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1.
The Saccharomyces cerevisiae mnn10 mutant is defective in thesynthesis of N-linked oligosaccharides (Ballou et al., 1989).This mutation has no effect on O-linked sugars, but resultsin the accumulation of glycoproteins that contain severely truncatedN-linked outer-chain oligosaccharides. We have cloned the MNN10gene by complementation of the hygromycin B sensitivity conferredby the mutant phenotype. Sequence analysis predicts that Mnn10pis a 46.7 kDa type II membrane protein with structural featurescharacteristic of a glycosyltransferase. Subcellular fractionationdata indicate that most of the Mnn10 protein cofractionateswith Golgi markers and away from markers for the endoplasmicreticulum (ER), suggesting Mnn10p is localized to the Golgicomplex. A comparison of the Mnn10 protein sequence to proteinsin the two different databases identified five proteins thatare homologous to Mnn10p, including a well characterized Schizosaccharomycespombe  相似文献   

2.
Plasma membrane sheets from Xenopus oocytes have been isolated for use in fluorescence resonance energy transfer (FRET) measurements. This system has the following advantages: 1) fluorescent recordings from a large surface area to maximize the signal-to-noise ratio, 2) reduction in background fluorescence from proteins retained in intracellular compartments, and 3) access to the cytoplasmic surface of the plasma membrane for rapid solution changes. To demonstrate the utility of this approach, we have examined a previously published FRET-based Ca2+ sensor, namely, the Cameleon-PM. This construct targets to the plasma membrane and, upon various Ca2+ additions to the cytoplasmic face of the membrane, shows ratiometric FRET changes. From the ratiometric changes recorded, an apparent Ca2+ affinity of 1.65 µM was determined. Thus preparation of Xenopus oocyte plasma membrane sheets and FRET measurements demonstrates all three of the advantages outlined above. fluorescence resonance energy transfer  相似文献   

3.
The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum. small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation  相似文献   

4.
The isolation of Golgi membranes from suspension-cultured cellsof rice (Oryza sativa L.) was attempted by linear glycerol densitygradient centrifugation. When "burst" membranes in the pelletobtaind after differential centrifugation at 100,000 ? g weresuspended in 20% (w/w) glycerol in 50 mM malate-NaOH (pH 6.0)and loaded onto a linear density gradient of glycerol, whichextended from 30 to 80% (w/w) in 1 mM EDTA in 50 mM glycylglycine-NaOH(pH 7.5), IDPase, a marker enzyme for Golgi membranes, was separatedfrom other membrane markers on the glycerol gradient. In addition,UDPase and GDPase activities overlapped with the peak fractionof IDPase activity. Furthermore, membrane glycoproteins in eachfraction were characterized by lectin-peroxidase staining. ConcanavalinA and lentil lectin, which have the ability to bind to the high-mannosetype of oligosaccharide, bound to glycoproteins distributedin ER membrane fractions, while wheat germ lectin, castor beanlectin, peanut lectin, and Ulex europaeus lectin-I which recognizethe complex type and/or the mucin type of oligosaccharides interactedwith glycoproteins in the Golgi membrane fractions but not withthose in the ER membrane. These results strongly suggest thatthe oligosaccharide structures of glycoproteins in the ER membraneare of the high-mannose type, whereas glycoproteins in the Golgimembrane have modified N-linked and/or O-linked oligosaccharidechains. (Received November 9, 1988; Accepted October 17, 1989)  相似文献   

5.
The surface patterns of winged seeds of Paulownia fortunei,P. tomentosa and P. fortuneixP. tomentosa were examined by scanningelectron microscopy. The pattern of reticulation on the wingsand seed coat of P. fortunei and the hybrid are comparable,while that on P. tomentosa is different and more elongated.Also, the wings are more extended at the oblong ends of theseeds in the former when compared to the wings of P. tomentosa.Distinct random amplified polymorphic DNA (RAPD) patterns wereobtained for the three taxa and P. kawakamii with five differentrandom oligonucleotide primers, suggesting that the method canyield genetic markers for differentiating the taxa. Also, Southernblot analyses of the RAPD products of the hybrid and the twoparent species revealed shared (inherited) genetic polymorphisms.Copyright 1999 Annals of Botany Company Paulownia species and hybrid, seed surface architecture, reticulated thickening, RAPD markers, Scrophulariaceae.  相似文献   

