Kinetics and equilibria of Ga(III)---thiocyanate complex formation. Mechanism of ligand substitution reactions of Ga(III) in aqueous solution

The kinetics and equilibria of complex formation by Ga(III) with NCS⁻ in aqueous solution have been measured over a range of acidities and temperatures, the contributing paths to the reaction resolved, and their rate constants and activation parameters determined. The hydrolysis equilibria required to carry out this resolution of kinetic behaviour have also been measured.

Unlike the other reported complexation reactions of Ga(III) in aqueous solution, the separate reaction pathways can be assigned with no ambiguity. At 25 °C and ionic strength 0.5 M, the observed forward rate constant for the complex formation is described by {k₁ + k₂K_1h/[H⁺] + k₃K_1hK_2h/[H⁺]²} M⁻¹ s⁻¹. For these conditions, the first and second successive hydrolysis constants of Ga(H₂O)₆³⁺ are given by pK_1h = 3.69 ± 0.01 and pK_2h = 3.74 ± 0.04. The rate constants corresponding to the reactions of the species Ga(H₂O)₆³⁺, Ga(H₂O)₅(OH)²⁺ and Ga(H₂O)₄(OH)₂⁺ with NCS⁻ are k₁ = 57 ± 4 M^{−1 −1}, k₂ = (1.08 ± 0.01) × 10⁵ M⁻¹ s⁻¹ and k₃ = 3 × 10⁶ M⁻¹ s⁻¹ respectively. The complexation equilibrium quotient [GaNCS²⁺]/([Ga³⁺][NCS⁻]) has been independently determined by spectrophotometric titration to be 20.8 ± 0.3 M⁻¹ at 25 °C and ionic strength 0.5 M.

These kinetic results lead to an interpretation of the data, and a reinterpretation of other data for aquo-Ga(III) complex formation kinetics from the literature which support the assignment of a dissociative interchange mechanism for these reactions rather than the associative activation mode sometimes proposed.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Catalysis of plastocyanin electron self-exchange by redox-inert multivalent cations

Electron self-exchange in solutions of the ‘blue’ copper protein plastocyanin is catalysed by the redox-inert multivalent cations Mg²⁺ or Co(NH₃)³⁺₆. Measurements of specific ¹H-NMR line broadening with 50% reduced solutions in the presence of these cations show that electron exchange proceeds through encounters of cation-protein complexes which dissociate at high ionic strength. In the presence of 8mM (5 equivalents/total protein) Co(NH₃)³⁺₆, with 10 mM cacodylate (pH^*6.0) as background electrolyte, the bimolecular rate constant at 25°C is 7 × 10⁴ M⁻¹·s⁻¹. For comparison, the ‘electrostatically screened’ rate constant measured in 0.1 M KCl in the absence of added multivalent cations is ˜ 4 × 10³ M¹·s⁻¹.

Plastocyanin Electron self-exchange NMR Protein-protein interaction Multivalent cation Blue copper protein     

Model reactions of a carbonylation catalyst: phosphite induced migratory CO insertion in [MeIr(CO)2I3]

Carbonylation of the anionic iridium(III) methyl complex, [MeIr(CO)₂I₃]⁻ (1) is an important step in the new iridium-based process for acetic acid manufacture. A model study of the migratory insertion reactions of 1 with P-donor ligands is reported. Complex 1 reacts with phosphites to give neutral acetyl complexes, [Ir(COMe)(CO)I₂L₂] (L = P(OPh)₃ (2), P(OMe)₃ (3)). Complex 2 has been isolated and fully characterised from the reaction of Ph₄As[MeIr(CO)₂I₃] with AgBF₄ and P(OPh)₃; comparison of spectroscopic properties suggests an analogous formulation for 3. IR and ³¹P NMR spectroscopy indicate initial formation of unstable isomers of 2 which isomerise to the thermodynamic product with trans phosphite ligands. Kinetic measurements for the reactions of 1 with phosphites in CH₂Cl₂ show first order dependence on [1], only when the reactions are carried out in the presence of excess iodide. The rates exhibit a saturation dependence on [L] and are inhibited by iodide. The reactions are accelerated by addition of alcohols (e.g. 18× enhancement for L = P (OMe)₃ in 1:3 MeOH-CH₂Cl₂). A reaction mechanism is proposed which involves substitution of an iodide ligand by phosphite, prior to migratory CO insertion. The observed rate constants fit well to a rate law derived from this mechanism. Analysis of the kinetic data shows that k₁, the rate constant for iodide dissociation, is independent of L, but is increased by a factor of 18 on adding 25% MeOH to CH₂Cl₂. Activation parameters for the k₁ step are ΔH^≠ = 71 (±3) kJ mol⁻, ΔS^≠ = −81 (±9) J mol⁻¹ K⁻¹ in CH₂Cl₂ and ΔH^≠ = 60(±4) kJ mol⁻¹, ΔS^≠ = −93(± 12) J mol⁻¹ K⁻¹ in 1:3 MeOH-CH₂Cl₂. Solvent assistance of the iodide dissociation step gives the observed rate enhancement in protic solvents. The mechanism is similar to that proposed for the carbonylation of 1.     

