Measurement of the electrical resistance of plasmodesmata and membranes of corn suspension-culture cells

Electrophysiological investigations of intercellular communication and membrane resistance in higher plants have been hampered by the difficulty in measuring these quantities independently. Uncertainty about the position of an electrode inserted into vacuolate tissue has further complicated such measurement. To overcome these problems sister cell pairs of a Zea mays L. Black Mexican Sweet suspension culture were used and dye was injected from the current-injecting electrode to determine the location of the electrode tip in each experiment. Of the impalements, 72% were cytoplasmic. The presence of plasmodesmata was fully incorporated into the electriccircuit model for the cell, and the resistance of the membrane of the current-injected cell was calculated, separate from the plasmodesmata resistance. This avoided some of the confusion resulting from work on multicellular tissue in which the position of the electrode and the extent of intercellular coupling is not determined. Using this technique, plasma-membrane resistivity was measured as 0.65 ·m², the resistivity of the tonoplast and plasma membrane in series was 1.35 ·m², and the resistance of a single plasmodesma was calculated to be 53 ± 11 G.Abbreviations BMS Black Mexican Sweet - PD potential difference - R_j resistance of the plasmodesmata in the junction between cells - R_m resistance of the plasma membrane of the current-injected cell - R_t resistance of the tonoplast - V₁, V₂ membrane PDs of sister cells This work was funded by an Australian Research Council grant to R.L.O. We are grateful to Dr. Maret Vesk (Electron Microscope Unit, The University of Sydney) for assistance with the preparation of EM sections, and to Dr. Richard Brettell (C.S.I.R.O. Division of Plant Industry) for assistance with the BMS culture.     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

CCCP activation of the reconstituted NaK-pump

Summary In the NaK-ATPase proteoliposomes (PLs), the NaK-pump activity, Na⁺ uptake, and ATP hydrolysis were apparently enhanced by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and other ionophores without ion gradients. These ionophore effects were not cation specific. Without ionophores, the PL's ATPase activity fell to its steady-state value within 3 sec at 15°C. This decrease in activity disappeared in the presence of CCCP. Since CCCP is believed to enhance proton mobility across the lipid bilayer and dissipate membrane potential (V _m ), we postulated that aV _m build-up partially inhibits the PLs by changing the conformation of the NaK-pump, and that CCCP eliminated this partial inhibition. Since this activation required extracellular K⁺ and high ATP concentration in the PLs, CCCP must affect the conversion between the phosphorylated forms of NaK-ATPase (EP); this step has been suggested by Goldschlegger et al. (1987) to be the voltage-sensitive step (J. Physiol. (London) 387:331–355). Although cytoplasmic K⁺ accelerated the change of ADP-and K⁺-sensitive EP (E^*P) to K⁺-sensitive ADP-insensitive EP (E₂P), CCCP did not compete with cytoplasmic K⁺ when cytoplasmic Na⁺ was saturated. When the PLs were phosphorylated with 20 m ATP and 20 m palmitoyl CoA instead of with high concentration of ATP, CCCP increased the E^*P content and decreased the ADP-sensitive K⁺-insensitive EP (E₁P). The results described above suggest that CCCP affects the E₁P to E^*P change in the E₁PE^*PE₂P conversion and that this reaction step is inhibited byV _m .     

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg²⁺-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Con A concanavalin A - Cyt cytochrome - LysoPC lysophosphatidylcholine - Pi orthophoshate - PP_iase pyrophosphatase - IDPase inosine-5-diphosphatase We thank Pr. Robert Dargent and André Moisan (Laboratoire de Cryptogamie, Toulouse, France) for use of their electron-microscope facilities. This work was supported by the Centre National de la Recherche Scientifique and by a grant Dynamique du fonctionnement de la vacuole from the Ministère de la Recherche et de la Technologie.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na⁺, K⁺ and Cl^– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP _K>P _Cl>P _Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na⁺–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.     

Electrical characteristics of sensory neurons of Hirudo medicinalis

During intracellular polarization of identified sensory neurons of the leech by square pulses of hyperpolarizing current electrical parameters of the cell membranes were determined: input resistance of the neuron R_n, time constant of the membrane , the ratio between conductance of the cell processes and conductance of the soma , the resistance of the soma membrane r_s, the input resistance of the axon r _a , capacitance of the membrane C_s, and resistivity of the soma membrane R_s. The results obtained by the study of various types of neurons were subjected to statistical analysis and compared with each other. Significant differences for neurons of N- and T-types were found only between the values of , C_s, and R_s (P<0.01). These parameters also had the lowest coefficients of variation. The surface area of the soma of the neurons, calculated from the capacitance of the membrane (the specific capacitance of the membrane was taken as 1 µF/cm²) was 7–10 times (N-neurons) or 4–6 times (T-neurons) greater than the surface area of a sphere of the same diameter. The resistivity of the soma membrane R_s was 35.00 k·cm² for cells of the N-type and 19.50 k·cm² for T-neurons. The reasons for the relative stability of this parameter compared with the input resistance of the cell (coefficient of variation 22–7 and 53–31% respectively) are discussed. The possible effects of electrical characteristics on the properties of repeated discharges in neurons of different types also are discussed.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol.7, No.3, pp.295–301, May–June, 1975.     

