The bacterial microbiota in the ceca of Capercaillie (Tetrao urogallus) differs between wild and captive birds

The diet of wild capercaillie differs strongly between seasons. Particularly during winter, when energy demands are high and the birds forage solely on coniferous needles, microbial fermentations in the ceca are considered to contribute significantly to the energy requirement and to the detoxification of the resinous diet. Here, we present the first cultivation-independent analysis of the bacterial community in the cecum of capercaillie, using the 16S rRNA gene as a molecular marker. Cloning and fingerprinting analyses of cecum feces show distinct differences between wild and captive birds. While certain lineages of Clostridiales, Synergistetes, and Actinobacteria are most prevalent in wild birds, they are strongly reduced in individuals raised in captivity. Most striking is the complete absence of Megasphaera and Synergistes species in captive capercaillie, which are characterized by a large abundance of Gammaproteobacteria closely related to members of the genus Anaerobiospirillum, bacteria that are commonly connected with intestinal dysfunction. The community profiles of cecum content from wild birds differed between summer and winter season, and the cecum wall may be an important site for bacterial colonization. Our results corroborate the hypothesis that the bacterial community in the ceca of tetraonid birds changes in response to their highly specialized seasonal diets. Moreover, we propose that the observed differences in community profiles between wild and captive capercaillie reflects a disturbance in the bacterial microbiota that compromises the performance of the cecum and may be responsible for the high mortality of captive birds released into nature.     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Isolation and characteristics of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Phospholipase A₂ was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 20,000. The optimum pH and temperature of the enzyme were at around pH 9.0 and 50°C, respectively, and the activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The enzyme had no fatty acid specificity. Starfish phospholipase A₂ hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine.     

Ca2+ Uptake Through Voltage-gated L-type Ca2+ Channels by Polarized Enterocytes from Atlantic Cod Gadus morhua

The presence and localization of voltage-gated Ca²⁺ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca²⁺ probe, fure-2/AM and changes in intracellular Ca²⁺ concentrations ([Ca²⁺] _i ) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 μm), nifedipine (5 μm) or ω-conotoxin (1 μm). L-type Ca²⁺ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca²⁺] _i . BAY K-8644 increased the [Ca²⁺] _i by 7.2%. Cells in a Ca²⁺-free buffer increased [Ca²⁺] _i after addition of 10 mm Ca²⁺, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by ω-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca²⁺ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca²⁺ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca²⁺ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca²⁺ into the enterocytes. This suggests that the L-type Ca²⁺ channels are involved in the transcellular Ca²⁺ entry into the enterocytes. Received: 21 August 1997/Revised: 15 April 1998     

Caco-2 cells express a combination of colonocyte and enterocyte phenotypes

Caco-2 cells are derived from a human colonic adenocarcinoma, but differentiate into small intestinal-like cells after confluence. While this enterocytic differentiation has been well studied, the presumed parallel loss of colonocyte function has not been as thoroughly examined. To follow the phenotype for both tissues, Western blots were performed using antisera recognizing liver/bone/kidney alkaline phosphatase and surfactant-like particle proteins found in normal human colon, along with antisera against the small bowel representatives of the same proteins. Antisera against proteins enriched in either enterocytes (α₁-antitrypsin) or colonocytes (surfactant protein A) were also evaluated. Alkaline phosphatase activity increased from 3 to 18 days post-confluence. Activity at 3 days post-confluence derived substantially from both isomers. Thereafter, the colonic (liver/bone/kidney) isomer declined to low levels as the content of the enterocytic isomer rose. A similar pattern was found with colonic (decreasing expression) and enterocytic (increasing expression) surfactant-like particle proteins. In particular, the content of larger enterocytic particle proteins (97 and 116 kDa) increased with time in culture. Expression of α₁-antitrypsin increased early and remained high, whereas surfactant protein A generally declined after the third day post-confluency. In summary, undifferentiated Caco-2 cells express very low levels of proteins characteristic of either colonocytes or enterocytes. Immediately after confluence, they expressed proteins characteristic of both cell types. Thereafter, the content of colonocyte-specific proteins decreased, whereas those specific for the enterocyte increased. The timing and degree of this phenotypic switch have implications for the interpretation of experiments using Caco-2 cells as a model of small intestinal function. J. Cell. Physiol. 174:362–369, 1998. © 1998 Wiley-Liss, Inc.     