Rapid scavenging of peroxynitrous acid by monohydroascorbate

The reaction of peroxynitrous acid with monohydroascorbate, over the concentration range of 250 μM to 50 mM of monohydroascorbate at pH 5.8 and at 25°C, was reinvestigated and the rate constant of the reaction found to be much higher than reported earlier (Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. The kinetics of oxidation of L-ascorbic acid by peroxynitrite. Free Radic. Biol. Med. 18:85–92; 1995; Squadrito, G. L.; Jin, X.; Pryor, W. A. Stopped-flow kinetics of the reaction of ascorbic acid with peroxynitrite. Arch. Biochem. Biophys. 322:53–59; 1995). The new rate constants at pH 5.8 are k₁ = 1 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 500 s⁻¹ for 25°C and k₁ = 1.5 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 1 × 10³ s⁻¹ for 37°C. These values indicate that even at low monohydroascorbate concentrations most of peroxynitrous acid forms an adduct with this antioxidant. The mechanism of the reaction involves formation of an intermediate, which decays to a second intermediate with an absorption maximum at 345 nm. At low monohydroascorbate concentrations, the second intermediate decays to nitrate and monohydroascorbate, while at monohydroascorbate concentrations greater than 4 mM, this second intermediate reacts with a second monohydroascorbate to form nitrite, dehydroascorbate, and monohydroascorbate. EPR experiments indicate that the yield of the ascorbyl radical is 0.24% relative to the initial peroxynitrous acid concentration, and that this small amount of ascorbyl radicals is formed concomitantly with the decrease of the absorption at 345 nm. Thus, the ascorbyl radical is not a primary reaction product. Under the conditions of these experiments, no homolysis of peroxynitrous acid to nitrogen dioxide and hydroxyl radical was observed. Aside from monohydroascorbate's ability to “repair” oxidatively modified biomolecules, it may play a role as scavenger of peroxynitrous acid.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Inhibition of Escherichia coli glucosamine synthetase by novel electrophilic analogues of glutamine--comparison with 6-diazo-5-oxo-norleucine

A series of electrophilic glutamine analogues based on 6-diazo-5-oxo-norleucine has been prepared, using novel synthetic routes, and evaluated as inhibitors of Escherichia coli. glucosamine synthetase. The γ-dimethylsulphonium salt analogue of glutamine was found to be one of the most potent inactivators of this enzyme yet reported, with an apparent second order rate constant (k₂/K_i) of 3.5×10⁵ M⁻¹ min⁻¹.     

Structure of 7S seed globulin from Phaseolus vulgaris L. in solution

The structure of the 7S globulin from Phaseoulus vulgaris L in dilatue solutions has been studied by small angle X-ray scattering (SAXS), by quasi-elastic light scattering (Q ELS), by circular dichroism spectroscopy (c.d.), and by precise density measurements. The molar mass, the radius of gyration, the volume, the maximum dimension and the diffusion coefficient were determined as M = 1.45 × 10⁵ g mol⁻¹, R_G = 4.05 nm, V = 300- nm³, L = 13.0 nm and D_20,w⁰ = 4.5 × 10⁻⁷ cm² s⁻¹, respectively. The molecule has an asymmetrical shape with the dimensions 12.5 × 12.5 × 3.75 nm. The secondary structure of the 7S globulin is characterized by a small portion of -helical structure (14%) and a marked content of β-structure (18%).     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.     

