Subunit-subunit interactions in TF1 as revealed by ligand binding to isolated and integrated α and β subunits

The binding of 3′-O-(1-naphthoyl)adenosinetriphosphate (1-naphthoyl-ATP), ATP and ADP to TF₁ and to the isolated α and β subunits was investigated by measuring changes of intrinsic protein fluorescence and of fluorescence anisotropy of 1-naphthoyl-ATP upon binding. The following results were obtained. (1) The isolated α and β subunits bind 1 mol 1-naphthoyl-ATP with a dissociation constant (K_D(1-naphthoyl-ATP)) of 4.6 μM and 1.9 μM, respectively. (2) The K_D(ATP) for α and β subunits is 8 μM and 11 μM, respectively. (3) The K_D(ADP) for α and β subunits is 38 μM μM and 7 μM, respectively. (4) TF₁ binds 2 mol 1-naphthoyl-ATP per mol enzyme with K_D = 170 nM. (5) The rate constant for 1-naphthoyl-ATP binding to α and β subunit is more than 5 · 10⁴ M⁻¹s⁻¹. (6) The rate constant for 1-naphthoyl-ATP binding to TF₁ is 6.6 · 10³ M⁻¹ · s⁻¹ (monophasic reaction); the rate constant for its dissociation in the presence of ATP is biphasic with a fast first phase (k^A₋₁ = 3 · 10⁻³s⁻¹) and a slower second phase (k^A₋₂ < 0.2 · 10⁻³s⁻¹). From the appearance of a second peak in the fluorescence emission spectrum of 1-naphthoyl-ATP upon binding it is concluded that the binding sites in TF₁ are located in an environment more hydrophobic than the binding sites on isolated α and β subunits. The differences in kinetic and thermodynamic parameters for ligand binding to isolated versus integrated α and β subunits, respectively, are explained by interactions between these subunits in the enzyme complex.     

A differential effect between the acute and chronic administration of ethanol on the endocytotic rate konstant,ke, for internalisation of asialoglycoproteins by hepatocytes

The endocytotic rate constant, k_e, originally described for the quantification growth factor by fibroblasts (Wiley, H.S. and Cunningham, D.D.(1982) J. Biol. Chem. 257, 4222–4229) has been adapted to measure receptor-mediated endocytosis of asialoglycoproteins by hepatocytes. A k_e value of 0.21 min⁻¹ was obtained for the internalisation of β-d-galactosyl bovine serum albumin by freshly isolated hepatocytes. The addition of ethaniol to the incubation medium had a biphasic effect on _e. The value of k_e was increased by up to 30% by low concentrations of ethanol, whereas higher concentrations progressively decreased k_e and in 500 mM ethanol the k_e value was 0.1 min⁻¹. The amount of ligand bound to the cell surface was independent of the extracellular concentration of ethanol and the changes in k_e were exclusively due to changes in the amount of internalised ligand. There was a progressive decrease in the value of k_e in hepatocytes prepared from rats that were maintained on an ethanol-impregnated liquid diet for up to 20 days. The decrease was already apparent by day 2 when blood alcohol levels were only 50 mg%, indicating that the effects of chronic alcoholism on endocytosis are manifested at an early stage.     

Intercellular synchronization of diffusively coupled Ca2+ oscillators

We examine the synchrony in the dynamics of localized [Ca^2 +]_i oscillations among a group of cells exhibiting such complex Ca^2 + oscillations, connected in the form of long chain, via diffusing coupling where cytosolic Ca^2 + and inositol 1,4,5-triphosphate are coupling molecules. Based on our numerical results, we could able to identify three regimes, namely desynchronized, transition and synchronized regimes in the (T − k_e) (time period-coupling constant) and (A − k_e) (amplitude-coupling constant) spaces which are supported by phase plots (Δϕ verses time) and recurrence plots, respectively. We further show the increase of synchronization among the cells as the number of coupling molecules increases in the (T − k_e) and (A − k_e) spaces.     

