Protection against suspended sand: the function of the branchial membrane in the blue mussel Mytilus edulis

Blue mussels (Mytilus edulis) living in estuaries have to cope with varying concentrations of suspended sand. Sand flowing through the inhalant siphons comes into the infrabranchial chamber. The inhalant siphon can be partially closed by the branchial membrane. As a result the inward flow decreases, and suspended sand sinks and can be eliminated. Experiments with mussels from three ecologically different locations showed about the same response of the branchial membrane on contact with suspended sand. The presence and function of the branchial membrane appears to be an adaptation of mussels to their estuarine environment.     

不同失水状态发菜类囊体膜蛋白的分离及鉴定

采用液氮研磨与超声波破碎相结合的方法,破碎不同失水状态的发菜藻体和细胞,用差速离心法制备发菜类囊体膜粗制品,蔗糖密度梯度高速离心纯化类囊体膜,SDS-PAGE电泳分离类囊体膜蛋白,并对膜蛋白进行了MALDI-TOF-TOF/MS质谱分析和鉴定。结果表明:(1)充分吸胀4h后失水6h的发菜(含水量51.2%)和失水24h的发菜(含水量14.9%),经过多步差速离心后再进行蔗糖密度梯度高速离心,可得到纯化的类囊体膜。(2)发菜类囊体膜蛋白SDS-PAGE电泳分离到14个条带,共鉴定出8种蛋白,根据其功能可分为4类——光合作用相关蛋白(光系统Ⅱ锰稳定蛋白PsbO,F1F0 ATP合成酶α亚基和β亚基)、结构域蛋白、选择性通道蛋白OprB、未知蛋白(hypothetical protein Npun_R1321、Npun_R3785、N9414_02186),它们在发菜的光合作用中具有重要作用。     

The somatic envelopes around the germ-line cells of polytrophic insect follicles: Structural and functional aspects

The polytrophic ovarioles of three insect species, the fruit fly Drosophila melanogaster, the fungus gnat Bradysia tritici, and the honeybee Apis mellifera, were compared morphologically and with respect to the cytological organization of the peripheral somatic layers. Staining with rhodaminyl-phalloidin revealed differences in the organization of the muscle strands of the epithelial sheath and the microfilament pattern in the basal part of the follicular epithelium (mid-vitellogenic stages). Also, the size of the intercellular space between the follicle cells differed considerably in the three analyzed species. The basement membrane of Drosophila and Bradysia follicles was partially digested using purified collagenase. The observed morphological changes indicated that in both species the basement membrane of the follicular epithelium plays an important role in shaping the follicles. The possible functional significance of the species-specific structural differences is discussed.     

Synthesis and Characteristics of Fluorescent BODIPY-Labeled Gangliosides

A series of new fluorescent-labeled gangliosides bearing the residues of acids labeled by 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene(BODIPY) in the polar or/and apolar moiety were synthesized. These are ganglioside GM1 labeled with the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5,7-dimethyl-s-indacenyl-3-propanoic (BODIPY-FL-propanoic) and -indacenyl-5-pentanoic (BODIPY-FL-pentanoic) acid in the oligosaccharide moiety of the molecule, and ganglioside GD1a labeled with two residues of BODIPY-FL-pentanoic acid in the oligosaccharide moiety and also with the residue of BODIPY-FL-pentanoic acid and the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5-octyl-s-indacenyl-5-pentanoic acid in the ceramide part of the molecule. Some spectral characteristics and the behavior in the model membrane systems of the synthesized probes were studied. In their emission spectra, the BODIPY-labeled gangliosides included into phosphatidylcholine liposomes at high concentrations (>1 mol %) exhibit a long-wavelength maximum (at 630 nm) in addition to the usual maximum (at 510–515 nm).     

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.     

Formation de la membrane de fécondation de l'oeuf d'Artemia salina

Résumé L'oeuf vierge d'Artemia salina n'est pas entouré de membranes exocellulaires. Le plasme sous-cortical ne contient pas d'organites spéciaux. Dès la fécondation, une membrane est secrétée par l'oeuf. La substance membranogène, contenue dans le reticulum endoplasmique lisse, passe par les éléments golgiens, où elle semble modifiée, et est expulsée dans des vésicules qui se détachent du Golgi. Retenue par un enduit granuleux, qui couvre le plasmolemme, et qui peut être un glycocoat ou du suc du tractus génital, elle s'étale en une membrane de fécondation, qui se soulève pour constituer l'espace périvitellin. Le processus est progressif et dure environ une heure et demi.

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Formation of the fertilization membrane of the egg inArtemia salina

Summary The unfertilized egg ofArtemia salina is not covered with any extracellular structure. No special organelles are found in the sub-cortical plasma. From the moment of fertilization, a membrane is progressively secreted by the egg. The membranogenous substance is first seen as large granules in the smooth endoplasmic reticulum, presumably transformed within Golgi elements and extruded in vesicles liberated from the Golgi apparatus. Retained by a glycocoat or by contact with the fluid of the genital tract, it spreads out into a fertilization membrane, soon surrounding a perivitelline space. The process lasts till 1 1/2 h after fertilization.

     

Surface morphology of peritrophic membrane formation in the cabbage looper,Trichoplusia ni

Summary The development of the peritrophic membrane in the cabbage looper, Trichoplusia ni, was investigated by scanning electron microscopy. The development of this membrane is characterized by a series of events suggested by the observations to be (1) secretion of material among and above the microvilli of the midgut epithelial cells, (2) maturation of this material into a randomly cross-linked fibrous matrix, and (3) aggregation of amorphous materials in and within the matrix. The membrane, possessing small discontinuities, remains intact in the midgut, but shows gross damage by the time it is passed from the insect, surrounding the feces.     

Measurement of the transmembrane electrical potential of Dunaliella acidophila by microelectrodes

A method was developed to determine electrical potential differences across the plasma membrane of the microalga Dunaliella by means of potential-sensitive microelectrodes. Special emphasis was put on the measurement of the membrane potential in the acidophilic Dunaliella acidophila (optimal growth at pH 1.0), but neutrophilic, halotolerant Dunaliella species were used as reference systems. For Dunaliella acidophila positive membrane potentials (cytoplasma relative to the medium), ranging from +30 to +65mV were measured. Illumination caused a decrease of the positive potential by about 10 mV. The ATPase inhibitor omeprazole caused an increase of the positive membrane potential ranging from +60 to +100 mV, whereas the ionophore gramicidin caused a decrease of the MP to +10 to +30 mV. The salt tolerant, neutrophilic Dunaliella parva and Dunaliella bardawil exhibited negative membrane potentials in the order of -40 to -60mV, and light caused a hyperpolarization of about 10 mV. A negative membrane potential was measured also in D. acidophila cells transferred to pH 7.0. The physiological significance of a positive membrane potential for acidophilic algae is discussed.Abbreviations E _m membrane potential - PM plasma membrane - TPB^– tetraphenylborone anion - TPP⁺ tetraphenyl-phosphonium cation - SCN^– isothiocyanate     

Inclusion membrane proteins of <Emphasis Type="Italic">Chlamydiaceae</Emphasis>

Inclusion membrane proteins (Inc-proteins) belong to the family of unique chlamydial proteins. Members of this family attract attention of scientists because Inc-proteins are localized in the inclusion membrane, they have been found in all chlamydial species, expression of the most part of their genes begins during the first hours after the infection of cell culture. Biological functions of Inc-proteins remain unknown, but these proteins are suggested to play a key role in process of the development of the chlamydial infection.     