Genetic analysis of resistance to whitebacked planthopper in twenty-one varieties of rice,Oryza sativa L.

Summary The inheritance of resistance to whitebacked planthopper Sogatella furcifera (Horvath) was studied in 21 rice varieties. Reactions of F₁; F₂ and F₃ progenies of the crosses of 21 resistant varieties with the susceptible variety TN 1 revealed that a single dominant gene governs resistance in Mushkan 41, Santhi, Siahnakidar 195, SM2-34, Tirisurkh 251, Zirijowaian 245, 18, 24A, 39, 76 S, 78, 180, 213 B, 267, 293, CI 6037-4, NP97, S39 JKW and Bansphul. In varieties 65 and 274 A, resistance is governed by one dominant and one recessive gene which segregate independently of each other. Tests for allelism with the Wbph 1 gene originally identified in N 22 revealed that the dominant gene present in all the test varieties is the same as Wbph 1. Further studies are required to determine the allelic relationships of the recessive gene found in varieties 65 and 274 A.     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁     

A reaction cycle for cytochrome c oxidase as an electron-transport-driven proton pump: The effect of electrochemical potential and slips

A detailed reaction cycle for cytochrome oxidase, an electron-transport-driven proton pump, has been presented earlier by our research group. The essential feature of the model is that both cytochrome a and Cu_A must be reduced in order to allow the transition from the electron and proton input state to the output state. The model is thus based on an indirect coupling between electron transfer and proton translocation.In this study, the same model is examined with respect to (1) intrinsic electron and proton leaks and (2) the effect of applying an electrochemical potential gradient on the pump incorporated in a membrane, both with respect to the electrical and chemical components.The model is successfully used to simulate various experimental results. Comparisons of experimental results with simulations based on the model support the existence of electron and proton leaks. The analysis of electron leaks suggests that electron gating is best achieved by varying the reorganization energy rather than by varying the reduction potentials.It is also suggested that both the electrical and chemical components of the electrochemical potential gradient are responsible for the regulation of the enzyme activity. Furthermore, an attempt is made to interpret the seemingly contradictory results obtained when measuring the pH dependence of the reduction potential of cytochrome a. In addition, the simulations support the assumption that protons are pumped by a mechanism that combines a membrane Bohr effect with the transition-state mechanism.Abbreviations R molar gas constant - k _B Boltzmann contant - F Faraday constant - e elementary charge - T absolute temperature - transmembrane electrochemical potential gradient - pH transmembrane pH difference - pH₁ and pH₂ inside (matrix) and outside (cytosol) pH, respectively - transmembrane electrical potential - E _m midpoint potential     

The effect of pH on the heat production and membrane resistance of Streptococcus bovis

Non-growing cultures of Streptococcus bovis JB1 which were incubated in 2-[N-moropholino] ethane-sulfonic acid (MES)-phosphate buffer (pH 6.8) and glucose (2 g/l) produced heat at a rate of 0.17 mW/mg protein, and this rate was proportional to the enthalpy change of the homolactic fermentation. Since the growth-independent heat production could be eliminated by dicyclohexylcarbodiimide (DCCD), an inhibitor of F₁F₀ ATPases, it appeared that virtually all of the energy was being used to counteract proton flux through the cell membrane. When the pH was decreased from 6.8 to 5.8, heat production and glucose consumption increased, the electrical potential () declined, the chemical gradient of protons (ZpH) increased, and there was a small increase in total protonmotive force (p). Further decreases in pH (5.8 to 4.5) caused a marked decrease in heat production and glucose consumption even though there was only a small decline in membrane voltage. Based on the enthalpy of ATP (4 kcal or 16.8 kJ/mol), it appeared that 38% of the wattage was passing through the cell membrane. The relationship between membrane voltage and membrane wattage or glucose consumption was non-linear (non-ohmic), and it appeared that the resistance of the membrane to current flow was not constant. Based on the electrical formula, resistance = voltage²/wattage and resistance = voltage/amperage, there was a marked increase in membrane resistance when the pH was less than 6.0. The increase in membrane resistance at low pH allowed S. bovis to maintain its membrane potential and expend less energy when its ability to ferment glucose was impaired.Abbreviations DCCD dicyclohexylcarbodiimide - MES 2-[N-moropholino] ethanesulfonic acid     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Electrical membrane potential and resistance in photoautotrophic suspension cells of Chenopodium rubrum L.