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (P^T-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors F_X, F_B and F_A, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of P^T-700 upon illumination is independent of the redox state of A and of F_X,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are k_x = 1100 s^?1 ± 10%, k_y = 1300 s^?1 ± 10% and k_z = 83 s^?1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A₀ and A₁, with A₀ preceding A₁. In LDS particles at temperatures below ?30°C only A₀ is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A^?₁ spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A₀A^?₁ must exceed 5 ms. We conclude that P^T-700 originates from charge recombination of P-700A^?₀, and that in Triton particles A₀ and A₁ are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF^?_X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.     

Reactions of iron-sulfur proteins with the aquated electron

The reaction of parsley 2Fe-2S ferredoxin in the normal oxidized state with e_aq^? generated by pulse radiolysis techniques has been studied at ～25°C, pH 7–8, I = 0.10 M (NaClO₄). Rate constants k_e (e_aq^? decay) and k_p (protein absorbance change) are the same, second-order rate constant 9.7 × 10⁹ M^?1 sec^?1. The reaction exhibits close to 100% efficiency. With 8Fe-8S ferredoxin from Clostridium pasteurianum under identical conditions it now appears that k_p (although sometimes significantly smaller) is equal to k_e. Varying efficiencies are also observed with this protein depending on the batch used. The reasons for such variable behavior are not fully understood. With oxidized and reduced forms of Chromatium v. high-potential iron-sulfur protein (HIPIP), k_e and k_p are essentially the same, but the highest efficiency observed is only ～50%. The prevailing pattern is therefore that rate constants k_e and k_p are generally in step for proteins having a single (or identical) active site(s). When the active site is buried as with HIPIP the efficiency of the reaction appears to decrease.     

Transport of nitrate and ammonium ions in a sandy loam soil treated with potassium zeolite – Evaluating equilibrium and non-equilibrium equations

The increased use of nitrogen fertilizers in agriculture would cause migration of nitrogen to surface and groundwater; accordingly, would lead to water resources contamination. The objective of this study is to investigate the effect of potassium zeolite on nitrate and ammonium ions sorption and retention in a saturated sandy loam soil in a laboratory condition. The study was conducted as a completely randomized block design with four treatments of 0, 2, 4 and 8 g zeolite per kilogram soil and three replications. Ammonium nitrate fertilizer with concentration of 10 g l⁻¹ was added to soil columns and then leaching was performed. The results show that increasing potassium zeolite to soil causes reduction to the mobility of both nitrate and ammonium and enhancement of the retention of ions in soil. Ions leaching were simulated with convection–dispersion-equation (CDE) and mobile–immobile model (MIM) using HYDRUS-1D code. The results indicate that ammonium ion sorption by soil followed the Freundlich isotherm model. Absorption isotherms and dispersion (D_e) coefficient were determined through the inverse modeling for both ions. Based on the results, optimized values of Freundlich isotherm were much less than the observed amounts. This shows that the HYDRUS-1D model underestimated the adsorption parameters to predict the ammonium ion mobility in soil macropores. Since soil has been disturbed, the prediction of CDE model was equal to MIM model approximately. Both models showed that as the amount of applied zeolite increases, the dispersion (D_e) coefficient of nitrate and ammonium ions in the soil increases.     

Studies on the reactions of PtCl2en,cis-Pt(NH3)2Cl2 and their aqua species with adenosine,deoxyadenosine and adenine using ion-pair HPLC