Radiation crosslinked psyllium and polyacrylic acid based hydrogels for use in colon specific drug delivery

In order to utilize the psyllium husk, a medicinally important natural polysaccharide, to develop the hydrogels meant for the drug delivery, we have prepared psyllium and polyacrylic acid based polymeric networks by radiation-induced crosslinked copolymerization. Polymeric networks (hydrogels) thus formed were characterized with SEMs, FTIR and swelling studies. Swelling behavior of the hydrogels was studied as a function of monomer concentration in the hydrogels and temperature, pH and [NaCl] of the swelling medium. This paper discusses the swelling kinetics of the hydrogels and release dynamics of anticancer model drug 5-fluorouracil from the hydrogels for the evaluation of swelling and drug release mechanisms. It has been observed from the release dynamics of drug that diffusion exponent ‘n’ have 0.7, 0.8 and 0.7 values and gel characteristics constant ‘k’ have 9.13 × 10⁻³, 6.22 × 10⁻³ and 9.01 × 10⁻³ values for the release of 5-fluorouracil, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer. The values of the diffusion exponent show that the release of drug from drug-loaded hydrogels has occurred through Non-Fickian diffusion mechanism. It has also been observed from the swelling and release of drug in the different pH buffer that the polymer matrix is pH responsive and can be exploited for the delivery of anticancer drug to the colon.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

Electrospray mass spectrometry of trans-[Ru(NO)Cl(dpaH)2] (dpaH=2,2′-dipyridylamine) and ‘caged NO’, [RuCl3(NO)(H2O)2]: loss of HCl and NO from positive ions versus NO and Cl from negative ions

The positive ion electrospray mass spectrometry (ESI-MS) of trans-[Ru(NO)Cl)(dpaH)₂]Cl₂ (dpaH=2,2′-dipyridylamine), obtained from the carrier solvent of H₂O–CH₃OH (50:50), revealed 1+ ions of the formulas [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ (m/z=508), [Ru^IIICl(dpaH)(dpa⁻)]⁺ (m/z=478), [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472), [Ru^III(dpa)₂]⁺ (m/z=442), originating from proton dissociation from the parent [Ru^II(NO⁺)Cl(dpaH)₂]²⁺ ion with subsequent loss of NO (17.4% of dissociative events) or loss of HCl (82.6% of dissociative events). Further loss of NO from the m/z=472 fragment yields the m/z=442 fragment. Thus, ionization of the NH moiety of dpaH is a significant factor in controlling the net ionic charge in the gas phase, and allowing preferential dissociation of HCl in the fragmentation processes. With NaCl added, an ion pair, {Na[Ru^II(NO)Cl(dpa)₂]}⁺ (m/z=530; 532), is detectable. All these positive mass peaks that contain Ru carry a signature ‘handprint’ of adjacent m/z peaks due to the isotopic distribution of ¹⁰⁴Ru, ¹⁰²Ru, ¹⁰¹Ru, ⁹⁹Ru, ⁹⁸Ru and ⁹⁶Ru mass centered around ¹⁰¹Ru for each fragment, and have been matched to the theoretical isotopic distribution for each set of peaks centered on the main isotope peak. When the starting complex is allowed to undergo aquation for two weeks in H₂O, loss of the axial Cl⁻ is shown by the approximately 77% attenuation of the [Ru^II(NO⁺)Cl(dpaH)(dpa)]⁺ ion, being replaced by the [Ru^II(NO⁺)(H₂O)(dpa)₂]⁺ (m/z=490) as the most abundant high-mass species. Loss of H₂O is observed to form [Ru^II(NO⁺)(dpa)₂]⁺ (m/z=472). No positive ion mass spectral peaks were observed for RuCl₃(NO)(H₂O)₂, ‘caged NO’. Negative ions were observed by proton dissociation forming [Ru^II(NO)Cl₃(H₂O)(OH)]⁻ in the ionization chamber, detecting the parent 1− ion at m/z=274, followed by the loss of NO as the main dissociative pathway that produces [Ru^IIICl₃(H₂O)(OH)]⁻ (m/z=244). This species undergoes reductive elimination of a chlorine atom, forming [Ru^IICl₂(H₂O)(OH)]⁻ (m/z=208). The ease of the NO dissociation is increased for the negative ions, which should be more able to stabilize a Ru^III product upon NO loss.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