On photoautotrophically grown, suspension-cultured cells of Chenopodium rubrum L. the electrical potential difference V _mand the electrical resistance across plasmalemma and tonoplast have been measured using one or two intracellular micro-electrodes. In a mineral test-medium of 5.8 mM ionic strength V _mvalues between 100 and 250 mV, 40% thereof between 170 and 200 mV, and a mean value (±S.E.M.) of 180.6±3.4 mV have been recorded. The average membrane input resistance R _mwas 269±36 M, corresponding to an average membrane resistivity r _mof 3.0 m². V _mand r _mare sensitive to light, temperature, and addition of cyanide, suggesting the presence of an electrogenic hyperpolarizing ion pump, and are ascribed essentially to the plasmalemma. A hexose-specific saturable electrogenic membrane channel is identified through a decrease of V _mand r _mupon addition of hexoses. The hexoseconcentration-dependent depolarization V _msaturates at 92 mV and returns half-saturating concentrations (apparent k _mvalues) of 0.16 mM galactose, 0.28 mM glucose, and 0.48 mM fructose. The magnitude of V _mand r _mwell agrees with pertinent data from mesophyll cells in situ (where only V _mdata are available) and from photoautotrophic lower plant cells. However, V _mis markedly higher than reported for heterotrophically grown suspension cells of different higher plants (with which r _mdata have not been reported so far). It is concluded from the present study and a companion paper on water transport (Büchner et al., Planta, in press) that photoautotrophically grown Chenopodium suspension cells closely resemble mesophyll cells as to cell membrane transport properties.Abbreviations V _m membrane potential(mV) - R _o input resistance () - R _m membrane input resistance () - r _m specific resistance (resistivity) of the membrane (m²)     

The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles

A fluorescence method for the direct measurement of Cl^- transport in isolated tonoplast vesicles is described. This technique utilises the Cl^--sensitive fluorescent compound, 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ). This is a water-soluble compound with excitation and emission wavelengths of 350 and 440 nm, respectively. Its fluorescence is quenched by Cl^-, Br^-, I^-, SCN^-, NO ₂ ^- and tetraphenylborate but not by NO ₃ ^- , SO ₄ ^2- , iminodiacetate or malate. These effects are independent of pH. This compound was loaded into tonoplast vesicles from red beet (Beta vulgaris L.) storage roots or from barley (Hordeum vulgare L.) roots by incubation at 37° C and the external probe was then removed by repeated centrifugation of the vesicles in SPQ-free medium. In this way a large proportion of the observed fluorescence signal was from the interior of the vesicles, and its quenching could be used to monitor, quantitatively, and in real time, the intravesicular Cl^- concentration. In this paper we describe some of the problems encountered in using this probe to measure Cl^- transport in tonoplast vesicles, how these were overcome and some characteristics of Cl^- transport at the tonoplast as measured by the probe.Abbreviations and symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino-propane - DTT dithiothreitol - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi inorganic pyrophosphate - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine     

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv RLM 198 from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal RLM 198 (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with RLM 198. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile RLM 198 and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.     

Effect of gene Lr34 in the enhancement of resistance to leaf rust of wheat

Summary Leaf rust resistance gene Lr34 is present in many wheat cultivars throughout the world that have shown durable resistance to leaf rust. Fourteen pair-wise combinations of Lr34 and seedling leaf rust resistance genes were developed by intercrossing near isogenic Thatcher lines. In both seedling and adult plant tests homozygous paired combinations of specific resistance genes with Lr34 had enhanced resistance relative to either parent to different numbers of isolates that were avirulent to the additional resistance genes. The TcLr34, 18 line also expressed enhanced resistance to specific isolates virulent to Lr18 in seedling and adult plant stages. In rust nursery tests, homozygous lines were more resistant than either parent, if the additional leaf rust gene conditioned an effective of resistance when present singly. The ability of Lr34 to interact with other genes conditioning effective resistance may contribute to the durability of leaf rust resistance in cultivars with Lr34. Contribution 1453 Agriculture Canada     

Testosterone and progesterone metabolism in the central nervous system: Cellular localization and mechanism of control of the enzymes involved

