The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: A high field proton NMR study

High field proton (¹H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O₂. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcystine (1.00 or 3.00 × 10^-3 mol · dm^-3) or cysteine (1.00, 2.00 or 5.00 × 10^-3 mol · dm^-3), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in ¹H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field ¹H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.     

High Field Proton NMR Investigations of the Metabolic Profiles of Multidrug-Sensitive and -Resistant Leukaemic Cell Lines: Evidence for Diminished Taurine Levels in Multidrug-Resistant Cells

High field proton (¹H) nuclear magnetic resonance (NMR) spectroscopy has for the first time been employed to investigate and compare the metabolic profiles of vinblastine-sensitive and -resistant T-lymphoid leukaemic cell lines (CCRF-CEM and CEM/VLB₁₀₀ respectively) and evidence is presented for a significantly lower taurine content in the CEM/VLB₁₀₀ resistant subline when expressed relative to that of its drug-sensitive parental counterpart. These data suggest differences in the nature and relative involvements of taurine biosynthetic pathways between the two cell lines, a phenomenon that may be related to their differing sensitivities towards chemotherapeutic agents such as adriamycin which promote the generation of cytotoxic reactive oxygen species (ROS) in vivo. However, the ¹H NMR data obtained provided no evidence for an increased metabolic consumption of hypotaurine (a metabolic precursor of taurine with powerful ·OH radical scavenging properties) in CCRF-CEM cells since differences observed in the hypotaurine: taurine concentration ratio between the drug-sensitive and -resistant cell lines were not statistically significant. Furthermore, hypotaurine is unlikely to compete with alternative endogenous ·OH radical scavengers present such as lactate since its level in either of the two cell lines investigated (ca. 6.0 × 10^-8mol./10⁸ cells) is insufficient for it to act as an antioxidant in this context. The biochemical and therapeutic significance of these results are discussed.     

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.     

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.     

The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products

Hypochlorous acid (HOCl) is an oxygen-derived species involved in physiological processes related to the defence of the organism that may cause adverse effects when its production is insufficiently controlled. In order to examine its reactivity with potential scavenging molecules from the non steroidal anti-inflammatory drugs (NSAIDs) family, a competition assay based on para-aminobenzoic acid (PABA) chlorination was developed. The original optimised in vitro fluorimetric procedure offered the possibility to determine rate constants (k_s) for the reaction with HOCl in physiologically relevant conditions. The specificity of the system was improved by a liquid chromatography (LC) which allows the separation of the drugs and their oxidation products. After determination of the rate constant for PABA chlorination by HOCl (mean±SD in M_-1 s_-1: 4.3±0.3×10³), the applied mathematical model for a chemical competition permits to obtain linear curves from competition studies between several NSAIDs and PABA. Their slopes provided the following rate constants for the different studied drugs: tenoxicam: 4.0±0.7×10³, piroxicam: 3.6±0.7×10³, lornoxicam: 4.3±0.7×10³, meloxicam: 1.7±0.3×10⁴, nimesulide: 2.3±0.6×10². Meloxicam therefore reacted significantly faster than the other oxicams and nimesulide, which is the weakest scavenger of the studied series. The identification of some of the oxidation products by NMR or MS permitted to explore the reaction mechanism and to examine some aspects of the structure/activity relationships for the molecules of the same chemical family.     

Kinetics of the reactions of nitrogen dioxide with glutathione,cysteine, and uric acid at physiological pH

Nitrogen dioxide (NO₂^•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO₂^• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO₂^• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO₂^• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO₂^• with glutathione, cysteine, and urate were estimated as 2 × 10⁷, 5 × 10⁷, and 2 × 10⁷ M⁻¹ s⁻¹, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO₂^• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10⁷ M⁻¹ s⁻¹. The implications of these values are: (i) the lifetime of NO₂^• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO₂^• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO₂^• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO₂^•; and (v) reactions between NO₂^• and thiols/urate severely limit the likelihood of reaction of NO₂^• with NO• to form N₂O₃ in the cytoplasm.     

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca]0-dependent and independent release of [H]dopamine in rat striatal slices

Release of [³H]dopamine ([³H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [³H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca²⁺]₀-independent release of [³H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 ± 4.15 to 6.98 ± 1.83 mol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5′-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C. under glucose-free conditions was significantly reduced from 0.73 ± 0.04 to 0.44 ± 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 ± 0.11 mol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 ± 0.37 nmol/mg protein. The resting release was enhanced in a [Ca²⁺]₀-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 μM ouabain, inhibitor of Na⁺/K⁺-activated ATPase, enhanced the release of [³H]DA at rest in a [Ca²⁺]₀-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na⁺/K⁺-ATPase that results in reversal of the uptake processes and in [Ca²⁺]₀-independent [³H]DA release from the axon terminals.     