6.
Degree of unsaturation of sn-2 located fatty acyl side-chainsin typical membrane phospholipids has a marked effect on surfaceten si on-associated parameters of monolayers of these lipidsover aqueous sub-phases. For a fixed area monolayer in a completelyexpanded state, an increase in the number of cis double bondswas found to cause a concomitant increase in surface tension.For the same fatty acids incorporated into phosphatidylcholinemolecules, surface pressure/molecular area isotherms show thatsn-2 linoleoyl monolayers manifest markedly higher surface pressuresthan sn-2 oleoyl ones in the fully compressed state. Furthermore,the isotherms and monolayer collapse points indicate a greaterrigidity of the oleoyl species monolayer. The strong correlationof these effects with molecular radius, rotational diffusionconstant and total molecular peripheral properties of the monolayermay be determined by rotational micro viscosity experiencedby the molecules. This in turn is determined by molecular radiusthrough the total molecular peripheral length. It is suggestedthat a contributing factor to membrane bioregulation may besuch changes in surface tension-associated parameters arisingfrom the structure of the unsaturated fatty acyl phospholipidside-chains. In a biological membrane the surface tension would,therefore, be an average of headgroup effects and of degreeof fatty acyl side-chain unsaturation. Key words: Fatty acyl unsaturation, membrane, phosphatidylcholine  相似文献   

7.
Stimulation ofpancreatic acinar cells induces the release of digestive enzymes viathe exocytotic fusion of zymogen granules and activates postfusiongranule membrane retrieval and receptor cycling. In the present study,changes in membrane surface area of rat single pancreatic acinar cellswere monitored by cell membrane capacitance(Cm)measurements and by the membrane fluorescent dye FM1-43. When measuredwith the Cmmethod, agonist treatment evoked a graded, transient increase in acinarcell surface area averaging 3.5%. In contrast, a 13% increase insurface area was estimated using FM1-43, corresponding to the fusion of48 zymogen granules at a rate of 0.5 s1. After removal of FM1-43from the surface-accessible membrane, a residual fluorescence signalwas shown by confocal microscopy to be localized in endosome-likestructures and confined to the apical regions of acinar cells. Thedevelopment of an optical method for monitoring the membrane turnoverof single acinar cells, in combination with measurements ofCm changes,reveals coincidence of exocytotic and endocytotic activity in acinarcells after hormonal stimulation.

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8.
The coenocytic endosperm of Euphorbia peplus contains a complexsystem of flat, tightly-stacked membrane saccules which havea sinuous profile and a smooth or smooth-rough surface. Theyare joined by membrane-to-membrane links. The usual ergastoplasmiccisternae are an integral component of this system which canessentially be regarded as a sui generis from of endoplasmicreticulum.Copyright 1995, 1999 Academic Press Euphorbia peplus, TEM, ultrastructure, endosperm, endoplasmic reticulum, cell organelles  相似文献   

9.
Epithelium--the primary building block for metazoan complexity   总被引:2,自引:0,他引:2  
In simplest terms, the complexity of the metazoan body arisesthrough various combinations of but two tissue types: epitheliumand mesenchyme. Through mutual inductions and interactions,these tissues produce all of the organs of the body. Of thetwo, epithelium must be considered the default type in the Eumetazoabecause it arises first in embryonic development and becausemesenchyme arises from it by a switching off of the mechanismsthat underly differentiation and maintenance of epithelial cells.In the few model metazoans whose epithelia have been studiedby molecular techniques (largely Drosophila, Caenorhabditis,mouse), the molecular mechanisms underlying differentiationof epithelia show remarkable similarity. Extrapolating fromthese studies and from comparisons of the morphology of epitheliain lower metazoans, I propose how epithelia arose in the stemmetazoan. Steps in epithelial differentiation include 1) establishmentof cell polarity by molecular markers confined to either apicalor basolateral domains in the plasma membrane; 2) aggregationof cells into sheets by localization of cell-adhesion moleculeslike cadherin to the lateral membrane; 3) formation of a zonulaadherens junction from the cadherins by their localization toa discrete belt; 4) cell-to-cell linking of certain transmembraneproteins (primitively in the septate junction) to produce gatesthat physiologically isolate compartments delimited by the cells;and 5) synthesis of a basal lamina and adaptation of receptors(integrins) to its components. Despite morphological differencesin the variety of cell junctions evident in various epithelia,the underlying molecular markers of these junctions are probablyuniversally present in all eumetazoan epithelia.  相似文献   

