Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Crystal and microparticle effects on MDCK cell superoxide production: oxalate-specific mitochondrial membrane potential changes

We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O₂⁻) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O₂⁻ was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [¹⁴C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O₂⁻ in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O₂⁻ or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm². Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O₂⁻ (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O₂⁻, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ_m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca²⁺ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca²⁺ from internal stores. Thus, COM-induced mitochondrial O₂⁻ requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O₂⁻ production, which is probably regulated by Δψ_m.     

Differentiated Staining of Osmium Tetroxide-Fixed, Vestopal-w-Embedded Tissue in thin Sections

Tissues were fixed at 20° C for 1 hr in 1% OsO₄, buffered at pH 7.4 with veronal-acetate (Palade's fixative), soaked 5 min in the same buffer without OsO₄, then dehydrated in buffer-acetone mixtures of 30, 50, 75 and 90% acetone content, and finally in anhydrous acetone. Infiltration was accomplished through Vestopal-W-acetone mixtures of 1:3, 1:1, 3:1 to undiluted Vestopal. After polymerisation at 60° C for 24 hr, 1-2 μ sections were cut, dried on slides without adhesive, and stained by any of the following methods. (1) Mayer's acid hemalum: Flood the slides with the staining solution and allow to stand at 20°C for 2-3 hr while the water of the solution evaporates; wash in distilled water, 2 min; differentiate in 1% HCl; rinse 1-2 sec in 10% NH,OH. (2) Iron-trioxyhematein (of Hansen): Apply the staining solution as in method 1; wash 3-5 min in 5% acetic acid; restain for 1-12 hr by flooding with a mixture consisting of staining solution, 2 parts, and 1 part of a 1:1 mixture of 2% acetic acid and 2% H₂SO₄ (observe under microscope for staining intensity); wash 2 min in distilled water and 1 hr in tap water. (3) Iron-hematoxylin (Heidenhain): Mordant 6 hr in 2.5% iron-alum solution; wash 1 min in distilled water; stain in 1% or 0.5% ripened hematoxylin for 3-12 br; differentiate 8 min in 2.5%, and 15 min in 1% iron-alum solution; wash 1 hr in tap water. (4) Aceto-carmine (Schneider): Stain 12-24 hr; wash 0.5-1.0 min in distilled water. (5) Picrofuchsin: Stain 24-48 hr in 1% acid fuchsin dissolved in saturated aqueous picric acid; differentiate for only 1-2 sec in 96% ethanol. (6) Modified Giemsa: Mix 640 ml of a solution of 9.08 gm KH₂PO₄ in 1000 ml of distilled water and 360 ml of a solution of 11.88 gm Na₂HPO₄-2H₂O in 1000 ml of distilled water. Soak sections in this buffer, 12 hr. Dissolve 1.0 gm of azur I in 125 ml of boiling distilled water; add 0.5 gm of methylene blue; filter and add hot distilled water until a volume of 250 ml is reached (solution “AM”). Dissolve 1.5 gm of eosin, yellowish, in 250 ml of hot distilled water; filter (solution “E”). Mix 1.5 ml of “AM” in 100 ml of buffer with 3 ml of “E” in 100 ml of buffer. Stain 12-24 hr. Differentiate 3 sec in 25 ml methyl benzoate in 75 ml dioxane; 3 sec in 35 ml methyl benzoate in 65 ml acetone; 3 sec in 30 ml acetone in 70 ml methyl benzoate; and 3 sec in 5 ml acetone in 95 ml methyl benzoate. Dehydrated sections may be covered in a neutral synthetic resin (Caedax was used).     

Effects of selenite on O2 consumption, glutathione oxidation and NADPH levels in isolated hepatocytes and the role of redox changes in selenite toxicity

Isolated hepatocytes incubated with selenite (30–100 μM) exhibited changes in the glutathione redox system as shown by an increase in O₂ consumption, oxidation of glutathione and loss of NADPH. Selenite (50 μM) raised O₂ consumption within the 1 h and induced an partial depletion of thiols with a concomitant increase in oxidized glutathione, as well as a decrease in NADPH levels within 2 h. With 100 μM selenite more pronounced effects were obtained such as a total depletion of thiols. This concentration of selenite also lysed cells within 3 h. Arsenite, HgCl₂ and KCN prevented the increase in O₂ uptake, counteracted loss of thiols and delayed selenite induced lysis. p-Tert-butylbenzoic acid, an inhibitor of gluconeogenesis, decreased selenite dependent O₂ consumption and potentiated the effect on NADPH levels as well as the toxic effect. Finally, methionine further enhanced O₂ consumption by selenite and also delayed loss of thiols and potentiated selenite toxicity. These results indicated that selenite catalyzed a reduction of O₂ in glutathione dependent redox cycles with NADPH as an electron donor. With subtoxic concentrations of selenite (50 μM) there were indications that O₂ reduction was terminated by selenite biotransformation to methylated metabolites. With toxic concentrations of selenite (100 μM) it appeared that O₂ reduction was eventually limited by the capacity of the cell to regenerate NADPH. It is suggested that a depletion of NADPH mediated the observed cytotoxicity of selenite.     

Laser-induced reactions of P700 and cytochrome f in a blue-green alga, Plectonema boryanum

Kinetics of the absorption change of P700 (blue band) and cytochrome f in whole cells of a blue-green alga, Plectonema boryanum, have been studied by Q-switched ruby-laser flash excitation (694 nm; approx. 20 nsec) to elucidate the sequential relationship of these two components in photosynthetic electron transport. “P700” was photooxidized within 2 μsec and recovered in two phases t_1/2 10 μsec and 200 μsec). Under the same conditions cytochrome f was oxidized with a half time of 15 μsec. The magnitude of the fast phase of “P700” recovery, however, diminished at lower laser intensity while the cytochrome f change remained unaffected. The result suggests that cytochrome f and P700 may not be on the same electron-transport chain.     

Use of Vital Dyes in Conjunction with the Semivitro Technic for the Detection of Pollen Tube Sperm Nuclei

The technique we describe here is a modification of that used by Hough et al. (1985), combined with “semivitro” pollen tube observations. With the semivitro technique, pollen tubes grow from the cut ends of pollinated styles (Brewbaker and Majumder 1961). Pollen of Nicotiana alata was presoaked for 15 min in simplified medium (Brewbaker and Kwack 1963) (10% sucrose, 300 ppm Ca(NO₃)₂, 100 ppm H₃BO₃ with the addition of 0.5 mg/ml of Hoechst 33258 stain from Serva Biochemicals, Heidelberg, Control H, purchased June 1983). (For germination of Nicotiana alata pollen in vitro, we use this same solution, except with 12% sucrose). After this prestaining, the pollen suspension was centrifuged for 5 min at 1200 × g, the pellet resuspended in control Brewbaker medium (i.e., no stain), recentrifuged and used to pollinate detached pistils. The pistils were then incubated at 25 C in a water-saturated atmosphere for 20 hr. At this time, the styles were cut just ahead of the front of the growing pollen tubes (Mulcahy and Mulcahy 1985) and the cut stylar ends each dipped in fresh control Brewbaker medium. Twelve to 24 hours later, tubes growing out of the cut styles were viewed by fluorescence microscopy (exciter filter, BG 12 + KV 418, beam splitter, 500 nm, and barrier filter OG 515). A distinct green fluorescence was seen in the generative and vegetative nuclei (Fig. 1).     

Studies on the “facilitated flux” of oxygen through hemoglobin solutions using a polarographic method

The phenomenon of hemoglobin-facilitated O₂ diffusion was studied by a polarographic method.

Polarograms relative to the reduction process of O₂ have been obtained at pH 7.2 (phosphate buffer, 30°) in the presence of various hemoglobin concentrations (Hb_tot*) and at various O₂ partial pressures (from 8 to 360 mm Hg).

Analogous experiments were performed at pH 6.4 and 8.1 (at constant ionic strength). Graphs of the limiting current values (at E = −1.5 V versus the saturated calomel electrode), relative to the overall reduction process of oxygen, plotted versus P_O2 (at Hb_tot* = constant), show some characteristic trends. The influence of pH on the features of the experimental curves is discussed.

Experimental results suggest that the diffusions of O₂, oxyhemoglobin and hemoglobin, as well as the kinetics of dissociation and association of O₂ with hemoglobin, are effective in determining the “facilitated flux”.

The corresponding nonlinear differential system is solved under some simplifying assumptions, and an expression for the flux, and consequently for the current, is obtained which is consistent with the experimental findings.

Furthermore, it is shown that the dissociation curve of oxyhemoglobin can be obtained from these polarographic experiments on the basis of this theory. Agreement with tensiometric data was satisfactory.     

Self-reduction of the iron(III)-doxorubicin complex

The Fe³⁺-doxorubicin complex undergoes reactions that suggest that the complex self-reduces to a ferrous oxidized-doxorubicin free radical species. The Fe³⁺-doxorubicin system is observed to reduce ferricytochrome c, consume O₂ and react with 2,2′-bipyridine. Bipyridine acts as a “ferrous ion scavenger” as it reacts with the ferrous ion produced by Fe³⁺-doxorubicin self-reduction. In the absence of O₂, a ferrous doxorubicin complex accumulates. In the presence of oxygen, Fe²⁺ recycles back to Fe³⁺. The rates of these reactions were measured and the Fe³⁺-doxorubicin self-reduction was determined to be the rate-determining step. The Fe³⁺-doxorubicin induced inactivation of cytochrome c oxidase and NADH cytochrome c reductase on beef heart submitochondrial particles occurs at a rate similar to Fe³⁺-doxorubicin self-reduction. Thus the rate at which damage to these mitochondrial enzymes occurs may be controlled by a nonezymatic Fe³⁺-doxorubicin self-reduction.     

Fluorescence characteristics of lyophilized maize chloroplasts suspended in buffer

Fluorescence transients were measured in lyophilized maize chloroplasts (suspended in Tris-maleate buffer (pH 6.6)) after extraction with heptane. (The fluorescence characteristics before extraction were qualitatively similar to those in the fresh chloroplasts.) The initial fluorescence level (m) in the (dry) heptane-extracted sample remained the same as in the unextracted material, but the variable fluorescence (Δm) was drastically diminished. A portion of variable fluorescence, however, could be restored by adding Na₂S₂O₄. If the heptane extraction was made in the presence of water (wet), the m level was almost as high as (or higher than) the final level (M) of the unextracted sample, and Δm was reduced. The “jet” of O₂ (that measures the pool size of the intersystem intermediate A) and the “microjet” (that measures the pool size of the reaction center complex E), present in the unextracted samples, were absent in both types of extracted samples. Some of the above data may be interpreted in a hypothesis in which two quenchers (Q₁ and Q₂) control the fluorescence (O → P) of chloroplasts — the reduction of Q₁ being responsible for the rapid and that of Q₂ for the slow fluorescence rise.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

The reduction of fungal laccase at high pH

1. 1. The nature and mechanism of the reduction of fungal laccase (p-diphenol: O₂ oxidoreductase, EC 1.10.3.2) obtained on an increase in pH have been studied by optical and electron paramagnetic resonance (EPR) spectroscopy and by measurements of O₂ concentration.

2. 2. The decreases in the optical absorption and the EPR signal of the “blue” Type 1 Cu²⁺ at high pH indicate that this ion is reduced. This is confirmed by oxidation with hexachloroiridate(IV) which restores the blue color. The “nonblue” Type 2 Cu²⁺ remains divalent over the pH range studied, as seen from the EPR spectra.