The reactions of PtCl₂en or cis-Pt(NH₃)₂Cl₂ and their aqua species with adenine and adenosine were studied by means of ion-pair HPLC. From the chromatograms, it was found that the first binding site of Pt(II) was the N(7) site of adenine under both acidic and neutral conditions. The rates of Pt(II) binding at the (N7) site of adenosine and deoxyadenosine were measured. The rate constants, k₁, were obtained for the reactions of PtCl₂en or cis-Pt(NH₃)₂Cl₂ with adenosine and deoxyadenosine at pH 3 and 7 over the temperature range 9–25 °C. The k₁ values were 6.8–7.7 × 10⁻⁴ dm³ mol⁻¹ s⁻¹ at 25 °C. For the aqua species, the rate of [cis-Pt(NH₃)₂ClH₂O]⁺ with adenosine N(7) was measured. The rate constants, k₂ which were found to be smaller than those of hydrolysis, k_h, were calculated at pH 3 over the temperature range 25–40 °C. The k₂ value obtained at 25 °C was 1.1 × 10⁻² dm³ mol⁻¹ s⁻¹, 15 time larger than k¹. The activation parameters were also calculated.     

Design,synthesis and evaluation of benzo[a]thieno[3,2-g]quinolizines as novel l-SPD derivatives possessing dopamine D1, D2 and serotonin 5-HT1A multiple action profiles

A novel scaffold derived from l-SPD with a substituted thiophene group in the D ring were designed, synthesized, and evaluated for their binding affinities at dopamine (D₁, D₂ and D₃) and serotonin (5-HT_1A and 5-HT_2A) receptors. Most of the tetracyclic compounds exhibited higher affinities for D₂ and 5-HT_1A receptors than l-SPD, while compound 23e showed the highest K_i value of 7.54 nM at D₂ receptor which was 14 times more potent than l-SPD. Additionally, compounds 23d and 23e were more potent than l-SPD at D₃ receptor. According to the functional assays, 23d and 23e were demonstrated as full antagonists at D₁ and D₂ receptors and full agonists at 5-HT_1A receptor. Since the combination of D₂ antagonism and 5-HT_1A agonism is considered effective in treating both the positive and negative symptoms of schizophrenia, these novel compounds are implicated as potential therapeutic agents.     

Kinetics and mechanism of malonate replacement in bis(malonato)oxovanadate(IV) by oxalate

The kinetics of malonate replacement in bis- (malonato)oxovanadate(IV), [VO(mal)₂H₂O]²⁻(hereafter water molecule will be omitted), by oxalate has been studied by the stopped-flow method. The reaction was found to consist of two consecutive steps (k₁ and k₂: first-order rate constants) passing through a mixed ligand complex, [VO(mal)(ox)]²⁻. The rates for each step depended linearly on the concentrations of free oxalate species, Hox⁻ and ox²⁻. The second-order rate constants for the replacement by ox²⁻ were much larger in the k₁ step than in the k₂ step and the activation parameters were determined as follows: ΔH^≠= 43.5 ± 5.6 kJ mol⁻¹, ΔS±-53 ± 19 J K⁻¹ mol⁻¹ and ΔH≠= 43.6 ± 0.5 kJ mol⁻¹, δS≠ = -62 ± 2 J K^−l mol⁻¹ for the k₁ and k₂ steps, respectively. The volume of activation was determined to be -0.65 ± 0.75 cm³ mol⁻¹ at 20.2 °C by the high-pressure stopped-flow method for the apparent rate constants.     

Effects of elevated CO2 and O3 on N-cycling and N2O emissions: a short-term laboratory assessment

Background and aims

Elevated atmospheric CO₂ (eCO₂) and tropospheric O₃ (eO₃) can alter soil microbial processes, including those underlying N₂O emissions, as an indirect result of changes in plant inputs. In this study, effects of eCO₂ and eO₃ on sources of N₂O in a soybean (Glycine max (L.) Merr.) agroecosystem in Illinois (SoyFACE) were investigated. We hypothesized that increases in available C and anaerobic microhabitat under eCO₂ would stimulate N₂O emissions, with a proportionally larger increase in denitrification derived N₂O (N₂O_D) compared to nitrification plus nitrifier denitrification derived N₂O (N₂O_N+ND). We expected opposite effects under eO₃.