10.
A field experiment was conducted with a water-stressed treatmentand well-watered control using eight maize (Zea mays L.) cultivars.Effects of water deficits on cell membrane stability (CMS) measuredby the polyethylene glycol (PEG) test, leaf surface wax content,and relative growth rate were investigated. Cytoplasmic lipidcontent was also analysed. Cell membrane stability and leaf surface wax content increasedwith the degrees of stress. Tolerance to drought evaluated asincrease in CMS under water deficit conditions was well differentiatedbetween cultivars and was well correlated with a reduction inrelative growth rate under stress. A negative correlation wasfound between percentage injury in the PEG test and leaf surfacewax content. High phospholipid contents were observed in tissuesof drought tolerant cultivars under water deficit conditions. Key words: Cell membrane stability, cytoplasmic lipid, drought tolerance, leaf surface wax, relative growth rate  相似文献   

11.
Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Zcal) based on the model agreed well with the experimental measurement (Zexp), and the percentage error defined as |(ZcalZexp)/Zexp| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin  相似文献   

12.
The spermatozoid of Dictyota is shown to be structurally morelike a vegetative zoospore than was the case in Fucus. The mitochondria,golgi, overall shape of the nucleus, nuclear membrane, fat bodies,and miscellaneous vesicles are as in a zoospore; there is, however,a vestigial chromatophore without an eyespot and the ciliaryapparatus is specialized. In spite of the single flagellum thereare two basal bodies inside the cell, one of which is apparentlyvestigial, ending blindy in the cytoplasm; both the a similarinternal structure with nine fibres in the wall. A fibrous ‘root’,possibly homologous with the ‘proboscis’ of Fucus,arises near the outer side of the functional basal body; itis a band of about eight fibres passing through the cell withouttouching the nucleus but closely pressed to the inner facesof at least two mitochondria before ending in an unknown mannerat the cell surface. The internal structure of the flagellumis normal but the fact that the median strand is marked by arowspines has been used to demonstrate is a conclusive way thefacts of bilateral symmetry in the whole organ; Dictyota agreesexactly with Fucus and a previous error based on incompleteinformation has been corrected. The process of unwinding ofthe flagellum from the surface of the body after liberationfrom the antheridium is described and used to illustrate someunexpected properties of the surface membrane.  相似文献   

13.
The role of the molybdenum cofactor (Mo cofactor) in the translocationof dimethyl sulfoxide (DMSO) reductase to the periplasmic spacewas studied in vivo by isolating chlorate-resistant mutantsof Rhodobacter sphaeroides f. sp. denitrificans. More than 50%of the chlorate-resistant mutants isolated were defective inthe biosynthesis of the Mo cofactor and all of these mutantsaccumulated the precursor form of the enzyme. About 45% of themutants contained the same level of Mo cofactor as the parentstrain and exhibited normal levels of DMSO reductase and nitratereductase activities when chlorate was absent from the medium,but the activities of these enzymes were depressed when chloratewas present. Much of the accumulated precursor form of the enzymein a Mo cofactor-deficient mutant was bound to the cytoplasmicmembrane and was sensitive to treatment with proteinase K fromthe periplasmic side of the membrane, an indication that theprecursor was exposed on the periplasmic surface of the membrane.The precursor accumulated on the membrane of the parent strainwhen molybdate was removed from the medium or upon additionof tungstate and this precursor was also sensitive to the treatmentwith proteinase K from the periplasmic side. These results suggestthat the Mo cofactor is necessary for proteolytic processingof the precursor to the mature enzyme on the periplasmic sideof the membrane, whereas binding of the precursor to the membraneand translocation across it can occur in the absence of thecofactor. Almost all of the Mo cofactor available for directreconstitution in vitro of nitrate reductase activity from thenit-l mutant of Neurospora crassa was present in the cytoplasmicfractions. (Received December 11, 1991; Accepted March 25, 1992)  相似文献   

