Bacterial supersystem for alginate import/metabolism and its environmental and bioenergy applications

Distinct from most alginate-assimilating bacteria that secrete polysaccharide lyases extracellularly, a gram-negative bacterium, Sphingomonas sp. A1 (strain A1), can directly incorporate alginate into its cytoplasm, without degradation, through a "superchannel" consisting of a mouth-like pit on the cell surface, periplasmic binding proteins, and a cytoplasmic membrane-bound ATP-binding cassette transporter. Flagellin homologues function as cell surface alginate receptors essential for expressing the superchannel. Cytoplasmic alginate lyases with different substrate specificities and action modes degrade the polysaccharide to its constituent monosaccharides. The resultant monosaccharides, α-keto acids, are converted to a reduced form by NADPH-dependent reductase, and are finally metabolized in the TCA cycle. Transplantation of the strain A1 superchannel to xenobiotic-degrading sphingomonads enhances bioremediation through the propagation of bacteria with an elevated transport activity. Furthermore, strain A1 cells transformed with Zymomonas mobilis genes for pyruvate decarboxylase and alcohol dehydrogenase II produce considerable amounts of biofuel ethanol from alginate when grown statically.     

Structure and function of bacterial super-biosystem responsible for import and depolymerization of macromolecules

Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a "superchannel", as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.     

Effect of starvation on glucose transport and membrane fluidity in rat intestinal epithelial cells

Studies on the surface area of microvilli (MV), fluidity of brush border membranes (BBM) and -glucose uptake were carried out in rat intestinal epithelial cells (IEC) during progressive starvation and under re-feed conditions. The surface area of MV, fluidity of BBM and -glucose transport through IEC membranes showed an increase during starvation when compared to well-fed controls. Re-feeding experiments restored the control values of all the three parameters within a short time. The results showed that the increase in -glucose transport through IEC membranes during starvation is due to increased surface area of MV and increased fluidity of BBM.     

Divergent actions of monomeric, dimeric, and tetrameric concanavalin A on lymphocyte traffic

The autoradiographic analysis of the localization of [³H]adenosine-labeled cells exposed to concanavalin A (Con A) in vitro has confirmed that the altered migration of Con A-treated lymphocytes is a consequence of their slower rate of migration and delay in normal areas of traffic (5, 6). The mechanisms through which Con A alters cell migration were further investigated by studying the effects of several derivatives of Con A on the distribution of ⁵¹Cr-labeled lymph node cells. The results obtained show that the monomeric and dimeric forms of Con A were unable to modify cell traffic, a condition that was partially reversed when succinyl Con A-treated cells were exposed to divalent antibodies to Con A. This suggests that Con A may alter lymphocyte recirculation by actively modifying the membrane fluidity or the surface lateral transport of the lymphocyte. Whatever the exact mechanisms responsible for the altered migration are, they probably involve complex active processes that can be related to the heterogeneity of Con A receptors, the existence of subsets of cells with different reactivities to the lectin, or simply the result of a passive phenomenon dependent on the presence of Con A on the cell surface.     

Ion and water transport across multicellular membranes through extracellular space by chemiperistaltic waves

It is assumed that waves of configurational change of cell surface proteins can pass over the length of columnar cells in a multicellular membrane, thus passing across the membrane. It is expected that waves of change in fluidity of surface water and of Na⁺ vs K⁺ complexing preference by cell surface proteins will result from the waves of change in surface protein configuration. The entire wave process is called a chemiperistaltic wave, and is a natural extension of the concepts embodied in the association-induction hypothesis of Ling. It is shown that chemiperistaltic waves may transport Na⁺ across multicellular membranes through extracellular space between cells in a manner which is consistent with the experiments of Cereijidoet al. (1968) on frog skin. Equations for transport of Na⁺ by chemiperistaltic waves are derived for an idealized membrane. It seems possible that Na⁺−K⁺ activated ATPase may represent the isolated form of the cell surface protein in which chemiperistaltic waves are propagated or that an actomyosin-like protein may be involved.     

Effects of an extremely low frequency electromagnetic field on the cell division rate and plasma membrane of Paramecium tetraurelia

The eukaryotic protozoan, Paramecium, was examined as a model for effects of pulsated electromagnetic fields (PEMF) on cells. A 72-Hz PEMF similar to fields employed clinically increased cell division rates in Paramecium by 8.5%. Two calcium transport mutants of these organisms showed differential responses to the same field. Verapamil, a calcium channel blocker, abolished any effect of PEMFs on cell division rates. A fluorescent probe that is thought to sense changes in membrane potential also manifested an altered response in the PEMF-exposed cells whereas a fluorescent lipid bilayer fluidity probe produced evidence of decreased membrane fluidity in the exposed cells. An effect of PEMFs on ion transport mediated by either a direct or indirect effect on the cell membrane is suggested by these studies.     