Methods

Isotopically labeled ¹⁵NH ₄ ¹⁴ NO₃ and ¹⁴NH ₄ ¹⁵ NO₃ were used to evaluate mineral N transformations, N₂O_D, and N₂O_N+ND in a 12-day incubation experiment.

Results

We observed minimal effects of eCO₂ and eO₃ on N₂O emissions, movement of ¹⁵?N through mineral N pools, soil moisture content and C availability. Possibly, altered C and N inputs by eCO₂ and eO₃ were small relative to the high soil organic C content and N-inputs via biological N₂-fixation, minimizing potential effects of eCO₂ and eO₃ on N-cycling.

Conclusion

We conclude that eCO₂ and eO₃ did not affect N₂O emissions in the short term. However, it remains to be tested whether N₂O emissions in SoyFACE will be unaltered by eCO₂ and eO₃ on a larger temporal scale under field conditions.     

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: We found that the charge recombination pathway of D⁺Q_AQ^?_B proceeds via the intermediate state D⁺Q^?_AQ_B, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product k_AD-times the fraction of reaction centers in the Q^?_AQ_B state) with the observed recombination rate, k^obs_{D⁺ →D}. The kinetic measurements were used to obtain the pH dependence (6.1 ? pH ? 11.7) of the free energy difference between the states Q^?_AQ_B and Q_AQ^?_B. At low pH (less than 9) Q_AQ^?_B is stabilized relative to Q^?_AQ_B by 67 meV, whereas at high pH Q^?_AQ_B is energetically favored. Both Q^?_A and Q^?_B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q^?_B provides the driving force for the forward electron transfer.     

Effect of temperature on the embryonic development of the plaice, Pleuronectes Platessa L. (teleostei)

In normalyears, eggs and prolarvae of the plaice (Pleuronectes platessa L.) in the southern North Sea develop within the temperature range 6.0–8.5 °C, although the water may at times be some degrees colder or warmer than this. The effect of temperature, t °C, on the embryonic development time, D days, has been investigated within the tolerated range 2.8–10.5 °C. Various models to express the observed curvilinear relationship between t and D have been considered, that giving the closest fit to the data being (t−t₀)(D−D₀) = k or $\text{D =}\text{k}\text{(t−t}{}_0\text{)+D}{}_0$. A method is given for the calculation of constants k, D₀, and t₀. The relationship may also be expressed by the equation D = a(t−t₀)^b where a and b are constants, but t₀ must in this case be found by iteration. At investigated temperatures in the range 4.1–10.5 °C the smallest eggs in a batch from a single source hatched first. Within the tolerated range, hatching prolarvae were substantially smaller at 10.5 °C than at the other temperatures. During the period of prolarval yolk utilization, growth is slower at the high temperatures, so that median temperatures of 6.5–8.0 °C are most efficient in terms of the relationship between growth in length and yolk utilization. Toward the end of the yolk-sac phase, the rate of yolk utilization declines unless a suitable external food source (e.g., Artemia nauplii) is provided.     

Electron-spin resonance studies of the bound iron-sulfur centers in Photosystem I II. Correlation of P-700 triplet production with urea/ferricyanide inactivation of the iron-sulfur clusters

Photosystem I charge separation in a subchloroplast particle isolated from spinach was investigated by electron spin resonance (ESR) spectroscopy following graduated inactivation of the bound iron-sulfur centers by urea-ferricyanide treatment. Previous work demonstrated a differential decrease in iron-sulfur centers A, B and X which indicated that center X serves as a branch point for parallel electron flow through centers A and B (Golbeck, J.H. and Warden, J.T. (1982) Biochim. Biophys. Acta 681, 77–84). We now show that during inactivation the disappearance of iron-sulfur centers A, B, and X correlates with the appearance of a spin-polarized triplet ESR signal with |D| = 279·10⁻⁴ cm⁻¹ and |E| = 39·10⁻⁴ cm⁻¹. The triplet resonances titrate with a midpoint potential of +380 ± 10 mV. Illumination of the inactivated particles results in the generation of an asymmetric ESR signal with g = 2.0031 and ΔH_pp = 1.0 mT. Deconvolution of the P-700⁺ contribution to this composite resonance reveals the spectrum of the putative primary acceptor species, A₀, which is characterized by g = 2.0033 ± 0.0004 and ΔH_pp = 1.0 ± 0.2 mT. The data presented in this report do not substantiate the participation of the electron acceptor A₁ in PS I electron transport, following destruction of the iron-sulfur cluster corresponding to center X. We suggest that A₁ is closely associated with center X and that this component is decoupled from the electron-transport path upon destruction of center X. The inability to photoreduce A₁ in reaction centers lacking a functional center X may result from alteration of the reaction center tertiary structure by the urea-ferricyanide treatment or from displacement of A₁ from its binding site.     