The versatile coordination chemistry of tricyanoethenolate, [(NC)2C=C(CN)O]

The reaction of meso-tetrakis (4-dimethoxyphenyl) porphinatomanganese(II), MnTP_OMeP, with TCNE (TCNE = tetracyanoethylene) leads to the formation of [MnTP_OMeP]⁺ [TCNE]⁻ and [MnTP_OMeP]⁺[OC(CN)C(CN)₂]⁻. The single-crystal X-ray structures of the latter as well as [Cu(bipy)₂Cl]⁺ [OC(CN)C(CN)₂]⁻ were determined. The former has a disordered [OC(CN)C(CN)₂]⁻ bridging via C and O between a pair of Mn^III sites, whereas the latter has an isolated [OC(CN)C(CN)₂]⁻ unbound to Cu^II. The IR characterization for μ₂-C,O bound [OC(CN)C(CN)₂]⁻ is at 2219m and 2196s (νCN) cm⁻¹ and at 1558s (νCO) cm⁻¹ while for unbound [OC(CN)C(CN)₂]⁻ it is at 2210m, 2203m, 2181m (νCN) cm⁻¹ and at 1583s (νCO) cm⁻¹.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

Kinetics of some electron-transfer reactions in biological Photosystems II. Study of intramolecular electron-transfer rates between ferredoxin-NADP reductase, NADP radical and oxidized ferredoxin

It has been shown by the pulse radiolysis technique that radiation-generated NADP free radicals (NADP·) first combine with ferredoxin-NADP reductase and then transfer the odd electron by a fast intramolecular process to the enzyme flavin moiety yielding the semiquinone (ferredoxin-NADP reductase, FNR-FADH·). The corresponding first-order rate constant k₁₅ varies with ionic strength from 2.6·10³ s⁻¹ at I = 0.66 M to 2.3·10⁴ s⁻¹ at I = 0.005 M In the presence of ferredoxin-NADP reductase-bound oxidized ferredoxin, the electron cascades, thus further reducing the ferredoxin. The transfer of the electron from the flavin semiquinone (ferredoxin-NADP reductase, FNR-FADH·) to the bound oxidized ferredoxin proceeds at a rate of k₁₈ = 2.36 s⁻¹. This process approaches an equilibrium condition which is in favor of the reverse reaction suggesting that k₋₁₈ > k₁₈.     

Synthesis, characterization and swelling studies of pH responsive psyllium and methacrylamide based hydrogels for the use in colon specific drug delivery

In order to utilize the psyllium husk a medicinally important natural polysaccharide and to develop the novel hydrogels meant for the colon specific drug delivery, we have prepared psyllium and methacrylamide based polymeric networks by using N,N′-methylenebisacrylamide (NN-MBAAm) as crosslinker and ammonium persulfate (APS) as initiator. To study various structural aspects of the polymeric networks thus formed psy-cl-poly(MAAm), these were characterized with SEMs, FTIR, TGA and swelling studies. The swelling studies of networks were carried out as a function of time, temperature, pH and [NaCl]. Equilibrium swelling has been observed to depend on both composition of the polymer and nature of swelling medium. Maximum percent swelling 1262 was observed for the polymeric network prepared with 19.45 × 10⁻³ mol/L of [NN-MBAAm] at 40 °C in 0.5 M NaOH solution. This article also discusses the release dynamics of tetracycline hydrochloride from the hydrogels, for the evaluation of the drug release mechanism and diffusion coefficients of drug from the polymer matrix. The effect of pH on the release pattern of tetracycline hydrochloride has been studied by varying the pH of the release medium. It has been observed from the release dynamics of drug from the hydrogels that the diffusion exponent ‘n’ have 0.477, 0.423 and 0.427 values and gel characteristic constant ‘k’ have 5.07 × 10⁻², 6.34 × 10⁻² and 6.38 × 10⁻² values, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer solution. The values the ‘n’ indicated that the Fickian type diffusion mechanism occurred for the release of tetracycline hydrochloride from drug loaded psy-cl-poly(MAAm) polymers in different release mediums. In Fickian type diffusion mechanism, the rate of polymer chain relaxation is more as compare to the rate of drug diffusion from these hydrogels and release behavior follows Fick’s law of diffusion. In each release medium, the values of the initial diffusion coefficient ‘D_i’ for the release of tetracycline hydrochloride was higher than the values of late time diffusion coefficient ‘D_L’ indicating that in the start, the diffusion of drug from the polymeric matrix was faster as compare to the latter stages.     