14.
Neurons aremechanically robust. During prolonged swelling, molluscan neurons cantriple their apparent membrane area. They gain surface area andcapacitance independent of extracellular Ca concentration([Ca]e), but it isunknown if an increase in intracellular Ca concentration([Ca]i) isnecessary. If Ca for stimulating exocytosis is unnecessary, it ispossible that swelling-induced membrane tension changes directlytrigger surface area readjustments. If, however, Ca-mediated but nottension-mediated membrane recruitment is responsible for surface areaincreases, swelling neurons should sustain elevated levels of[Ca]i. The purpose ofthis investigation is to determine if the[Ca]i in swellingneurons attains levels high enough to promote exocytosis and if anysuch increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethyleneglycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 µM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min)were done at either a standard[Ca]e or very low[Ca]e level (0.9 mM or0.13 µM, respectively). In neither case did the[Ca]i increase tolevels that drive exocytosis. We also monitored osmomechanically drivenmembrane dynamics [swelling, then formation and reversal ofvacuole-like dilations (VLDs)] with the[Ca]i clamped below 40 nM via1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]idid not change with swelling, and VLD behavior was unaffected,consistent with tension-driven,[Ca]i-independent surface area adjustments. In addition, neurons with[Ca]i clamped at 0.1 µM via an ionophore could produce VLDs. We conclude that, undermechanical stress, neuronal membranes are compliant by virtue ofsurface area regulatory adjustments that operate independent of[Ca]i. The findingssupport the hypothesis that plasma membrane area is regulated in partby membrane tension.

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15.
Glycosyl phosphatidylinositols (GPIs) are usedto anchor many proteins to the cell surface membrane and are utilizedin all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. Weisolated one of the components of this complex,mGPAA1 (murine GPI anchor attachment), by the signalsequence trap method. mGPAA1 cDNA is about 2 kb in lengthand encodes a putative 621 amino acid protein. The mGPAA1gene has 12 small exons and 11 small introns. mGPAA1 mRNA isubiquitously expressed in mammalian cells, and in situ hybridizationanalysis revealed that it is abundant in the choroid plexus, skeletalmuscle, osteoblasts of rib, and occipital bone in mouse embryos. Itsexpression levels and transamidation efficiency decreased withdifferentiation of embryonic stem cells. The 3T3 cell lines expressingantisense mGPAA1 failed to express GPI-anchored proteins onthe cell surface membrane.

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16.
Photosynthate movement within the coat of the developing seedof Vicia faba occurs radially inward from the restricted vascularsystem and laterally through the non-vascularized region ofthe seed coat prior to exchange to the seed apoplast. Thin-walledparenchyma/transfer cells line the entire inner surface of theseed coat and thus are located at the terminus of the photosynthatetransfer pathway. The principal cellular route of transfer withinthe seed coat and the role of the thin-walled parenchyma/transfercells in membrane exchange to the seed apoplast has been investigated.Sucrose fluxes, computed from estimates of the plasma membranesurface areas of the cell types of the pathway, the plasmodesmatalcross-sectional areas interconnecting contiguous cells and theobserved rate of sucrose delivery to the embryo indicate thatsieve element unloading and subsequent transfer to the thin-walledparenchyma/transfer cells is through the symplast. For the cellsof the ground tissue, plasmodesmatal density is consistentlyhigher on their anticlinal walls. This observation supportsthe reported pattern of lateral transfer through these tissuesin the non-vascular regions of the seed coat. Wall ingrowthsare initiated sequentially in the thin-walled parenchyma cellsto maintain 1–3 rows of thin-walled parenchyma/transfercells. The development of these wall ingrowths results in a58% increase in the plasma membrane surface area of these cellsand provides them with the capacity to act as the principalcellular site for membrane exchange of sucrose to the seed apoplast.This cellular route of symplastic transfer from the sieve elementsto the ground tissues where membrane exchange to the seed apoplastoccurs is consistent with that reported for Phaseolus vulgaris Key words: Cellular pathway, photosynthate transfer, transfer cell, Vicia seed coat  相似文献   