Partitioning of latent heat flux at a northern peatland

The partitioning of latent heat flux (Q_E) to vascular plant and moss surface components was assessed for a Sphagnum-dominated bog with a hummock–hollow surface having a sparse canopy of low shrubs. Results from porometry and eddy covariance measurements of Q_E showed evaporation from the moss surface ranged from greater than 50% of total Q_E early in the growing season to less than 20% after a dry period toward the end of the growing season. Both soil moisture and vapour pressure deficit (D_a) affected this partitioning with drier moss and peat, lower water table, and smaller D_a all reducing moss Q_E. Daily maximum moss Q_E ranged from greater than 200 W m⁻² early in the growing season to less than 100 W m⁻² during a dry period. In contrast, vascular contribution to total Q_E increased over the season from a daily maximum of about 150 W m⁻² to 250 W m⁻² due to increase in leaf area by leaf replacement and emergence and to drying of the moss surface. Porometry results showed average daily maximum conductance from bog shrubs was near 8 mm s⁻¹. These conductance values were smaller than those reported for vascular plants from more nutrient-rich wetlands. The effect of increases in D_a on vascular Q_E were moderated by decreases in stomatal conductance. At constant available energy, vascular leaf conductance was reduced by as much as 2 mm s⁻¹ and moss surface conductance was enhanced by up to 3 mm s⁻¹ by large D_a. Considering vascular and non-vascular water transport characteristics and frequency of water table position and given the observed variations of Q_E partitioning with water table location and moss and peat water content, it is suggested that modelling efforts focus on how dry hummocks and wet hollows each contribute to Q_E, especially as related to D_a and soil moisture dynamics.     

Kinetics and mechanism of aluminum(III)/siderophore ligand exchange: mono(deferriferrioxamine B)aluminum(III) formation and dissociation in aqueous acid solution

The kinetics and mechanism of a linear trihydroxamic acid siderophore (deferriferrioxamine B, H₄DFB⁺) ligand exchange with Al(H₂O)₆³⁺ to form mono(deferriferrioxamine B)aluminum(III) (Al(H₂O)₄H₃DFB)³⁺ have been investigated at 25 °C over the [H⁺] range 0.001−1.0 M and I = 2.0 M (HClO₄/NaClO₄) by ²⁷Al NMR. Kinetic results are consistent with Al(H₂O)₄(H₃DFB)³⁺ formation and dissociation proceeding through a parallel path mechanistic scheme involving Al(H₂O)₆³⁺(k₂/k₋₁) and Al(H₂O)₅(OH)²⁺(k₂/k₋₂) where k₁ = 0.13 M⁻¹ s⁻¹, k₋₁ = 8.7 × 10⁻³ M⁻¹ s⁻¹, k₂ = 2.7 × 10³ M⁻¹ s⁻¹, and k₋₂ = 9.6 × 10⁻⁴ s⁻¹. Relative complex formation rates at Al(H₂O)₆³⁺ and Al(H₂O)₅OH²⁺, and comparison with kinetic data for a series of synthetic hydroxamic acids, suggest that an interchange mechanism is operative. These results are also discussed in relation to kinetic data for the corresponding iron(III)-deferriferrioxamine B system.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Saxitoxin binding to neural membranes from pyrethroid susceptible and resistant tobacco budworm moths Heliothis virescens

