The Prevalence of Pores and Canals in Leaf Cuticular Membranes. 2. Supplemental Studies

Evidence is presented for the existence of discrete, naturalcuticular pores concomitant with anticlinally-oriented transcuticularcanals found in the mature leaf surfaces of 26 out of 37 taxaamong 19 families. This investigation is an extension of earlierobservations made on 32 other taxa among 14 families. Dewaxed,chemically isolated, adaxial and abaxial cuticular membranesin conjunction with transverse leaf sections were examined usingordinary staining techniques. The ubiquitous pores occur randomlywith no evidence of clustering. Pore and canal diameters averageapprox. 1 µm. Canal lengths are directly related to cuticlethickness. No correlations were found between cuticle thicknessesand either pore frequencies or pore and canal diameters. Evidenceis provided by light microscopy photomicrographs. Leaf cuticles, cuticular membranes, cuticular pores, transcuticular canals, cuticular flanges     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

Protein Arcs May Form Stable Pores in Lipid Membranes

Electron microscopy and atomic force microscopy images of cholesterol-dependent cytolysins and related proteins that form large pores in lipid membranes have revealed the presence of incomplete rings, or arcs. Some evidence indicates that these arcs are inserted into the membrane and induce membrane leakage, but other experiments seem to refute that. Could such pores, only partially lined by protein, be kinetically and thermodynamically stable? How would the lipids be structured in such a pore? Using the antimicrobial peptide protegrin-1 as a model, we test the stability of pores only partially lined by peptide using all-atom molecular dynamics simulations in POPC and POPE/POPG membranes. The data show that, whereas pure lipid pores close rapidly, pores partially lined by protegrin arcs are stable for at least 300 ns. Estimates of the thermodynamic stability of these arcs using line tension data and implicit solvent calculations show that these arcs can be marginally stable in both zwitterionic and anionic membranes. Arcs provide an explanation for the observed ion selectivity in protegrin electrophysiology experiments and could possibly be involved in other membrane permeabilization processes where lipids are thought to participate, such as those induced by antimicrobial peptides and colicins, as well as the Bax apoptotic pore.     

The Prevalence of Pores and Canals in Leaf Cuticular Membranes

Ubiquitous, visibly discrete, natural cuticular pores and transcuticularcanals were found in the dewaxed leaf (and one herbaceous stem)cuticular membranes of 27 out of 32 taxa among 14 families.Clear evidence for their existence is provided by light photomicrographs.Both adaxial and abaxial leaf surfaces were investigated usingthin transections and chemically isolated cuticular membranes,in conjunction with ordinary staining techniques and light microscopymethods. No correlations were found between cuticle thicknessesand either the frequency of pore or the pore and canal diameters. Leaf cuticles, cuticle morphology, cuticular pores, transcuticular canals, cuticular flanges     

The Electrical Resistance and Capacitance of the Membranes of Nitella translucens

The resistance and capacitance of the membranes of Nitella translucenshave been measured by direct current and alternating currentmethods. Current of the order of 10^-7 amp. was injected intothe cell by means of a conventional Ag, AgCl_-3N KCl glass microelectrodeinserted into the vacuole of the cell. The change of potentialacross the membrane was recorded by two other internal microelectrodeswhich had been inserted into the cell at known distances fromthe current-injecting electrode. In the direct-current experimentsthe input current was in the form of a square pulse, while sinusoidalcurrents of frequency 25 cycles per second were used in thealternating current experiments. The cell was treated as a shortlength of coaxial cable and from the measurements the followingparameters could be obtained: the space constant (), the membraneresistance (R_m) and the membrane capacitance (C_m). The valuesof R_m ranged from 6.7 to 36 K ohm cm.² (mean of 21.4 K ohm cm.²)and those of ranged from 1.5 to 5.7 cm. (mean of 2.6 cm.).The capacitance value was about I µF cm.^-2 These results are discussed within the framework of our knowledgeof these parameters for other cells, particularly plant cells.The measured electrical resistance is shown to be at least tentimes less than the value estimated from the passive fluxesof the principal ions K, Na, and Cl. It is suggested that thisdiscrepancy, which is usually attributed to non-independentmovement of these ions, could be partially explained on electro-osmoticgrounds. The value of the capacitance is very close to thatwhich is usually obtained for other cell membranes. One exceptionallylow value for Nitella has been quoted in the literature. Thereason for the gross error in this particular measurement isgiven.     