Summary This paper summarizes the most recent data obtained in the authors' laboratory on the metabolism of testosterone and progesterone in neurons and in the glia.1. The activities of 5-reductase (the enzyme that converts testosterone into dihydrotestosterone; DHT) and of 3-hydroxy steroid dehydrogenase (the enzyme that converts DHT into 5-androstane-3,17-diol; 3-diol) were first evaluated in primary cultures of neurons, oligodendrocytes, and type-1 and type-2 astrocytes, obtained from the fetal or neonatal rat brain. The formation of DHT and 3-diol was evaluated incubating the different cultures with labeled testosterone or labeled DHT as substrates. The results obtained indicate that the formation of DHT takes place preferentially in neurons; however, also type-2 astrocytes and oligodendrocytes possess considerable 5-reductase activity. A completely different localization was observed for 3-hydroxysteroid dehydrogenase; the formation of 3-diol appears to be prevalently, if not exclusively, present in type-1 astrocytes; 3-diol is formed in very low yields by neurons, type-2 astrocytes, and oligodendrocytes. Moreover, the results indicate that, in type 1 astrocytes, both 5-reductase and 3-HSD are stimulated by coculture with neurons and by the addition of neuron-conditioned medium, suggesting that secretory products released by neurons might intervene in the control of glial cell function.2. Subsequently it was shown that, similarly to what happens when testosterone is used as the substrate, 5-reductase, which metabolizes progesterone into 5-pregnane-3,20-dione, (DHP), shows a significantly higher activity in neurons than in glial cells; however, also type-1 and type-2 astrocytes as well as oligodendrocytes possess some ability to 5-reduce progesterone. On the contrary, 3-hydroxysteroid dehydrogenase, the enzyme which converts DHP into 5-pregnane-3-ol-20-one (THP), appears to be present mainly in type-1 astrocytes; much lower levels of this enzyme are present in neurons and in type-2 astrocytes. At variance with the previous results obtained using androgens as precursors, oligodendrocytes show considerable 3-hydroxysteroid dehydrogenase activity, even if this is statistically lower than that present in type-1 astrocytes. The existence of isoenzymatic forms of the enzymes involved in androgen and progesterone metabolism is discussed.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Genes determining leucine aminopeptidase and mildew resistance from the ornamental apple, ‘White Angel’

Mildew resistance in the ornamental apple White Angel was found to be determined by complementary genes. The gene R _w was found to be necessary for the expression of resistance controlled by the resistance gene Pl _w . The close linkage between the isoenzyme gene, Lap-2, for leucine aminopeptidase and P1 _w was confirmed. The efficiency of Lap-2 as a marker in screening for mildew resistance is limited, as it cannot account for susceptible plants with the r _w r _w P1 _w p1 _w genotype. It has, however, an important role to play in combining resistance genes from different sources. The genotypes of White Angel (R _w r _w , Pl _w pl _w , Lap-2an), Jester (R _w r _w , p1 _w p _w , Lap-2an) Katja (R _w r _w ,p1 _w p1 _w , Lap-2an) and Gloster 69 (r _w r _w , p1 _w p1 _w , Lap-2an) were determined. It also appeared that R _w might influence Lap-2 activity in young seedlings.     

Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light

Responses of the rate of net CO₂ assimilation (A) to the intercellular partial pressure of CO₂ (p _i ) were measured on intact spinach (Spinacia oleracea L.) leaves at different irradiances. These responses were analysed to find the value of p _i at which the rate of photosynthetic CO₂ uptake equalled that of photorespiratory CO₂ evolution. At this CO₂ partial pressure (denoted ), net rate of CO₂ assimilation was negative, indicating that there was non-photorespiratory CO₂ evolution in the light. Hence was lower than the CO₂ compensation point, . Estimates of were obtained at leaf temperatures from 15 to 30°C, and the CO₂/O₂ specificity of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (E.C. 4.1.1.39) was calculated from these data, taking into account changes in CO₂ and O₂ solubilities with temperature. The CO₂/O₂ specificity decreased with increasing temperature. Therefore we concluded that temperature effects on the ratio of photorespiration to photosynthesis were not solely the consequence of differential effects of temperature on the solubilities of CO₂ and O₂. Our estimates of the CO₂/O₂ specificity of RuBP carboxylase/oxygenase are compared with in-vitro measurements by other authors. The rate of nonphotorespiratory CO₂ evolution in the light (R _d ) was obtained from the value of A at . At this low CO₂ partial pressure, R _d was always less than the rate of CO₂ evolution in darkness and appeared to decrease with increasing irradiance. The decline was most marked up to about 100 mol quanta m^-2 s^-1 and less marked at higher irradiances. At one particular irradiance, however, R _d as a proportion of the rate of CO₂ evolution in darkness was similar in different leaves and this proportion was unaffected by leaf temperature or by [O₂] (ambient and greater). After conditions of high [CO₂] and high irradiance for several hours, the rate of CO₂ evolution in darkness increased and R _d also increased.Abbreviations and symbols A rate of net CO₂-assimilation - CO₂ compensation point - CO₂ compensation point in the absence of R _d - p _i intercellular partial pressure of CO₂ - R _d (day respiration) rate of non-photorespiratory CO₂ evolution in the light - R _n (night respiration) rate of CO₂ evolution in darkness - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase