External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential E_m of −42 ± 2 mV and a membrane resistance R_m of 15 ± 5 MΩ; the membrane was more permeable to Cl⁻ than to K⁺. The oocyte spontaneously hyperpolarized and E_m gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K⁺. However, the hyperpolarization occured only if Mg²⁺ was present in the seawater; external Ca²⁺ was not required. Long-term incubation without external Mg²⁺ depolarized the membrane and increased membrane resistance. After preincubation in Mg²⁺-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg²⁺ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.     

Voltage clamp studies of fertilization in sea urchin eggs: I. Effect of clamped membrane potential on sperm entry,activation, and development

To analyze the role of the activation potential (a positive shift of the membrane potential which occurs following sperm attachment) in fertilization and development of the sea urchin egg, unfertilized Lytechinus variegatus eggs were voltage clamped at membrane potentials (E_m) from +20 to ?90 mV, and then inseminated. Either a fast two electrode voltage clamp, or a single electrode switched voltage clamp was used. The clamp was maintained for 3 to 15 min after initiation of a conductance increase. At E_m more positive than +18 mV, even though many sperm may attach, the egg remains completely inert (Jaffe, Nature (London)261, 68–71, 1976). At E_m from +17 to ?90 mV, all inseminated eggs elevate normal fertilization envelopes, although substantially increased concentrations of sperm are required at E_m from +17 to +12 mV. Whether cleavage occurs depends on the clamped E_m. When clamped at E_m from +17 to ?25 mV, 100% of activated eggs cleave. However, when clamped at E_m from ?26 to ?75 mV the percentage of activated eggs which cleave progressively decreases. At clamped E_m between ?76 and ?90 mV, none of the activated eggs cleave. All monospermic voltage clamped eggs that cleave develop to normal swimming blastulae. In all eggs that fail to cleave (clamped at E_m more negative than ?30 mV), sperm penetration is blocked, the sperm is lifted off the egg surface as the fertilization envelope rises, and a sperm aster never forms. Preventing formation of the fertilization envelope by prior disruption of the vitelline layer with dithiothreitol does not promote entry of the sperm. In conclusion, preventing the depolarization normally associated with fertilization suppresses sperm entry in the sea urchin egg, yet activation proceeds. Present evidence suggests an effect of the electrical field across the plasma membrane in suppressing sperm entry.     

Fertilization in crabs: IV. The fertilization potential consists of a sustained egg membrane hyperpolarization

The electrophysiological properties of immature and mature oocytes of two crabs were analyzed. Growing immature oocytes of Carcinus maenas and fully grown immature oocytes of Maia squinado had essentially K⁺ dependent resting potentials, Em, of ?61 ? 1 mV, n=19, and ?67.3 ± 0.5 mV, n=68, respectively. Fully grown immature oocytes of Carcinus maenas showed an Em of ?40 ± 1.5 mV, n=19, that was k⁺ and Cl^? dependent. In mature oocytes of both species, the plasma membrane became exclusively permeable to cl^? and the Em attained–41 ± 1 mV, n=49 and ?34 ± 1.5 mV, n=27 for Carcinus maenas and Maia squinado, respectively. After in vitro insemination, a dramatic increase in egg membrane permeability to K⁺ was observed. This instantaneously caused a sustained hyperpolarization constituting, for these crabs, the fertilization potential. We observed that concurrently with this electrical response to fertilization, sperm reinitiated the oocyte meiotic maturation previously arrested at the first metaphase. The triggering mechanism of the fertilization potential as well as the possible occurrence of a physiological polyspermy are discussed.     

Some electrophysiological and permeability properties of the mouse egg

Certain electrophysiological and ionic properties of the mouse egg (CF-1 and BDF 12–18 hr post ovulation) have been investigated. Membrane potential (?14 ± 0.4 mV, ± SE, inside negative), membrane resistance (2610 ± 38 ohm·cm²), and membrane capacitance (1.6 ± 0.03 μF cm^?2) have been determined by means of intracellular microelectrode recording techniques. Membrane potential and related parameters are stable for extended periods of time upon impalement and the magnitude of the cell membrane potential has been demonstrated to be sensitive to alteration in external sodium. The electrophysiological studies in conjunction with measurements of unidirectional potassium fluxes using isotope tracer-techniques have allowed determination of membrane permeability to potassium (8 × 10^?8 cm sec^?1) and membrane potassium conductance (25 μmho cm^?2). Furthermore, the use of tracer flux techniques has indicated that the exchangeable fraction of intracellular potassium is 204 ± 14 mM. This represents the bulk of egg potassium (222 ± 19 mM as determined from flame photometry). Studies of unidirectional potassium efflux have indicated that its movement out of the egg is made up of at least two components; an external potassium-independent potassium efflux and external potassium-dependent efflux, the latter possibly representing a potassium exchange mechanism. The combined electrophysiological and tracer-flux data indicate that only a small portion of the total membrane conductance is composed of potassium conductance at this stage of development. This and the fact that the membrane potential is far from the potassium equilibrium potential are similar to observations made on mature eggs of several other species.     

Developmental changes in membrane transport and permeability in the early mouse embryo

Electrophysiological and isotope tracer flux experiments were performed to measure the membrane ion permeability and transport properties of the two-cell mouse embryo. The results show that the internal exchangeable Na and K are 151 and 130 mM, respectively, and their membrane permeabilities are (P_Na) 16 × 10^?8 cm sec^?1 and (P_K) 21 × 10^?8 cm sec^?1. These values predict a membrane potential of ?24 mV (inside negative) which agrees well with ?19 mV measured with microelectrodes. Ouabain-sensitive isotope fluxes demonstrate a Na/K pump mechanism with a stoichiometry of 1.7:1 (Na:K). An external-Na-dependent Na efflux is demonstrated by the reduction of unidirectional Na efflux in Na-free medium, but there is no evidence for a similar mechanism of K efflux at this stage of development. These results are compared with the values reported for the mouse oocyte [Powers, R. D., and Tupper, J. T. (1974). Develop. Biol.38, 320–331; (1975). Exp. Cell Res.91, 413–421]. The hyperpolarization of the membrane potential as compared with the oocyte (?13 mV) results primarily from the increased $\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ ratio. A similar phenomenon has been noted in other developing embryos. The increase in pump-mediated K influx at the two-cell stage is accompanied by a decrease of similar magnitude in the external-K-dependent K efflux which is found in the oocyte. This suggests that the KK exchange mechanism may be converted to an active pump. Because of the changes in ion concentrations and movements and the unusual metabolic requirements of the mouse embryo, the effect of external Na on the uptake of glucose and pyruvate in the oocyte and two- and eight-cell stage was examined. No Na-dependent carbohydrate transport could be found at these stages.     

Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 °K) and potentiometric titration.Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix “N”) resolves into two single electron titrations with E_{m 7.2} values of ?380±20 mV and ?240±20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g_// = 2.03, g_⊥ = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with g_z = 2.03, g_y = 1.94 and g_x = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g_// = 2.05 and g_⊥ = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high E_{m 7.2} value (?20±20 mV).Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with E_{m 7.2} = ? 240±20 mV is defined as Center N-3 (g_z = 2.10, (g_y = 1.93?), g_x = 1.87); the ?405±20 mV component as Center N-4 (g_z = 2.11, (g_y = 1.93?), g_x = 1.88). At temperatures close to 4.2 °K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with E_{m 7.2} = ?260±20 mV. The multiplicity of peaks suggests the presence of at least two different ironsulfur centers having similar E_{m 7.2} values (?260±20 mV); hence, tentatively assigned as N-5 and N-6.Consistent with the individual E_{m 7.2} values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the E_{m 7.2} of Center N-4 lies much closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. E_{m 7.2} values and EPR lineshapes for the other ironsulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show E_{m 7.2} values around -250 mV; Center N-2 exhibits a high E_{m 7.2} (?20±20 mV), while Center N-1a shows a very low E_{m 7.2} (?380±20 mV).     

A study of the current-voltage relationships of electrogenetic active and passive membrane elements in riccia fluitans

Potassium- and proton-dependent membrane potential, conductance, and current-voltage characteristics (I–V curves) have been measured on rhizoid cells of the liverwort Riccia fluitans. The potential difference (E_m) measured with microelectrodes across plasmalemma and tonoplast is depolarized to the potassium-sensitive diffusion potential (E_D) in the presence of 1 mM NaCN, 1 mM NaN₃, or at temperatures below 6°C. Whereas the temperature change from 25°C to 5°C decreases the membrane conductance (g_m) from 0.71 to 0.43 S ? m^?2, 1 mM NaCN increases g_m by about 25%. The membrane displays potassium-controlled rectification which gradually disappears at temperatures below 5°C. The potassium pathway can be described by an equivalent circuit of a diode and an ohmic resistor in parallel. In the potential interval of E_D ± 100 mV the measured I-V curves roughly fit the theoretical curves obtained from a modified diode equation. ⁸⁶Rb⁺(K⁺)-influx is voltage sensitive: In the presence of 1 mM NaCN, ⁸⁶Rb⁺-influx follows a hyperbolic function corresponding to a low conductance at low [K⁺]_o and high conductance at high [K⁺]_o. On the contrary ⁸⁶Rb⁺-influx is linear with [K⁺]_o when pump activity is normal. It is believed that there are two K⁺-transport pathways in the Riccia membrane, one of which is assigned to the low conductance (0.2 S · m^?2), the other to a temperature-dependent facilitated diffusion system with a higher conductance (7.7 S · m^?2). The electrogenic pump essentially acts as a current source and consumes about 39% of the cellular ATP-turnover. In the presence of 30 μM CCCP the saturation current of 0.1 A · m^?2 is doubled to about 0.2 A · m^?2, and the electromotive force of ?360 mV switches to ?250 mV. It is suggested that this may be due to a change in stoichiometry from one to two transported charges per ATP hydrolyzed.     

Electrical properties of the Drosophila egg membrane

Electrical properties of the egg membrane of Drosophila melanogaster were examined using intracellular microelectrodes. Unfertilized eggs and fertilized eggs for the period up to the syncytial blastoderm stage were used. Among Na, K, and Cl, K was most permeant to the membrane. The K permeability, however, did not completely determine the membrane potential. The resting potential in standard solution was ?63.5 ± 8.0 mV (mean ± SD) in unfertilized eggs collected within 2–3 days after virgin flies started to lay eggs. The resting potential in fertilized eggs was ?27.0 ± 8.4 mV within 20 min after egg deposition, while it was ?55.1 ± 6.5 mV at the syncytial blastoderm stage. These changes at different developmental stages were associated with changes in the K-dependence of the membrane. The larger amplitude of the resting potential was suggested to be due to increased K permeability but not to decreased nonspecific leakage. The current-voltage relation was linear throughout the stages examined. Action potentials, such as those in eggs of other animals, were not observed. High Ca media significantly increased the amplitude of the resting potential associated with increase in the membrane resistance. A remarkable nonlineality in the I–V relation appeared in high Ca media, which caused continuously increasing hyperpolarization during sustained inward current. Eggs of temperature-sensitive mutants, shi^ts1 and para^ts1 showed properties similar to those in wild-type eggs with transient temperature changes.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Effect of oxidation-reduction potential on light-induced cytochrome and bacteriochlorophyll reactions in chromatophores from the photosynthetic green bacterium Chlorobium

The reaction center bacteriochlorophyll of Chlorobium thiosulfatophilum has a midpoint oxidation-reduction potential (E_m) of +330 mV. Its photooxidation is unaffected by oxidation-reduction potentials in the range from +260 mV to ?70 mV but on further reduction is attenuated to zero in a one-electron transition with an E_m of ?130 mV.A c-type cytochrome with an E_m of +220 mV and absorption maxima at 551–552 nm (α-band) and 420 nm (γ-band) is present in Chlorobium chromatophores and undergoes photooxidation. Cytocrome c photooxidation is attenuated to zero in two 1-electron steps with E_m of +30 mV and ?130 mVPossible roles for +30 mV and ?130 mV components in photosynthetic electron transport in Chlorobium are discussed.     

The tyrosinase inhibitory effects of isoxazolone derivatives with a (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold

Thirteen (Z)-4-(substituted benzylidene)-3-phenylisoxazol-5(4H)-ones were designed to confirm the geometric effect of the double bond of the β-phenyl-α, β-unsaturated carbonyl scaffold on tyrosinase inhibitory activity. Compounds 1a–1m, which all possessed the (Z)-β-phenyl-α, β-unsaturated carbonyl scaffold, were synthesized using a tandem reaction consisting of an isoxazolone ring formation and a Knoevenagel condensation, and three starting materials, ethyl benzoylacetate, hydroxylamine and benzaldehydes. Some of the compounds showed inhibitory activity against mushroom tyrosinase as potent as compounds containing the “(E)”-β-phenyl-α, β-unsaturated carbonyl scaffold. Compounds 1c and 1m showed greater inhibitory activity than kojic acid: IC₅₀?=?32.08?±?2.25?μM for 1c; IC₅₀?=?14.62?±?1.38?μM for 1m; and IC₅₀?=?37.86?±?2.21?μM for kojic acid. A kinetic study indicated that 1m inhibited tyrosinase in a competitive manner and that it probably binds to the enzyme’s active site. In silico docking simulation supported binding of 1m (?7.6?kcal/mol) to the active site of tyrosinase with stronger affinity than kojic acid (?5.7?kcal/mol). Similar results were obtained using cell-based assays, and in B16F10 cells, compound 1m dose-dependently inhibited tyrosinase activity and melanogenesis. These results indicate the anti-melanogenic effect of compound 1m is due to the inhibition of tyrosinase and (Z)-isomer of the β-phenyl-α, β-unsaturated carbonyl scaffold can, like its congener the (E)-isomer, act as an excellent scaffold for tyrosinase inhibition.     

Electron spin resonance characterization of Chromatium D hemes,non-heme irons and the components involved in primary photochemistry

The combination of redox potentiometry with low temperature electron spin resonance (ESR) spectroscopy has led to further characterization of electron transfer components of Chromatium D. These include the readily buffer-soluble cytochromes c₅₅₃ and c′ and the high-potential iron-sulfur protein in the isolated state and associated with the chromatophore membrane. Buffer-insoluble cytochrome c₅₅₃, cytochro—me c₅₅₅, bacteriochlorophyll and the primary electron acceptor have been characterized both in the chromatophore membrane and also in a sodium dodecylsulfate detergent-solubilized subchromatophore preparation. Two iron-sulfur proteins have been revealed which are present in the chromatophore membrane but are released on treatment with sodium dodecylsulfate. They have central g values at 1.90 and 1.94 and have estimated midpoint potentials at pH 7.4 (E_m7·4) at +280 mV and ?100 mV, respectively, when associated with the chromatophore.In the membrane associated state the apparent E_m of cytochrome c′ is approximately 200 mV more positive than the E_m values reported for the free state; this implies either that the reduced form of cytochrome c′ binds to the membrane (or to a component therein) to a degree which is > 10³ times greater than that of the oxidized form or that the E_m shift results from membrane solvation. In the case of the high-potential iron-sulfur protein however, its E_m when associated with the chromatophore membrane is similar to that reported in the isolated state. The light-induced oxidation of the high-potential iron-sulfur protein at room temperature appears to be linked only to the oxidation of cytochrome c₅₅₅; it could serve as an electron pool in equilibrium with cytochrome c₅₅₅ in the cyclic electron flow system.The redox component defined in the reduced state by its g_y = 1.82 and g_x = 1.62 ESR spectrum satisfies the following criteria for its identification as the primary electron acceptor of P883. (a) The E_m7·4 value of the g = 1.82 component is ?120 ± 25mV. (b) At ?70 mV, where the g = 1.82 component is mainly oxidized in the dark, brief illumination at low temperature which causes the irreversible oxidation of one cytochrome c₅₅₃ heme, also induces the permanent reduction of the g = 1.82 component; the extent of reduction after brief illumination, given by the g = 1.82 signal height, is the same as that induced chemically at ?270 mV showing it to be fully reduced by the receipt of a single electron. (c) At more positive potentials where cytochrome c₅₅₃ is oxidized and is not involved in low-temperature reactions, the light-induced low-temperature kinetics of the g = 1.82 signal are reversible; the flash-induced g = 1.82 formation and subsequent dark decay are the same as those for the flash-induced P⁺883 (g = 2) formation and dark decay. We suggest that until a full physical-chemical characterization is completed this g = 1.82 component be designated “photoredoxin”.     

Oxidation-reduction potentials of cytochromes P-450 and ferredoxin in the bovine adrenal: Their modification by substrates and inhibitors

The midpoint reduction potentials of the haem iron in bovine adrenal cytochrome P-450 and its associated iron-sulphur protein, adrenal ferredoxin, have been measured, using EPR spectroscopy to monitor the high and low spin ferric haem iron and reduced adrenal ferredoxin signals as a function of potential, in mitochondrial and microsomal suspensions.In mitochondria the high spin (substrate-bound) cytochrome P-450 showed single-component one-electron plots under most conditions; at pH 6.65 cholesterol side-chain cleavage cytochrome P-450 (P-450_scc) had a midpoint E_m = ?305 mV; at pH 8.0 11β-hydroxylase cytochrome P-450 (P-450_11β) had E_m = ?335 mV. Low spin cytochrome P-450 showed more complex titration curves under all conditions, which could be most simply interpreted in terms of two one-electron components with midpoint potentials approx. ?360 and ?470 mV, with varying intensities. During treatments that caused substrate binding, only the ?470 mV component was reduced in magnitude. On sonication and removal of adrenal ferredoxin, the ?470 mV low spin component was converted to higher potential. The potentials could also be altered by the cytochrome P-450 inhibitors aminoglutethimide and metyrapone. In the microsomes, a high spin component of cytochrome P-450 (E_m ≈ ?290 mV) was observed even at pH 8.0, suggesting the binding of an endogenous substrate, while the low spin P-450 showed a predominance of the ?360 mV component. The midpoint potential of membrane-bound adrenal ferredoxin under these various conditions was found to be ?248 mV ± 15 mV.     