番茄成熟过程中果皮细胞核孔变化及其与乙烯生成的关系

应用冰冻蚀刻技术探讨了番茄(Lycopersiconesculentum Mill 大红品种)成熟过程中果皮细胞核孔的变化。成熟开始发动时(发白期前,IEC>0.1 ppm)核孔就开始扩大,起初核孔扩大比IEC增加更快,转包期(IEC约10 ppm)达到最大,以后不再变化。核孔扩大先于ACC含量增加。核孔的数量从转色期增加,粉红期最大,成熟后又减少。     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

Palmitic and Stearic Acids Bind Ca2+ with High Affinity and Form Nonspecific Channels in Black-Lipid Membranes. Possible Relation to Ca2+-Activated Mitochondrial Pores

A mitochondrial hydrophobic component that forms Ca²⁺-induced nonspecific ion channels in black-lipid membranes (Mironova et al., 1997) has been purified and its nature elucidated. It consists of long-chain saturated fatty acids—mainly palmitic and stearic. These fatty acids, similar to the mitochondrial hydrophobic component, bind Ca²⁺ with high affinity in comparison with unsaturated fatty acids, saturated fatty acids with shorter aliphatic chains, phospholipids, and other lipids. Ca²⁺-binding is inhibited by Mg²⁺ but not by K⁺. For palmitic acid, the K _d for Ca²⁺ was 5 M at pH 8.5 and 15 M at pH 7.5, with the B _max of 0.48 ± 0.08 mmol/g. This corresponds to one Ca²⁺ ion for eight palmitic acid molecules. The data of IR spectroscopy confirm that Ca²⁺ does not form ionic bonds with palmitic and stearic acids under hydrophobic conditions. It has been found that in the presence of Ca²⁺, palmitic and stearic acids, but not unsaturated FFA induce a nonspecific permeability in black-lipid membranes. Addition of Ca²⁺ in order to induce the permeability transition, increases the extractable amount of palmitic and stearic acids, the effect being prevented by a phospholipase A₂ inhibitor. The possible involvement of palmitic and stearic acids in the mitochondrial nonspecific permeability is discussed.     

Model Pores of Molecular Dimension: The Preparation and Characterization of Track-Etched Membranes

Extremely uniform pores of near molecular dimension can be formed by the irradiation-etching technique first demonstrated by Price and Walker. The technique has now been developed to the stage where it can be used to fabricate model membranes for examining the various steric, hydrodynamic, and electrodynamic phenomena encountered in transport through molecular-size pores. Methods for preparing and characterizing membranes with pores as small as 25 A (radius) are described in this paper. Results on pore size determination via Knudsen gas flow and electrolyte conduction are compared. Pore wall modification by monolayer deposition is also discussed.     

Further Studies in Magnetotropism

Studies have been mode of the growth curvatures induced in theroots of Lepidium sativum, the coleoptiles of Avena saliva andthe sporangiophores of Phycomyces blakesleeanus by exposureto an intense magnetic gradient during continuous rotation ona slow klinostat (0.5 rpm). Attempts have been made to determinethe relationships of the curvature responses to the parametersof the magnetic field (H, dH/dx, and H. dH/dx). Analysis ofthe data was by a progressive regression analysis up to thethird order coupled with an analysis of variance to test theimprovement of fit at each stage. Response is characterizedby an initial lag (reaction time, T) followed by a constantrate of curvature down the magnetic gradient for the first 20or 30? of curvature. The reciprocal of T was the most satisfactorymeasure of response (i. e. a measure of the rate of inductionof the response while the organ was in a constant magnetic environment)and in Avena and Phycomyces was best fitted by linear regressionson log_edH/dx and on log_eH. dH/dx. For Lepidium, with much morevariable data, the best fit was with a linear regression onlog_eH. The significance of these results is discussed.     

Biochemical and Molecular Responses to Loss of Renal Mass: Membranes and Protein Synthesis: Further Studies on a Renotropic System in Rats

Based upon many investigations, the existence of short-lived, specific, circulating substances which incite and/or regulate compensatory renal growth has been proposed. In our studies, we find that sera and plasma from unilaterally nephrectomized rats compared to sera and plasma from sham-operated rats stimulate the incorporation of ³H-thymidine monophosphate, ³H-thymidine and ¹⁴C-uridine into the DNA of incubating rat kidney fragments. While extracts from growing rat kidneys are not excitatory, they produce a relative enhancement to incorporation of isotope into DNA when combined with sera from uninephrectomized rats—more than the sera do alone. The above is found also for the incorporation of ¹⁴C-uridine into RNA of incubating rat kidney fragments. Sera from uninephrectomized rats fail to stimulate DNA synthesis in liver slices from rats but do so in the presence of extracts from growing kidneys. Renotropic factors in sera and extracts do not appear to work by diluting the isotopes, by enhancing transport, or by effecting overall metabolism of the renal cells. The above described serum and liver factors may play a role in compensatory renal growth.