1. [³H]Saxitoxin, a sodium channel probe, was found to bind reversibly with high affinity to a single class of noninteracting sites in membrane preparations from whole head homogenates of tobacco budworm, Heliothis virescens (F.), moths from a pyrethroid susceptible strain and from a pyrethroid resistant strain with nerve insensitivity and metabolic resistance mechanisms.2. No significant interstrain differences were detected in saturation (K_D, B_max) and kinetic (k₊₁, k₋₁) experiments.3. Also, five pyrethroids, two formamidines, and DDT had no significant effect on [³H]saxitoxin binding when tested at concentrations of 1 × 10⁻⁴ and 1 × 10⁻⁸ M.4. It appeared that a decreased density in sodium channels in resistant strain moths as compared with that in susceptible strain moths was not a factor in pyrethroid-induced resistance in this particular strain of tobacco budworm.     

Reversible linkage isomerization of pentaamminecobalt(III) complexes of urea and its N-methyl derivatives

The (NH₃)₅CoOC(NH₂)₂³⁺ ion is consumed in water according to the rate law k(obs.) = k₁ + k₂[OH⁻], where k₁ = 4.0 × 10⁻⁵ s⁻¹ and k₂ = 14.2 M⁻¹ s⁻¹ (0–0.1 M [OH⁻];μ = 1.1 M, NaClO₄, 25 °C). A hitherto unrecognized intramolecular O- to N- linkage isomerization reaction has been detected. In strongly acid solution only aquation to (NH₃)₅CoOH₂³⁺ is observed, but in 0.1–1.0 M [OH⁻], 7% of the directly formed products is the urea-N complex (NH₃)₅CoNHCONH₂²⁺ which has been isolated. In the neutral pH region a much greater proportion (25%) of the products is the urea-N species. These results are interpreted in terms of an urea-O to urea-N linkage isomerization reaction competing with hydrolysis for both spontaneous (k₁) and base-catalyzed (k₂) pathways; the rearrangement is not observed in strongly acidic solution (pH ⩽ 1) because the protonated N-bonded isomer (pK′_a ≈ 3) is unstable with respect to the O-bonded form. The appearance of the isomerization pathway as the pH is raised in the 0–6 region is commensurate with a rate increase which cannot be attributed to a contribution from the base catalysis term k₂[OH⁻]. It is argued that this observation establishes, for the spontaneous pathway, that hydrolysis and linkage isomerization are separate reaction pathways — there is no common intermediate. The product distribution and rate data lead to the complete rate law, k(obs.) = k₁ + k₂[OH⁻] = (k_s + k_ON) + (k_OH + k′_ON) [OH⁻] for the reactions of the O-bonded isomers, where k_s, k_OH are the specific rates for hydrolysis, and k_ON, k′_ON are the specific rates for O- to N-linkage isomerization, by spontaneous and base-catalyzed pathways respectively; k_ON = 1.3 × 10⁻⁵ s⁻¹ and k′_ON = 1.1 M⁻¹ s⁻¹ (μ = 1.0 M, NaClO₄, 25 °C). The O- to N- linkage isomerization has been observed also for complexes of N-methylurea, N,N-dimethylurea and N-phenylurea, but not for the N,N′-dimethylurea species. There is an approximately statistical relationship among the data for −NH₂ capture (versus H₂O), while −NHR and −NR₂ do not compete with water as nucleophiles for Co(III) in either the spontaneous or base-catalyzed hydrolysis processes. For each urea-O complex, O- to N-isomerization is a more significant parallel reaction in the spontaneous as opposed to the base-catalyzed pathway. This is interpreted as being indicative of more associative character in the spontaneous route to products, a conclusion supported by other evidence. Some activation parameter data have been recorded and the effect of the N-substitution on the rates of solvolysis (H₂O, Me₂SO) is discussed. The urea-N complexes have been isolated as their deprotonated forms, [(NH₃)₅CoNHCONRR′](ClO₄)₂·xH₂O (R,R′ = H, CH₃). They are kinetically inert in neutral to basic solution but in acid they protonate (H₂O, pK′_a 2–3; μ = 1.0 M, 25 °C) and then isomerize rapidly back to their O-bonded forms. Some solvolysis accompanies this N- to O-rearrangement in H₂O and Me₂SO. Specific rates and activation parameters are reported. The kinetic data follow a rate law of the form k_NO(obs.) = (k + k_NO)[H⁺]/(K′_a + [H⁺]) and the active species in the reaction is the protonated form; k, k_NO are the specific rates for hydrolysis and isomerization, respectively. Proton NMR data establish that the site of protonation (in Me₂SO) is the cobalt-bound nitrogen atom. For the unsubstituted urea species (NH₃)₅CoNH₂CONH₂³⁺, diastereotopic exo-NH₂ protons arising from restricted rotation about the CN bond are observed. The relevance to the mechanism of the linkage isomerization process is considered. ¹³C and ¹H NMR and electronic absorption spectral data are presented, and distinctions between linkage isomers and the solution structures (electronic and conformational) are discussed. The urea-N/urea-O complex equilibrium is governed by the relation K′_NO(obs.) = K′_NO[H⁺]/[H⁺](K′_a), where K′_NO is the equilibrium constant = [(NH₃₅Co(urea-O)³⁺]/[(NH₃)₅Co(urea-N)³⁺]. Values for K′_NO(=k_NO/k_ON = 260 and pK′_a ≈ 3 for the NH₂CONH₂ system are consistent with the stability of the N-isomer in feebly acidic to basic solution (e.g. pH 6, K′_NO(obs.) = 2.6 × 10⁻²) and instability in acid solution (e.g. pH 1, K′_NO(obs.) = 240). The equilibrium data for this and other urea complexes of (NH₃)₅Co(III) are contrasted with the result for the analogous Rh(III)NH₂CONH₂ system K′_NO ≈ 1).     

Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, k_ti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The k_ti values ranged between 10⁻¹⁰ and 10⁻¹⁵ ml cells⁻¹ h⁻¹, when estimated under various conditions. Moderate liquid agitation had a favorable effect on k_tf but agitation rates higher than 33 s⁻¹ (intergrated shear force) greatly decreased the value of k_ti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h⁻¹. The rate constant k_ti at temperatures of 10–15°C was as low as the detection limit (10⁻¹⁵ ml cells⁻¹ h⁻¹).     

Asymmetric total synthesis and identification of tetrahydroprotoberberine derivatives as new antipsychotic agents possessing a dopamine D1, D2 and serotonin 5-HT1A multi-action profile

An effective and rapid method for the microwave-assisted preparation of the key intermediate for the total synthesis of tetrahydroprotoberberines (THPBs) including l-stepholidine (l-SPD) was developed. Thirty-one THPB derivatives with diverse substituents on A and D ring were synthesized, and their binding affinity to dopamine D₁, D₂ and serotonin 5-HT_1A and 5-HT_2A receptors were determined. Compounds 18k and 18m were identified as partial agonists at the D₁ receptor with K_i values of 50 and 6.3 nM, while both compounds act as D₂ receptor antagonists (K_i = 305 and 145 nM, respectively) and 5-HT_1A receptor full agonists (K_i = 149 and 908 nM, respectively). These two THPBs compounds exerted antipsychotic actions in animal models. Further electrophysiological studies employing single-unit recording in intact animals demonstrated that 18k-excited dopaminergic (DA) neurons are associated with its 5-HT_1A receptor agonistic activity. These results suggest that these two compounds targeted to multiple neurotransmitter receptors may present novel lead drugs with new pharmacological profiles for the treatment of schizophrenia.