Cytochemical and ultrastructural study of anoikis and secondary necrosis in enterocytes detached in vivo

Detachment-induced apoptosis of enterocytes (anoikis) has not been investigated in vivo. Here we describe anoikis of fish enterocytes following detachment in a septicemia by Photobacterium damselae subsp. piscicida, or following injection of its exotoxin. The in vivo study was complemented with an ex vivo time-lapse analysis using conditions duplicating the in vivo situation. Linings of enterocytes detached from intestine mucosa dissociate into isolated enterocytes which undergo caspase 3-mediated anoikis with cell rounding, loss of polarization, condensation of chromatin and fragmentation of the nuclear envelope, early swelling of mitochondria with rupture of the outer membrane, and brush border disappearance. One mechanism for brush border loss was shedding of apoptotic bodies incorporating the apical part of the enterocyte. Brush border disappearance was also associated with disassembly of the F-actin microvillar core and involved re-absorption into the cell, or expansion and vesiculation followed by shedding of microvillar fragments. The enterocyte anoikis terminates by secondary necrosis and lysis due to lack of elimination by phagocytosis of apoptosing enterocytes. The conditions prevailing in vivo in the gut lumen accelerate enterocyte secondary necrosis. Our results underscore the importance of analyzing anoikis under conditions similar to those occurring in vivo.     

Extracellular Production of a Serratia marcescens Serine Protease in Escherichia coli

The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH₂-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH₂-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an “intramolecular chaperone,” in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane.     

Significant role of the nitrogen recycling system through the ceca occurs in protein-depleted chickens

This study focuses on the role of the ceca in nitrogen nutrition in chickens (Gallus domesticus). Urea is a very good nitrogen tracer for these studies. Little urea is synthesized by chickens due to the absence of carbamyl phosphate synthetase, an essential enzyme initiating the urea cycle. Urea is utilized by chickens when crystalline amino acid diets low in nonessential nitrogen or diets containing low concentrations of intact protein are fed, and most ureolytic activity is found in the ceca. Dietary urea was absorbed intact from the upper intestine of the chicken. The absorbed urea was excreted into ureteral urine that refluxed from the cloaca into the colon and ceca where urea was degraded to ammonia. Presumably the ammonia was incorporated into amino acids by cecal microorganisms and some urea, amino acids and proteins were absorbed from the ceca. These were utilized by the chickens. A beneficial role of ceca in the nitrogen metabolism in the chicken is, therefore, conservation of urinary nitrogen in protein-depleted chickens.     

Pressure effects on the GTPase activity of brain membrane G proteins of deep-living marine fishes

In marine fishes, heterotrimeric guanyl nucleotide binding proteins (G proteins), which couple cell surface membrane receptors to their effector elements, are sensitive to hydrostatic pressure. The intrinsic high affinity GTPase activity of the α subunits of G proteins in three signaling systems coupled to adenylyl cyclase, the A₁ adenosine receptor, the muscarinic cholinergic receptor and the β-adrenergic receptor, was tested at pressures up to 340 atm. Brain membrane preparations from four members of the deep-sea teleost fish family Macrouridae were studied. Coryphaenoides armatus, C. filifer, C. rupestris and Macrourus berglax have depth distributions which together span 100–5810 m. Increased pressure inhibited basal GTPase activity only in M. berglax, which of the four species has the shallowest center of abundance. Increased hydrostatic pressure did not alter the response of GTPase activity to the β-adrenergic receptor agonist isoproterenol. Increased pressure decreased the stimulation of GTPase activity by the A₁ adenosine receptor agonist cyclopentyladenosine (CPA) in C. armatus and M. berglax, and by the muscarinic cholinergic receptor agonist carbamyl choline in C. armatus, C. filifer and M. berglax. Decreased agonist-stimulation of the GTPase activity at elevated pressure may result from pressure-induced changes in conformational states or inhibition of agonist binding. The binding of the non-hydrolyzable GTP analog guanosine 5′-[γ-thio]triphosphate (GTP[S]) in response to CPA was determined at 5 °C and atmospheric pressure. Six macrourid species and a morid were studied. The halftime (t_1/2) values for GTP[S] binding, ranging from 20.8 to 40.9 min, are similar to values previously reported for two other cold-adapted fishes.     