Protein kinase A-regulated Cl− channel in ML-1 human hematopoietic myeloblasts

Using the inside-out patch clamp technique, we identified a Cl^? channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl^? channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (?40 mV). Biophysical studies of the Cl^? channel revealed that the current-voltage (I-V) relationship of the Cl^? channel was outwardly rectifying in symmetrical 142 mm Cl^? solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at ?60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl^? channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC⁵⁰) for 9-AC and DIDS were 174 ± 20 and 70±16 μm, respectively. Blockade of the Cl^? channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl^? channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses.     

Development and morphological characterization of the immature stages of Tetrastichus giffardianus Silvestri (Hymenoptera: Eulophidae)

The gregarious endoparasitoid Tetrastichus giffardianus Silvestri (Hymenoptera: Eulophidae) is a natural enemy of fruit flies. This parasitoid was previously used to successfully control Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in Hawaii, USA. Despite its importance in the control of fruit fly pests, little is known about the development or characteristics of its preimaginal stages. The aim of this study was to observe the development and morphologically characterize the immature stages of Tetrastichus giffardianus. Tetrastichus giffardianus individuals were reared on C. capitata larvae/pupae under laboratory conditions at a temperature of 25 ± 2 °C, relative humidity of 60 ± 10%, and 12-h photophase. Third-instar C. capitata larvae were exposed to parasitism for 24 h. After parasitism, the pupae were dissected every 24 h to evaluate the stage of development attained by T. giffardianus, and to record their morphological characteristics. A stereomicroscope was used to observe all the immature stages of T. giffardianus. The complete development of T. giffardianus under these conditions was completed within 14 days as follows: egg (duration ? 1 day); first (? 1 day), second (? 1 day), and third (? 2 days) larval instars; pre-pupa (? 2 days); and pupa (? 7 days). The immature stages of T. giffardianus differed sufficiently in their shape, color, and size to allow morphological characterization.     

Energetics of the one-electron steps in the NAD+/NADH redox couple

The one-electron reduction potential of NAD⁺ has been determined using pulse radiolysis to study electron-transfer equilibria between it and a low potential bipyridylium compound. The determined value E¹₇ (NAD⁺/NAD^.) = ?922 ± 8 mV (NHE scale) is used to calculate E²₇ (NAD^./NADH) = +282 mV. E¹₇ for 1-methylnicotinamide, E¹₇ (MeN⁺/MeN^.) = ?918 ± 7 mV.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

Electrophysiology of phagocytic membranes: intracellular K+ activity and K+ equilibrium potential in macrophage polykaryons

The role of K⁺ as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca²⁺ injections into macrophage polykaryons is studied. The intracellular K⁺ activity (a_K) and the K⁺ equilibrium potential (E_K) are measured using ion-sensitive microelectrodes. The mean value of a_K is 84 ± 5 mM in a culture medium containing 5.3 mM K⁺, but increases to 100 ± 8 mM when the extracellular K⁺ concentration is raised to 30.3 mM. Under the same conditions the values of E_K obtained from the Nernst equation are −81 ± 2 mV and −40 ± 2 mV, respectively. The reversal potentials (E_R) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean E_R values obtained are −74 ± 8 mV in the presence of low K⁺ concentration and −37 ± 3 mV for the high K⁺ medium. These values are significantly smaller than the estimated E_K for the corresponding situations. Evidence for the existence of an electrogenic (Na⁺ + K⁺)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.     

Partial purification and characterization of two soluble c-type cytochromes from Chromatium vinosum

Two c-type cytochromes from Chromatium vinosum have been partially purified and characterized. Cytochrome c₅₅₀, which appears to function as an electron carrier in the cyclic electron transport chain of this photosynthetic purple sulfur bacterium, has a molecular weight of approximately 15,000 and an oxidation-reduction midpoint potential (E_m) of + 240 mV at pH 7.4. It has (in the reduced form) an α band at 550 nm and a β band at 520 nm. Cytochrome c₅₅₁ is characterized by absorbance maxima at 354 and 409 nm in the oxidized form and 418, 523, and 551 nm in the reduced form. The reduced cytochrome reacts with CO. Cytochrome c₅₅₁ is a monomeric protein with a molecular weight of 18,800 ± 700 and E_m = ?299 ± 5 mV (pH independent between pH 6.3 and 8.0). It appears to lack a methionine axial ligand as indicated by the absence of an absorbance band at 695 nm in the oxidized form.