3. 3. Approximately one equivalent of hexachloroiridate(IV) is sufficient to restore the color of a pH-bleached protein which suggests that the reduction involves a single electron. A comparison between the optical spectra at pH 5 and 8 shows that the two-electron accepting unit, which at pH 5.5 is reduced concomitantly with the Type 1 Cu²⁺, remains oxidized in the protein brought to high pH. This unit can be reduced at pH 8.3 by octacyanotungstate(IV), as shown by the fact that this reductant in anaerobic titrations is found to add about two electrons (and no more) to a protein already having the Type 1 copper reduced. Thus, an increase in pH introduces a difference in the reduction behavior of the electron acceptors in fungal laccase.

4. 4. Oxygraph experiments show that there is no production of O₂ with an increase in pH, as would occur if water was oxidized by laccase. On the contrary, there is a continuous consumption of O₂ at both pH 5 and 8, indicating that the protein preparation contains a reducing substance which is responsible for the pH-dependent reduction.

The chemical microenvironment of the symbiotic planktonic foraminifer Orbulina universa

Microsensor measurements of CO₂, O₂, pH and Ca²⁺ in the vicinity of the symbiont-bearing planktonic foraminifer Orbulina universa showed major light-modulated changes in the chemical microenvironment due to symbiont photosynthesis, respiration of the holobiont, and precipitation of the calcite shell. Under saturating light conditions, microprofiles measured towards the shell surface showed an O₂ increase of up to 220% air saturation, a decrease in CO₂ concentration to 4.9 μM, and a pH increase to 8.8 due to symbiont photosynthesis. The Ca²⁺ concentration decreased to ∼9.6 mM in two specimens, while it increased to 10.2-10.8 mM in three other specimens kept in light. In darkness, the respiration of the community decreased the O₂ concentration to 82% of air saturation, CO₂ increased up to 15 μM, the pH decreased to 8.0, and the Ca²⁺ concentration increased up to 10.4 mM. These data, and derived calculations of the distribution of HCO₃^- and CO₃^2- near the shell, showed that the carbonate system in the vicinity of O. universa was significantly different from conditions in the surrounding seawater, both in light and darkness, due to the metabolism of the foraminifer and its associated algae. Experimental light-dark cycles indicated a sufficient CO₂ supply sustaining high carbon fixation rates of the symbiotic algae via conversion of HCO₃^- or via CO₂ release from calcification and host respiration. Our findings on irradiance-dependent CO₂ and pH changes in the vicinity of symbiont-bearing planktonic foraminifera give direct experimental evidence for the predictions of isotope fractionation models used in palaeoclimatology stating that metabolic processes affect the isotopic carbon signal (δ¹³C) in foraminifera.     

Examination of the Inhibitory Effect of Norathyriol in Formylmethionyl-Leucyl-Phenylalanine-Induced Respiratory Burst in Rat Neutrophils

Norathyriol, aglycone of a xanthone C-glycoside mangiferin isolated from Tripterospermum lanceolatum, concentration dependently inhibited the formylmethionyl-leucyl-phenylalanine (fMLP)-induced superoxide anion (O₂^˙−) generation and O₂ consumption in rat neutrophils. In cell-free oxygen radical generating system, norathyriol inhibited the O₂^˙− generation during dihydroxyfumaric acid (DHF) autoxidation and in hypoxanthine-xanthine oxidase system. fMLP-induced transient elevation of [Ca²⁺]_i and the formation of inositol trisphosphate (IP₃) were significantly inhibited by norathyriol (30 μM) (about 30 and 46% inhibition, respectively). Norathyriol concentration dependently suppressed the neutrophil cytosolic phospholipase C (PLC). In contrast with the marked attenuation of fMLP-induced protein tyrosine phosphorylation (about 70% inhibition at 10 μM norathyriol), norathyriol only slightly modulated the phospholipase D (PLD) activity as determined by the formation of phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt). Norathyriol did not modulate the intracellular cyclic AMP level. In the presence of NADPH, the phorbol 12-myristate 13-acetate (PMA)-activated particulate NADPH oxidase activity was suppressed by norathyriol in a concentration-dependent manner and the inhibition was noncompetitive with respect to NADPH. Norathyriol inhibited the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free NADPH oxidase system at the same concentration range as those used in the suppression of PMA-activated particulate NADPH oxidase activity. Taken together, these results suggest that the scavenging ability of norathyriol contributes to the reduction of generated O₂^˙−, however, the inhibition of O₂^˙− generation from neutrophils by norathyriol is attributed to the blockade of PLC pathway, the attenuation of protein tyrosine phosphorylation, and to the suppression of NADPH oxidase through the interruption of electrons transport.     