Relevance of drug uptake,cellular distribution and cell membrane fluidity to the enhanced sensitivity of Down's syndrome fibroblasts to anticancer antibiotic-mitoxantrone

Sensitivity of human fibroblasts derived from Down's syndrome (DS) individuals (S-240, T-158, T-74, T-164) and normal donors (S-126, WA-1) to anticancer antibiotic-mitoxantrone (1,4-dihydroxy-5,8-bis((2-((2-hydroxy-ethyl)amino)ethyl)amino)-9,10-anthracenedione dihydrochloride; MIT) and its relationship to the transport rate, cellular distribution and interaction with cell membrane were studied. The survival assay showed that MIT was more toxic to trisomic fibroblast lines than to normal cells. Studies of transport kinetics indicated that the amount of drug taken up and extruded by DS cells was diminished, compared to control cells. In contrast, the cellular level of MIT associated with DNA was greater in trisomic than in normal cells. The fluorescence anisotropy measurements of TMA-DPH and 12-AS demonstrated that the fluidity of the polar region of the outer lipid monolayer of DS cell membrane was decreased in comparison with normal cells. MIT treatment decreased fluidity of the inner hydrophobic region of plasma membrane, but only slightly influenced the fluidity of the outer surface of the cell membrane. Finally, we concluded that lowered membrane fluidity, diminished amount of MIT extruded by cells and the enhanced level of the drug associated with DNA could be responsible for the enhanced sensitivity of DS fibroblasts to the MIT treatment.     

Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes

Sphingomyelin pathway has been linked with insulin signaling through insulin-dependent GLUT-4 glucose transporter, but a relationship between sphingomyelin and the GLUT-1 transporter responsible for the basal (insulin-independent) glucose transport has not been clearly established. As GLUT-1 is mainly distributed to the cell surface, we explored the effects of changes in membrane sphingomyelin content on glucose transport through GLUT-1. The addition of exogenous sphingomyelin or glutathione (an inhibitor of endogenous sphingomyelinase) to the culture medium increased membrane sphingomyelin and cholesterol contents. Basal glucose uptake was enhanced and positively correlated to sphingomyelin (SM), cholesterol (CL) and SM/CL ratio. The exposure of 3T3-L1 preadipocytes to sphingomyelinase (SMase) significantly increased basal glucose uptake, membrane fluidity and decreased membrane sphingomyelin and cholesterol contents 60 min after SMase addition. There was no significant change in the abundance of GLUT-1 at the cell surface. The membrane sphingomyelin and cholesterol contents, fluidity and basal glucose transport returned to baseline levels within 2 h. The basal glucose uptake was negatively correlated with cholesterol contents and positively with SM/CL ratio. The SM/CL ratio might represent an important parameter controlling basal glucose uptake and a mechanism by which insulin resistance might be induced.     

ESR studies on membrane fluidity of Chinese hamster ovary cells grown on microcarriers and in suspension

Electron spin resonance (ESR) spin label methods were used to study membrane fluidity of Chinese hamster ovary (CHO) cells grown on microcarriers and in suspension using 5-doxylstearic acid spin label as a probe. CHO cells grown on microcarriers had a more rigid cell membrane compared to CHO cells grown in suspension culture. CHO cells removed from the surface of the microcarriers by either trypsinization, EDTA treatment or osmotic shock had a membrane fluidity similar to that of CHO cells grown in suspension culture. Conversely, when the cells grown in suspension culture were attached and flattened on the surface of the microcarriers the fluidity decreased. Moreover, membrane fluidity of CHO cells grown on microcarriers changed as a function of the population density, whereas that of the cells in suspension did not. This implies that cell adhesion and/or cell-cell interactions influence the fluidity of the cell surface membrane.     

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.     

Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells

Cholesterol and phospholipid are the two major lipids of the red cell membrane. Cholesterol is insoluble in water but is solubilized by phospholipids both in membranes and in plasma lipoproteins. Morever, cholesterol exchanges between membranes and lipoproteins. An equilibrium partition is established based on the amount of cholesterol relative to phospholipid (C/PL) in these two compartments. Increases in the C/PL of red cell membranes have been studied under three conditions: First, spontaneous increases in vivo have been observed in the spur red cells of patients with severe liver disease; second, similar red cell changes in vivo have been induced by the administration of cholesterol-enriched diets to rodents and dogs; third, increases in membrane cholesterol have been induced in vitro by enriching the C/PL of the lipoprotein environment with cholesterol-phospholipid dispersions (liposomes) having a C/PL of >1.0. In each case, there is a close relationship between the C/PL of the plasma environment and the C/PL of the red cell membrane. In vivo, the C/PL mole ratio of red cell membranes ranges from a normal value of 0.9–1.0 to values which approach but do not reach 2.0. In vitro, this ratio approaches 3.0. Cholesterol enrichment of red cell membranes directly influences membrane lipid fluidity, as assessed by the rotational diffusion of hydrophobic fluorescent probes such as diphenyl hexatriene (DPH). A close correlation exists between increases in red cell membrane C/PL and decreases in membrane fluidity over the range of membrane C/PL from 1.0 to 2.0; however, little further change in fluidity occurs when membrane C/PL is increased to 2.0–3.0. Cholesterol enrichment of red cell membranes is associated with the transformation of cell contour to one which is redundant and folded, and this is associated with a decrease in red cell filterability in vitro. Circulation in vivo in the presence of the slpeen further modifies cell shape to a spiny, irregular (spur) form, and the survival of cholesterol-rich red cells is decreased in the presence of the spleen. Although active Na-K transport is not influenced by cholesterol enrichment of human red cells, several carrier-mediated transport pathways are inhibited. We have demonstrated this effect for the cotransport of Na + K and similar results have been obtained by others in studies of organic acid transport and the transport of small neutral molecules such as erythritol and glycerol. Thus, red cell membrane C/PL is sensitive to the C/PL of the plasma environment. Increasing membrane C/PL causes a decrease in membrane fluidity, and these changes are associated with a reduction in membrane permeability, a distortion of cell contour and filterability and a shortening of the survival of redcells in vivo.     

How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl2 is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0 degrees C42 degrees C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42 degrees C0 degrees C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Cargo surface fluidity can reduce inter-motor mechanical interference,promote load-sharing and enhance processivity in teams of molecular motors

In cells, multiple molecular motors work together as teams to carry cargoes such as vesicles and organelles over long distances to their destinations by stepping along a network of cytoskeletal filaments. How motors that typically mechanically interfere with each other, work together as teams is unclear. Here we explored the possibility that purely physical mechanisms, such as cargo surface fluidity, may potentially enhance teamwork, both at the single motor and cargo level. To explore these mechanisms, we developed a three dimensional simulation of cargo transport along microtubules by teams of kinesin-1 motors. We accounted for cargo membrane fluidity by explicitly simulating the Brownian dynamics of motors on the cargo surface and considered both the load and ATP dependence of single motor functioning. Our simulations show that surface fluidity could lead to the reduction of negative mechanical interference between kinesins and enhanced load sharing thereby increasing the average duration of single motors on the filament. This, along with a cooperative increase in on-rates as more motors bind leads to enhanced collective processivity. At the cargo level, surface fluidity makes more motors available for binding, which can act synergistically with the above effects to further increase transport distances though this effect is significant only at low ATP or high motor density. Additionally, the fluid surface allows for the clustering of motors at a well defined location on the surface relative to the microtubule and the fluid-coupled motors can exert more collective force per motor against loads. Our work on understanding how teamwork arises in cargo-coupled motors allows us to connect single motor properties to overall transport, sheds new light on cellular processes, reconciles existing observations, encourages new experimental validation efforts and can also suggest new ways of improving the transport of artificial cargo powered by motor teams.     

Cholinergic signaling inhibits oxalate transport by human intestinal T84 cells

Urolithiasis remains a very common disease in Western countries. Seventy to eighty percent of kidney stones are composed of calcium oxalate, and minor changes in urinary oxalate affect stone risk. Intestinal oxalate secretion mediated by anion exchanger SLC26A6 plays a major constitutive role in limiting net absorption of ingested oxalate, thereby preventing hyperoxaluria and calcium oxalate urolithiasis. Using the relatively selective PKC-δ inhibitor rottlerin, we had previously found that PKC-δ activation inhibits Slc26a6 activity in mouse duodenal tissue. To identify a model system to study physiologic agonists upstream of PKC-δ, we characterized the human intestinal cell line T84. Knockdown studies demonstrated that endogenous SLC26A6 mediates most of the oxalate transport by T84 cells. Cholinergic stimulation with carbachol modulates intestinal ion transport through signaling pathways including PKC activation. We therefore examined whether carbachol affects oxalate transport in T84 cells. We found that carbachol significantly inhibited oxalate transport by T84 cells, an effect blocked by rottlerin. Carbachol also led to significant translocation of PKC-δ from the cytosol to the membrane of T84 cells. Using pharmacological inhibitors, we observed that carbachol inhibits oxalate transport through the M(3) muscarinic receptor and phospholipase C. Utilizing the Src inhibitor PP2 and phosphorylation studies, we found that the observed regulation downstream of PKC-δ is partially mediated by c-Src. Biotinylation studies revealed that carbachol inhibits oxalate transport by reducing SLC26A6 surface expression. We conclude that carbachol negatively regulates oxalate transport by reducing SLC26A6 surface expression in T84 cells through signaling pathways including the M(3) muscarinic receptor, phospholipase C, PKC-δ, and c-Src.     