Composition of the type VII secretion system membrane complex

Pathogenic mycobacteria require type VII secretion (T7S) systems to transport virulence factors across their complex cell envelope. These bacteria have up to five of these systems, termed ESX‐1 to ESX‐5. Here, we show that ESX‐5 of Mycobacterium tuberculosis mediates the secretion of EsxN, PPE and PE_PGRS proteins, indicating that ESX‐5 is a major secretion pathway in this important pathogen. Using the ESX‐5 system of Mycobacterium marinum and Mycobacterium bovis BCG as a model, we have purified and analysed the T7S membrane complex under native conditions. blue native‐PAGE and immunoprecipitation experiments showed that the ESX‐5 membrane complex of both species has a size of ～ 1500 kDa and is composed of four conserved membrane proteins, i.e. EccB₅, EccC₅, EccD₅ and EccE₅. Subsequent limited proteolysis suggests that EccC₅ and EccE₅ mostly reside on the periphery of the complex. We also observed that EccC₅ and EccD₅ expression is essential for the formation of a stable membrane complex. These are the first data on a T7S membrane complex and, given the high conservation of its components, our data can likely be generalized to most mycobacterial T7S systems.     

Effect of entomocidal proteins from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> on ion permeability of apical membranes of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> larvae gut epithelium

Effects of entomocidal Cry-type proteins, δ-endotoxins Cry3A and Cry11A produced by Bacillus thuringiensis, on ion permeability of the apical membranes of intestinal epithelium from Tenebrio molitor larvae midgut were studied. Using potential-sensitive dyes safranine O and oxonol VI and δpH indicator acridine orange, it was shown that placing brush border membrane vesicles (BBMV) (loaded with Mg²⁺ during their preparation) into a salt-free buffer medium resulted in spontaneous generation of transmembrane electric potential on the vesicular membrane (negative inside the vesicles) accompanied by acidification of the aqueous phase inside the vesicles. The generation of transmembrane ion gradients on the vesicular membrane was a result of an electrogenic efflux of Mg²⁺ from the vesicles as shown by abolishing of the membrane potential by such agents as MgSO₄ or CaCl₂ in centimolar concentrations, a highly lipophilic cation tetraphenylphosphonium, and some blockers of cell membrane Ca²⁺-channels in submillimolar concentrations. A passive generation of membrane potential on the vesicular membrane (but positive inside the vesicles) was also observed upon addition of centimolar concentrations of K₂SO₄. Addition of δ-endotoxins Cry3A and Cry11A to the vesicle suspension in a salt-free buffer medium or in the same medium supplemented with centimolar concentrations of K₂SO₄ exerted a pronounced hyperpolarization of the vesicular membrane. This hyperpolarization was sensitive to the same agents, which abolished the membrane potential generation in the absence of δ-endotoxin. It is concluded that Cry proteins induced in BBMV from T. molitor opening pores or ion channels, which were considerably more permeable for alkaline- and alkaline-earth metal cations than for the accompanying anions.     