17.
Effects of fatty acids on BK channels in GH(3) cells   总被引:6,自引:0,他引:6  
Ca2+-activated K+ (BK) channels inGH3 cells are activated by arachidonic acid (AA). Becausecytosolic phospholipase A2 can produce other unsaturatedfree fatty acids (FFA), we examined the effects of FFA on BK channelsin excised patches. Control recordings were made at several holdingpotentials. The desired FFA was added to the bath solution, and thevoltage paradigm was repeated. AA increased the activity of BK channelsby 3.6 ± 1.6-fold. The cis FFA, palmitoleic, oleic,linoleic, linolenic, eicosapentaenoic, and the triple bond analog ofAA, eicosatetraynoic acid, all increased BK channel activity, whereasstearic (saturated) or the trans isomers elaidic,linolelaidic, and linolenelaidic had no effect. The cisunsaturated FFA shifted the open probability vs. voltage relationshipsto the left without a change in slope, suggesting no change in thesensitivity of the voltage sensor. Measurements of membrane fluidityshowed no correlation between the change of membrane fluidity and thechange in BK channel activation. In addition, AA effects on BK channelswere unaffected in the presence of N-acetylcysteine.Arachidonyl-CoA, a membrane impermeable analog of AA, activateschannels when applied to the cytosolic surface of excised patches,suggesting an effect of FFAs from the cytosolic surface of BK channels.Our data imply a direct interaction between cis FFA and theBK channel protein.

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18.
By following the movement of carbon particle markers on theexposed surface of a cultured tomato apex it has been shownthat a leaf primordium is formed by growth on the flank of theapex raising the tissue upwards and outwards to form the leafbuttress. The whole of the apical surface is in an active stateof cell division and expansion except in the axillary regionabove the primordium. The data provide direct estimates of therates of division in the outer layer of cells. The distribution of blocked metaphase figures following treatmentwith colchicine, shows that in the early stages of primordiumformation cell divisions are concentrated in what appears tobo a ‘growth centre’ in the corpus to one side ofthe apical dome. As the bulge of the primordium develops, thegrowth centre spreads out and splits into two parts continuingthe growth of the dome (proximal side) and the primordium (distalside). Between these two regions of active division there arisesa small pocket of cells in the axil, whose rate of divisionrapidly declines. Cuts made in the apical surface in the early stages of primordiumformation immediately gape widely, apparently as a result ofpressure exerted on the outer layers from within by divisionsin the corpus. Once the upper surface of the primordium becomesraised above the dome, the axillary cells seem to become compressedbetween the two zones of active division. In the axil at thisstage (a) cuts do not gape but close up after exuding cell sapand (b) the carbon particle markers move slightly together.  相似文献   

19.
Intron length polymorphisms (ILPs) have been used as geneticmarkers in some studies. However, a systematic investigationand large-scale exploitation of ILP markers has not been reported.In this study, we performed a genome-wide search of ILPs betweentwo subspecies (indica and japonica) in rice using the draftgenomic sequences of cultivars 93-11 (indica) and Nipponbare(japonica) and 32 127 full-length cDNA sequences of Nipponbareobtained from public databases. We identified 13 308 putativeILPs. Based on these putative ILPs, we developed 5811 candidateILP markers via electronic-PCR with primers designed in flankingexons. We further conducted experiment to verify the candidateILP markers. Out of 215 candidate ILP markers tested on 93-11,Nipponbare and their hybrid, we successfully exploited 173 codominantILP markers. Further analyses on 10 rice accessions showed thatthese ILP markers were widely applicable and most (71.1%) exhibitedsubspecies specificity. This feature suggests that ILPs wouldbe useful for the studies of genome evolution and inter-subspeciesheterosis and for cross-subspecies marker-assisted selectionin rice. In addition, by testing 51 pairs of the ILP primerson five Gramineae plants and three dicot plants, we found anotherdesirable characteristic of rice ILP markers that they havehigh transferability to other plants.  相似文献   

20.
SYNOPSIS. Carcinonemertes errans is a nemertean worm, the juvenilesof which are found as epibionts on the Dungeness crab, Cancermagister, in close association with the arthrodial membranesof the crabs. The juvenile nemerteans appear to have no meansof taking in particulate food but survive for many months onthe surface of the host. We show that the juvenile C. erransare capable of removing amino acids from dilute solution insea water, that the water near the arthrodial membranes wherethe worms are found contains high concentrations of primaryamines, and that there is a low resistance pathway for low molecularweight amino acids across the arthrodial membrane examined invitro.  相似文献   

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