Membrane structure and surface coat of Entamoeba histolytica. Topochemistry and dynamics of the cell surface: cap formation and microexudate

Treatment of living entamoeba histolytica cells with low concentrations of concanavalin A (con A) and peroxidase results in redistribution of the plasma membrane con A receptors to one pole of the cell where a morphologically distinct region--the uroid--is formed. Capping of con A receptors is not accompanied by parallel accumulation of ruthenium red-stainable components. In capped cells, the pattern of distribution of acidic sites ionized at pH 1.8 (labeled by colloidal iron) at the outer surface and of membrane particles (integral membrane components revealed by freeze-fracture) is not altered over the uroid region. Cytochemistry of substrate-attached microexudate located in regions adjacent to E. histolytica cells demonstrates the presence of con A binding sites and ruthenium red- and alcian blue-stainable components and the absent of colloidal iron binding sites. In a previous report we demonstrated that glycerol-induced aggregation of the plasma membrane particles is accompanied by a discontinuous distribution of colloidal iron binding sites, while con A receptors and acidic sites ionized at pH 4.0 remain uniformly distributed over the cell surface. Taken together, our experiments show that, in E. histolytica cells, peripheral membrane components may move independently of integral components and, also, that certain surface determinants may redistribute independently of others. These results point to the complexity of the membrane structure-cell surface relationship in E. histolytica plasma membranes relative to the membrane of the erythrocyte ghost where integral components (the membrane-intercalated particles) contain all antigens, receptors, and anionic sites labeled so far. We conclude that fluidity of integral membrane components (integral membrane fluidity) cannot be inferred from the demonstration of the mobility of surface components nor, conversely, can the fluidity of peripheral membrane components (peripheral membrane fluidity) be assumed from demonstration of the mobility of integral membrane components.     

Band 3 tyr-phosphorylation in normal and glucose-6-phospate dehydrogenase-deficient human erythrocytes

Artificial transformation of Escherichia coli with plasmid DNA in presence of CaCl₂ is a widely used technique in recombinant DNA technology. However, exact mechanism of DNA transfer across cell membranes is largely obscure. In this study, measurements of both steady state and time-resolved anisotropies of fluorescent dye trimethyl ammonium diphenyl hexatriene (TMA-DPH), bound to cellular outer membrane, indicated heat-pulse (0°C→42°C) step of the standard transformation procedure had lowered considerably outer membrane fluidity of cells. The decrease in fluidity was caused by release of lipids from cell surface to extra-cellular medium. A subsequent cold-shock (42°C→0°C) to the cells raised the fluidity further to its original value and this was caused by release of membrane proteins to extra-cellular medium. When the cycle of heat-pulse and cold-shock steps was repeated, more release of lipids and proteins respectively had taken place, which ultimately enhanced transformation efficiency gradually up to third cycle. Study of competent cell surface by atomic force microscope showed release of lipids had formed pores on cell surface. Moreover, the heat-pulse step almost depolarized cellular inner membrane. In this communication, we propose heat-pulse step had two important roles on DNA entry: (a) Release of lipids and consequent formation of pores on cell surface, which helped DNA to cross outer membrane barrier, and (b) lowering of membrane potential, which facilitated DNA to cross inner membrane of E. coli.     

Development of beta-amino acid transport in the kidney

This study focuses on the maturation of the renal beta-amino acid transport system and uses dietary manipulation as a probe. The epithelial surface of the renal proximal tubule is responsible for the conservation of ions and organic solutes including beta-amino acids. This beta-amino acid transport system is stimulated during periods of reduced dietary intake and permits increased excretion following dietary excess. We have examined transport of the sulfur-containing beta-amino acid, taurine, as a measure of this renal adaptive response to fluctuations in dietary sulfur amino acid intake and as a substrate for the beta-amino acid transport system. A precession of taurine uptake values by brush border membrane vesicles (BBMV) prepared from nursing rats from youngest to oldest was evident. However, these membranes demonstrate the full renal adaptive response to altered sulfur amino acid intake after the first week of life. This adaptive response is expressed at the brush border surface by transport changes in both directions ("up regulation" and "down regulation"), through changes in the initial rate (15 sec) of Na+-taurine cotransport. No alterations in the lipid microenvironment of the membrane, as detected by altered membrane fluidity, were uncovered. Although vesicles from 7-day-old pups demonstrate adaptation and accumulate taurine to a limited extent, the accumulation of Na+, which energizes uptake, may be altered, thereby preventing full expression of the adaptive response and of transport capacity at this age.     

Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)