Identification and comparsion of bile acid-binding polypeptides in ileal basolateral membrane

Summary Bile acid-binding polypeptides were examined using basolateral membrane vesicles and enterocytes isolated from rat ileum. The uptake of a photolabile taurocholate derivative, (7,7,-azo-3, 12-dihydroxy-5[3-³H]cholan-24-oyl)-2-aminoethanesulfonate, 7,7-azo-TC, in ileal vesicles preloaded with paraaminohippurate (PAH) was stimulated with respect to uptake in unpreloaded vesicles. The PAH-transstimulated uptake of 7,7-azo-TC was inhibited by taurocholate and vice versa. Irradiation of membrane vesicles in the presence of 7,7-azo-TC irreversibly inhibited PAH-transtimulated taurocholate uptake. Photoaffinity labeling of basolateral membrane vesicles directly with [³H] 7,7-azo-TC and separation of proteins by SDS-PAGE revealed incorporation of radioactivity into several polypeptides. Photoaffinity labeling of vesicles in the presence of taurocholate inhibited the labeling of 54,000 and 59,000 mol. wt. polypeptides. The efflux of taurocholate from ileal enterocytes wascis-inhibited by 7,7-azo-TC andtransstimulated by PAH. Irradiation of enterocytes in the presence of 7,7-azo-TC inhibited taurocholate efflux greater than the presence of 7.7-azo-TC in the dark. When enterocytes that were irradiated in the presence of [³H] 7,7-azo-TC were fractionated and the resultant basolateral membrane fraction was subjected to SDS-PAGE, incorporation of radioactivity into the 54,000 and 59,000 mol. wt. polypeptides was seen. In contrast, when the brush-border membrane fraction was subjected to SDS-PAGE, greatest incorporation of radioactivity was seen in the previously described 99,000 mol. wt. polypeptide. These studies suggest that 7,7-azo-TC shared transporters with natural bile acid and identified polypeptides that may be involved in bile acid and identified polypeptides that may be involved in bile acid transport across the basolateral membrane and differ from that seen in the brush-border membrane of the ileal epithelial cell.     

绞股蓝提取液对蛋白核小球藻的化感效应及影响机理研究

以中草药植物绞股蓝[Gynostemma pentaphyllum(Thunb.) Makino]为化感供体材料,研究其不同浓度的提取液(0、5、10、25、50 g/L)对蛋白核小球藻(Chlorella pyrenoidesa)生长及生理生化特征的化感效应。结果表明:(1)绞股蓝提取液对蛋白核小球藻生长均具有抑制作用,其抑制作用随提取液质量浓度增大和培养时间延长均呈增强趋势,且25 g/L绞股蓝提取液培养15 d时的抑制率达到最大(79.41%)。(2)各浓度绞股蓝处理组蛋白核小球藻细胞内的叶绿素a含量均低于对照组,且随着提取液浓度升高以及处理时间延长叶绿素a含量较对照的降低量越多,表明蛋白核小球藻光合作用受到的影响也越大。(3)绞股蓝处理组蛋白核小球藻细胞的膜透性(吸光度OD_(264))显著高于对照,且膜透性随着提取液浓度增大而增强;高浓度提取液处理下,藻细胞内部的可溶性蛋白质(OD_(280))及核酸(OD_(260))含量均显著高于对照,且随着处理时间延长,细胞膜透性增大,细胞内部的可溶性蛋白质及核酸向胞外渗透增多。研究发现,绞股蓝提取液能够抑制蛋白核小球藻生长,并随着提取液质量浓度增大而增强;绞股蓝提取液能促进藻细胞叶绿素分解、增加细胞膜透性,引起可溶性蛋白质和核酸向胞外渗透量升高,导致藻细胞结构受损,代谢功能紊乱,从而达到化感抑制作用。     

Ca++-transport across basal-lateral plasma membranes from rat small intestinal epithelial cells

Summary Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5mm MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 g/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na+K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 g/ml oligomicin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal-lateral membranes possess two distinct mechanisms for Ca-transport:a) ATP-driven Ca-transport andb) Na/Ca-exchange.     