Dopamine receptors in human foetal brains: Characterization, regulation and ontogeny of [H]spiperone binding sites in striatum

Eighteen corpora striata from normal human foetal brains ranging in gestational age from 16 to 40 weeks and five from post natal brains ranging from 23 days to 42 years were analysed for the ontogeny of dopamine receptors using [³H]spiperone as the ligand and 10 mM dopamine hydrochloride was used in blanks. Spiperone binding sites were characterized in a 40-week-old foetal brain to be dopamine receptors by the following criteria: (1) It was localized in a crude mitochondrial pellet that included synaptosomes; (2) binding was saturable at 0.8 nM concentration; (3) dopaminergic antagonists spiperone, haloperidol, pimozide, trifluperazine and chlorpromazine competed for the binding with IC₅₀ values in the range of 0.3–14 nM while agonists—apomorphine and dopamine gave IC₅₀ values of 2.5 and 10 μM, respectively suggesting a D₂ type receptor.

Epinephrine and norepinephrine inhibited the binding much less efficiently while mianserin at 10 μM and serotonin at 1 mM concentration did not inhibit the binding. Bimolecular association and dissociation rate constants for the reversible binding were 5.7 × 10⁸ M⁻¹ min⁻¹ and 5.0 × 10⁻² min⁻¹, respectively. Equilibrium dissociation constant was 87 pM and the K_D obtained by saturation binding was 73 pM.

During the foetal age 16 to 40 weeks, the receptor concentration remained in the range of 38–60 fmol/mg protein or 570–1080 fmol/g striatum but it increased two-fold postnatally reaching a maximum at 5 years Significantly, at lower foetal ages (16–24 weeks) the [³H]spiperone binding sites exhibited a heterogeneity with a high (K_D, 13–85 pM) and a low (K_D, 1.2–4.6 nM) affinity component, the former accounting for 13–24% of the total binding sites. This heterogeneity persisted even when sulpiride was used as a displacer. The number of high affinity sites increased from 16 weeks to 24 weeks and after 28 weeks of gestation, all the binding sites showed only a single high affinity.

GTP decreased the agonist affinity as observed by dopamine competition of [³H]spiperone binding in 20-week-old foetal striata and at all subsequent ages. GTP increased IC₅₀ values of dopamine 2 to 4.5 fold and Hill coefficients were also increased becoming closer to one suggesting that the dopamine receptor was susceptible to regulation from foetal life onwards.     

New herbicide-binding site in the photosynthetic electron-transport chain: Competitive herbicide binding at the photosystem I phylloquinone-(vitamin K1)-binding site

The binding of herbicides to the phylloquinone-(primary electron acceptor A₁)-binding site in green plant photosystem (PS) I reaction centers is shown. Dissociation constants (K_d) of various herbicides to the phylloquinone-binding site were estimated by analyzing their competitive inhibition of the reconstitution of the phylloquinone analogue, menadione (vitamin K₃), to the phylloquinone-extracted spinach PS I particles. The phylloquinone-binding site was found to bind o-phenanthroline (K_d = 1.2 × 10⁻⁴ M), but only weak binding was observed with atrazine (K_d > 10⁻² M), although both are known to bind specifically to the quinone-(Q_B)-binding site in reaction centers of purple photosynthetic bacteria or PS II. The inhibitors of the cytochrome b/c₁(ƒ) complex, myxothiazol (K_d=9.5 × 10⁻⁶ M) or antimycin A (K_d = 2.8 × 10⁻⁶ M), also strongly bound to the phylloquinone site. This is the first report showing that the PS I reaction center complex also has a herbicide-binding site, although the site is probably not sensitive in vivo to these herbicides due to its higher affinity for phylloquinone than herbicides. The inhibitor specificity of the PS I phylloquinone site is different from that of the other quinone-functioning sites in the photosynthetic or respiratory electron-transfer chain, suggesting it to have a unique structure.