitonin-cholesterol complexes in the fat cell plasma membrane. Possible ways in which this may results in inhibition of the effect of insulin are discussed.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Molecular organization of gap junction membrane channels

Gap junctions regulate a variety of cell functions by creating a conduit between two apposing tissue cells. Gap junctions are unique among membrane channels. Not only do the constituent membrane channels span two cell membranes, but the intercellular channels pack into discrete cell-cell contact areas formingin vivo closely packed arrays. Gap junction membrane channels can be isolated either as two-dimensional crystals, individual intercellular channels, or individual hemichannels. The family of gap junction proteins, the connexins, create a family of gap junctions channels and structures. Each channel has distinct physiological properties but a similar overall structure. This review focuses on three aspects of gap junction structure: (1) the molecular structure of the gap junction membrane channel and hemichannel, (2) the packing of the intercellular channels into arrays, and (3) the ways that different connexins can combine into gap junction channel structures with distinct physiological properties. The physiological implications of the different structural forms are discussed.     

Interaction of the lamB protein with the peptidoglycan layer in Escherichia coli K12

In Escherichia coli K12 the product of gene lamB is an outer membrane protein involved in the transport of maltose and maltodextrins and serving as a receptor for several bacteriophages including lambda. About 30 to 40% of this protein can be recovered associated to peptidoglycan when the cells are dissolved in sodium dodecyl sulfate in the presence of 2 mM Mg2+ ions. The bound protein can then be quantitatively eluted from peptidoglycan by incubating the complex in Triton X-100 and EDTA, or sodium dodecyl sulfate and NaCl. The protein eluted in such ways is still totally active in its phage-neutralizing activity. Two other membrane proteins known to behave similarly to the lamB protein are proteins Ia and Ib. However the binding of these proteins to peptidoglycan appears tighter, in several respects, than that of the lamB protein. The lamB protein may span the outer membrane since it appears to interact with the peptidoglycan on the inner side of this membrane while it is known to be accessible to both phages and antibodies at the cell surface.