Synthesis and properties of the dichloro [tetramethyl (trifluoromethyl)cyclopentadienyl]ruthenium dimer: X-ray structure of [M2(η-C5Me4CF3)2Cl2(μ-Cl)2] (M=Ru, Rh)

The reaction of RuCl₃(H₂O), with C₅Me₄CF₃J in refluxing EtOH gives [Ru₂(η⁵-C₅Me₁CF₂)₂ (μ-Cl₂] (20 in 44% yield. Dimer 2 antiferromagnetic (−2J=200 cm¹). The crystal structures of 2 (rhombohedral system, R3 space group, Z=9, R=0.0589) and [Rh₂(η⁵-C₅Me₄CF₃(₂Cl₂(μ-Cl)₂] (3) (rhombohedral system. space group, Z = 9, R = 0.0641) were solved; both complexes have dimeric structures with a trans arrangement of the η⁵-C₅Me₄CF₄ rings. Comparison of the geometry of 2 and 3 with those of the corresponding η⁵-C₅Me₅ complexes shows that lowering the ring symmetry causes significant distortion of the M₂(μ-Cl)₂ moiety. The analysis of the MCl₃ fragment conformations in 2 and 3 and in the η⁵-C₅ME₅ analogues shows that they are correlated with the M---M distances. The Cl atoms are displaced by Br on reaction of 2 with KBr in MeOH to give the diamagnetic dimer [Ru₂(η⁵-C₅Me₄CF₃)₂Br₂ (μ-Br₂] (4). Complex 2 reacts with O₂ in CH₂Cl₂ solution at ambient temperature to form a mixture of isomeric η⁶-fulvene dimers [Ru₂(η⁶-C₅Me₃CF₃ = CH₂)₂Cl₂(μ-Cl)₂] (5). Reactions of 5 with CO and allyl chloride give Ru(η⁵-C₅Me₃CF₃CH₂Cl)(CO)₂Cl (6) and Ru(η⁵-C₅Me₃CF₃CF₃CH₂Cl)(η³-C₃H₅)Cl₂ (7) respectively.     

Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Grimelius' Silver Stain for Endocrine Cell Granules, as Shown by Electron Microscopy

The silver impregnation method of Grimelius has been applied to 100-150 μ thick sections of tissues fixed 2 hr to 1 mo in mixtures containing formaldehyde, glutaraldehyde or picric acid. After silvering, the sections (partly postfixed in 1% OsO₄, for 0.5 hr) were processed for electron microscopy. Endocrine granules of pancreatic A cells, enter-ochromaffin and some nonenterochromaffin cells of the gastrointestinal mucosa, thyroid C cells and adrenal medullary cells were found to be selectively stained by silver grains 10-30 nm in diameter, either as a peripheral “halo” or covering the entire granule. At least in some cells, the reactive material should not be identified with the hormonal products known to be stored in the granules.     

A low-temperature-sensitive intermediate state between S2 and S3 in photosynthetic water oxidation deduced by means of thermoluminescence measurements

The temperature dependence of S-state transitions in Photosystem II was measured by means of thermoluminescence using two different protocols for low-temperature flash excitation: protocol A, “last flash at low temperature”, and protocol B, “all flashes at low temperature”. Comparison of the temperature-dependence curves obtained by these two protocols revealed a marked difference particular for the three-flash experiments. The difference was attributed to the formation of a low-temperature sensitive precursor state between S₂ and S₃. The state is formed by two flash illumination given at −5 to −50°C, spontaneously transforms to normal S₃ on dark warming, and is not converted to S₀ by the 3rd flash. The precursor state was tentatively assigned to an S₃ in which H⁺ release is not completed.     

From the {Cu(μ2-S)N}4 butterfly architecture to the {Cu(μ3-S)N}12 double wheel

Copper(I) complexes with {Cu(μ₂-S)N}₄ and {Cu(μ₃-S)N}₁₂ core portions of butterfly-shaped or double wheel architectures have been isolated in the reaction of Cu(I) with the Schiff base ligand C₆H₄(CHNC₆H₄S)₂, “iso-abt”, under different conditions. containing the tetranuclear electroneutral complex is formed by the reaction of CuI in acetonitrilic solution and recrystallization from DMF, whereas containing dodecanuclear wheels is accessible starting from CuBF₄. Complexes 2 and 4 represent the first examples of cyclic complexes with the same overall stoichiometry but different ring sizes. The ligand induces two different coordination environments around copper(I) by switching between μ₂- and μ₃-sulfur bridging modes.     

PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching

Recently, it has been shown that PKA-mediated phosphorylation of the β₂-adrenergic receptor (β₂-AR) by the cyclic AMP-dependent protein kinase (PKA) reduces its affinity for G_s and increases its affinity for G_i. Here we demonstrate that, like the β₂-AR, the β₁-AR is also capable of “switching” its coupling from G_s to G_i in a PKA-dependent manner. The β₁-AR is capable of activating adenylate cyclase via G_s, and can also activate the extracellular-regulated kinases, p44 and p42 (ERK1/2). In transfected CHO cells, the observed β₁-AR-mediated activation of ERK is both sensitive to pertussis toxin (PTX), indicating involvement of G_i/G_o, and to the PKA inhibitor, H-89. β₁-ARs with PKA phosphorylation sites mutated to alanines are unable to activate ERK. Mutating these same residues to aspartic acid, mimicking PKA phosphorylation, leads to a decrease in G_s-stimulated cAMP accumulation and an increase in PTX-sensitive ERK activation. These results strongly support the hypothesis that the β₁-AR, like the β₂-AR, can undergo PKA-dependent “G_s/G_i switching”.     

Wavelength-dependent quantum yields of chloroplast phosphorylation catalyzed by phenazine methosulphate

Studies of phenazine methosulphate (PMS)-catalyzed O₂ exchange and phosphorylation in spinach chloroplasts reveal that at short wavelengths (<680 mμ) PMS acts at a reduced quantum efficiency as an oxidant for O₂ evolution with concomitant phosphorylation. The quantum yield profile of phosphorylation obtained with PMS differs markedly from the yield profile of phosphorylation for normal chloroplasts with NADP⁺ or ferricyanide as oxidant. Between 525 and 680 mμ the quantum yield of phosphorylation (ATP) catalyzed by PMS is less than half the constant maximum ATP of the normal system. The maximum ATP value for the normal system is approx. 0.16 ATP/hv. With the PMS system a peak in the yield at 690 mμ is obtained approaching the ATP value of the normal system. This yield falls again at longer wavelengths (>700 mμ).

The addition of ascorbate to the PMS phosphorylating system decreases the short-wavelength (<680 mμ) phosophorylation activity but increases the long-wavelength (>690 mμ) phosphorylation activity. The quantum yield profile of this system, showing a long-wavelength rise in phosphorylation efficiency is obtained with or without the addition of 3(3,4-dichlorophenyl)-1, 1-dimethylurea.

These experiments have been interpreted as indicating two separate electrontransfer processes catalyzed by PMS, one in which PMS acts at a reduced efficiency as a Hill oxidant, and the other in which PMS acts as an electron donor and acceptor in a cyclic fashion in sensitizing and essentially long-wavelength phosphorylation process.