Activities of a Mechanosensitive Ion Channel in anE. Coli Mutant Lacking the Major Lipoprotein

Summary The activity of the mechanosensitive (MS) ion channels in membrane patches, excised fromE. coli spheroplasts, was analyzed using the patch-clamp technique. Outer membranes from a mutant lacking the major lipoprotein (Lpp) and its wildtype parent were examined. The MS-channel activities in the wild-type membrane rarely revealed substates at the time resolution used. These channels showed a stretch sensitivity indicated by the IISP (the suction for ane-fold increase in channel open probability) of 4.9 mm Hg suction. The MS-channel activities oflpp included a prominent substate and showed a weaker mechano-sensitivity with an 1/S _p of 10.0 mm Hg. Whereas small amphipaths (chlorpromazine, trinitrophenol) or a larger amphipath (lysolecithin) all activated the MS channel in the wild-type membrane under minimal suction, only the larger lysolecithin could activate the MS channel in thelpp membranes. After lysolecithin addition, thelpp membrane became more effective in transmitting the stretch force to the MS channel, as indicated by a steepening of the Boltzmann curve. We discuss one interpretation of these results, in which the major lipoprotein serves as a natural amphipath inserted in the inner monolayer and the loss of this natural amphipath makes the bilayer less able to transmit the gating force.     

Interaction of tubulin with the plasma membrane: tubulin is present in purified plasmalemma and behaves as an integral membrane protein

Using monoclonal antibodies we have studied the interaction of tubulin with the plasma membrane of leaves of Nicotiana sylvestris (Speg. et Comes) and tobacco suspension-culture cells. The results show that isolated plasma membranes contain tightly bound -tubulins. Their association with the plasma membrane is resistent to non-ionic detergent and to low and high ionic strength. Only extraction with sodium dodecyl sulfate is capable of dissociating these cytoskeletal proteins. It is unlikely that this membrane-bound tubulin is present in its polymeric form because electron-microscopical analysis does not reveal the presence of filaments, whereas treatment of membranes with oryzalin (which has been shown to destabilize microtubules in vitro) does not remove the tubulins from isolated plasma membrane. When living cells are treated with oryzalin, the amount of membrane-associated tubulin is drastically reduced, which could mean that its presence is related to in-vivo microtubule dynamics.Abbreviations Mes 2 (N-morpholino) ethane sulfonic acid - NP40 Nonidet P40     

Purification and characterization of a calcium-dependent protein kinase from beetroot plasma membranes

Several calcium-dependent protein kinases (CDPKs) are located in plant plasma membranes where they phosphorylate enzymes and transporters, like the H⁺-ATPase and water channels, thereby regulating their activities. In order to determine which kinases phosphorylate the H⁺-ATPase, a calcium-dependent kinase was purified from beetroot (Beta vulgaris L.) plasma membranes by anion-exchange chromatography, centrifugation in glycerol gradients and hydrophobic interaction chromatography. The kinetic parameters of this kinase were determined (V _max: 3.5 μmol mg⁻¹ min⁻¹, K _m for ATP: 67 μM, K _m for syntide 2: 15 μM). The kinase showed an optimum pH of 6.8 and a marked dependence on low-micromolar Ca²⁺ concentrations (K _d : 0.77 μM). During the purification procedure, a 63-kDa protein with an isoelectric point of 4.7 was enriched. However, this protein was shown not to be a kinase by mass spectrometry. Kinase activity gels showed that a 50-kDa protein could be responsible for most of the activity in purified kinase preparations. This protein was confirmed to be a CDPK by mass spectrometry, possibly the red beet ortholog of rice CDPK2 and Arabidopsis thaliana CPK9, both found associated with membranes. This kinase was able to phosphorylate purified H⁺-ATPase in a Ca²⁺-dependent manner.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

An estimation of the proton conductance of the inner membrane of turnip (Brassica napus L.) mitochondria

The permeability of the inner membrane of turnip mitochondria to H⁺ and OH^- ions has been investigated using an acid pulse technique. The rate of decay of a H⁺ pulse across the inner membrane is exponential having first-order kinetics and gives t 1/2 values of approx 54 s at neutral pH and at 25° C. Valinomycin or 1799 alone have little effect on t 1/2 values, whereas in combination, values of <15 s are observed. Nigericin produces a similar effect. The effective proton conductance of the inner membrane near pH 7 at 25° C is 0.27 nmol H⁺ min^-1 mg protein^-1 mV^-1. The results suggest that at neutral pH, the inner membrane of plant mitochondria is relatively impermeable to H⁺ and OH^- ions.     

Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles

Summary Membrane potential and resistance were recorded from parenchymal cells of oat (Avena) coleoptiles, using one and two intracellular electrodes. Membrane potential is largest (–100 mV) in impalements with low input resistance (2–4 M), and is less negative (–50 mV) in penetrations with high input resistance (> 20 m). The interpretation is that the electrode lodges in the vacuole which is positive to the cytoplasm (but still negative to the external solution), and that measurements of net membrane potential are compromised to varying degrees by leakage shunts introduced across the high resistance vacuolar membrane by the electrode. This conclusion is supported by several additional lines of evidence. (1) It is possible to convert large-R/small-V impalements into small-R/large-V penetrations by passing excess current through the electrode or by briefly ringing the capacitance neutralization circuit in the amplifier. The cells usually recover their resistance in a few minutes, with a concomitant decrease in the negativity of the membrane potential. (2) Changes in external [K] affect the measuree potential by an amount that is independent of the input resistance of the impalement. This is consistent with an effect of [K] _o on the potential of the plasma membrane and the occurrence of leakage shunts primarily at the tonoplast. (3) Quantitatively, the effects of a change in [K] _o on resistance indicate that nearly 90 percent of the input resistance of unshunted cells resides in the tonoplast. (4) The effects of metabolic inhibitors (DNP, CN^–) on potential are smaller in large-R than in small-R impalements. This observation suggests there are electrogenic pumps contributing to the membrane potential at both the plasmalemma and tonoplast. Finally, we conclude that with an electrode in the vacuole it is possible to record potentials that are dominated by the contribution of the plasma membrane, provided care is taken to select impalements combining both large, negative potential and low input resistance.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Theory of negative capacitance in membrane impedance measurements

Summary Three possible explanations of the negative capacitance seen in theChara corallina membrane impedance are critically examined. These explanations are based on: (1) voltage-dependent channel kinetics; (2) electro-osmosis; and (3) extracellular negative capacitance. It is shown that the first two can produce negative capacitance only with parameters which differ by several orders of magnitude from measured values. The last mechanism can produce a very large magnitude negative capacitance, in the appropriate frequency range. Possible experimental tests are discussed.     

Initial purification study of the cytochromeb 561 of bean hypocotyl plasma membrane

Summary The plasma membrane (PM) of higher plants contains a major ascorbate-reducible, high-potentialb-type cytochrome, named cytochromeb ₅₆₁ (cytb ₅₆₁). In this paper a rapid purification protocol for the cytb ₅₆₁ of bean hypocotyls PM is described. An almost 200-fold increase of cytb ₅₆₁ specific concentration was achieved with respect to the PM fraction, which contained about 0.2 nmol of ascorbate-reducible heme per mg protein. The procedure can be performed in one day starting from purified PMs obtained by the phase-partitioning procedure. However, cytb ₅₆₁ proved to be unstable during chromatographic purification and the amount of protein finally recovered was low. Purified cytb ₅₆₁ eluted as a 130,000 Da protein-detergent complex from gel-filtration columns. It was completely reduced by ascorbate and reduced-minus-oxidized spectra showed -, - and -bands at 561, 530, and 429 nm respectively, not unlike the spectra of whole PMs. This work represents an initial approach to the biochemical characterization of the cytb ₅₆₁ of higher plants, formerly suggested to be related to cytb ₅₆₁ of animal chromaffin granules.Abbreviations cytb ₅₆₁ cytochromeb ₅₆₁ - PM plasma membrane - UPV upper-phase vesicles - GSII glucan synthase II - CCR NADH-dependent cytochromec reductase - CCO cytochromec oxidase - TX-100R reduced Triton X-100     

Implications of cytochromeb 6/f location for thylakoidal electron transport

The cytochromeb ₆/f complex of higher plant chloroplasts is uniformly distributed throughout both appressed and nonappressed thylakoids, in contrast to photosystem II and photosystem I, the other major membrane protein complexes involved in electron transport. We discuss how this distribution is likely to affect interactions of the cytochromeb ₆/f complex with other electron transport components because of the resulting local stoichiometries, and how these may affect the regulation of electron transport.     

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H⁺-ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H⁺-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.