On insertion of pigment-associated polypeptides during membrane biogenesis in Rhodopseudomonas capsulata

Early stages in the formation of membranes and photosynthetic units were studied under growth-limiting phototrophic and chemotrophic conditions in cells of Rhodopseudomonas capsulata. The incorporation of polypeptides, forming bacteriochlorophyll-carotinoid-protein complexes in the membrane, was followed by use of pulse-labeling and immunoprecipitation techniques. The newly synthesized polypeptides were inserted into two distinct membrane fractions at both different rates and proportions. The two membrane fractions differed in sedimentation behavior, absorption spectra and activities of the respiratory chain. The individual pigment-associated proteins did not exhibit precursor-product relationship between the two membrane fractions. The data suggest that newly synthesized polypeptides were integrated both into cytoplasmic and pre-existing intracytoplasmic membranes, where the proteins and pigments were assembled to form reaction centers and light-harvesting pigment-protein complexes.Abbreviations Bchl bacteriochlorophyll - cpm counts per minute - M _r relative molecular mass - P 100 pellet of 100,000xg, 60 min - P300 pellet of 300,000xg, 90 min - pO₂ oxygen partial pressure - R Rhodopseudomonas - dodecyl sulfate sodium dodecyl sulfate. International standard units - Bq Becquerel (s^-1) - Pa Pascal (N/m²; 1 Torr=133,3 Pa)     

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)‐rich lipoprotein] assembly and secretion. The accumulation of circulating intestine‐derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose‐gradient centrifugation from differentiated Caco‐2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD‐associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC‐MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte‐specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl‐CoA synthetases) and for TAG hydrolysis. In differentiated Caco‐2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3‐β‐hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA‐IV (apolipoprotein A‐IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA‐IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high‐fat diet. Conclusions. We report for the first time the protein endowment of Caco‐2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell‐specific manner.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Electrical properties of the cellular transepithelial pathway inNecturus gallbladder: III. Ionic permeability of the basolateral cell membrane

Summary The ionic permeability of the basolateral membrane ofNecturus gallbladder epithelium was studied with intracellular microelectrode techniques. After removal of most of the subepithelial tissue (to reduce unstirred layer thickness), impalements were performed from the serosal side, and ionic substitutions were made in the serosal solution while a microelectrode was kept in a cell. Thus, it was possible to obtain continuous (and reversible) records of transepithelial and cell membrane potentials and to measure intermittently the transepithelial resistance and the ratio of cell membrane resistances. From these data and the mean value of the equivalent resistance of the cell membranes in parallel (obtained from cable analysis in a different group of tissues), absolute cell membrane and shunt resistances and equivalent electromotive forces (emf's) were calculated. From the changes of basolateral membrane emf (E _b ) produced by the substitutions, the conductance (G) and permeability (P) of the membrane for K, Cl and Na were estimated. Potassium-for-sodium substitutions produced large reductions of both cell membrane potentials, ofE _b , and of the resistance of the basolateral membrane (R _b ), indicating highG _K andP _K . Chloride substitution with isethionate or sulfate resulted in smaller changes of cell membrane potentials andE _b and in no significant change ofR _b , indicating small but measurable values ofG _Cl andP _Cl . Sodium substitutions with N-methyl-d-glucamine (NMDG) resulted in cell potential changes entirely attributable to the biionic potential produced in the shunt pathway (P _Na >P _NMDG ), and in no significant changes ofP _b orE _b , indicating thatG _Na andP _Na are undetectable. The question of the mechanism of Cl transport across the basolateral membrane was addressed by comparing the mean rate of transepithelial Cl transport (J _Cl ^net ) and the predicted passive Cl flux across the basolateral membrane (from the membrane Cl conductance, potential, and Cl equilibrium potential). The conclusion is that only a very small fraction of the Cl flux across the basolateral membrane can be electrodiffusional. Since the paracellular Cl conductance is also too low to account forJ _Cl ^net , these results suggest the presence of a neutral mechanism of Cl extrusion from the cells. This could be a NaCl pump, a downhill KCl transport mechanism, or a Cl–HCO₃ exchange mechanism.     

Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 °C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 μm and a radius of 0.4 μm with an osmotically inactive cell volume, V_b, of 0.61V_o, where V_o is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L_pg and activation energy, E_Lp). The predicted best-fit permeability parameters ranged from, L_pg = 0.021–0.038 μm/min-atm and E_Lp = 27.8–41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.