Thymus-dependency of the immune response of mice to a primary infection with the nematode Trichuris muris

Adult mice which had been thymectomized, irradiated and given stem-cell protection were incapable of making a self-cure response to a primary infection with the nematode Trichuris muris. The capacity to mount a self-cure response was restored by the injection of 2·5 × 10⁶ mesenteric lymph node lymphocytes or by 2·5 × 10⁸, but not 2·5 × 10⁷, thymocytes. Restoration of the ability to respond to sheep red blood cells was achieved with all three cell injections. Suppression of the immune response was also achieved by injection of ALS during the second week of infection and at intervals thereafter. The results of thymectomy and ALS treatment show that immunity to T. muris is dependent upon the presence of an intact thymus and thymus-dependent cell population.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2

Previously, we have shown that substitution of Pro² for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high μ-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [³H][(1S,2R)ACPC²]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [³H][(1S,2R)ACPC²]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K_d = 1.80 ± 0.21 nM and receptor density, B_max = 345 ± 27 fmol × mg protein⁻¹ at 25 °C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na⁺ and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [³H][(1S,2R)ACPC²]endomorphin-2 retained the μ-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of μ-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.     

Isolation and characterization of soluble sulfated polysaccharide from the red seaweed Gracilaria cornea

The composition, structure and rheological properties of a soluble sulfated polysaccharide from Gracilaria cornea (Brazilian red marine alga) were investigated. Agarocolloid yield, intrinsic viscosity, monosaccharide composition, sulfate and cation content as well as molecular weight were determined. The main polysaccharide components were 3,6-anhydrogalactose (24.7%) and galactose (64.6%). In addition, minor components such as 6-O-methyl-galactose (8.5%), glucose (1.5%), xylose (0.7%) and sulfated groups (4.8%) were detected. Comparison between sulfate contents determined by Fourier transform IR (FT-IR) spectroscopy and microelemental analysis was made. Data from ¹³C NMR and FT-IR provided evidence of sulfation in C-4 and C-6 of galactose. Sodium, calcium, magnesium and potassium cations were detected in the agarocolloid. The intrinsic viscosities were lower than typical values for agar in the same experimental conditions. No gelation in 1.5, 2.0 and 3.0% (w/v) aqueous solution was observed, even by cooling up to 4 °C. Gel permeation chromatography indicated two major polysaccharide fractions of M_pk 7.4×10⁴ and 1.8×10⁴ g/mol and a minor fraction of M_pk 2.1×10⁶ g/mol, probably a protein–polysaccharide complex.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Microbial characterization during composting of municipal solid waste

This study investigates the prevailing physico-chemical conditions and microbial community; mesophilic bacteria, yeasts and filamentous fungi, bacterial spores, Salmonella and Shigella as well as faecal indicator bacteria: total coliforms, faecal coliforms and faecal Streptococci, present in a compost of municipal solid waste. Investigations were conducted in a semi-industrial pilot plant using a moderate aeration during the composting process. Our results showed that: (i) auto-sterilization induced by relatively high temperatures (60–55°C) caused a significant change in bacterial communities. For instance, Escherichia coli and faecal Streptococci populations decreased, respectively, from 2×10⁷ to 3.1×10³ and 10⁷ to 1.5×10³ cells/g waste dry weight (WDW); yeasts and filamentous fungi decreased from 4.5×10⁶ to 2.6×10³ cells/g WDW and mesophilic bacteria were reduced from 5.8×10⁹ to 1.8×10⁷ bacteria/g WDW. On the other hand, the number of bacterial spores increased at the beginning of the composting process, but after the third week their number decreased notably; (ii) Salmonella disappeared completely from compost by the 25th day as soon as the temperature reached 60°C; and (iii) the bacterial population increased gradually during the cooling phase. While Staphylococci seemed to be the dominant bacteria during the mesophilic phase and at the beginning of the thermophilic phase, bacilli predominated during the remainder of the composting cycle. The appearance of gram-negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse. Compost sonication for about 3 min induced the inactivation of delicate bacteria, in particular gram-negatives. By contrast, gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in the compost.     

The effect of diamphenethide on protein synthesis by the liver fluke, Fasciola hepatica

The effect of the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 μg ml⁻¹) on the uptake and incorporation by adult Fasciola hepatica of radioactively labelled precursors of DNA, RNA and protein synthesis ([³H]thymidine, [³H]uridine and [³H] leucine, respectively) was measured by liquid scintillation counting. Comparison was made between the effects of DAMD and those of specific inhibitors of DNA, RNA and protein synthesis, namely, 5-fluorouracil, cordycepin and cycloheximide, respectively. DAMD caused a significant decrease in the overall uptake and incorporation of [³H]uridine by F. hepatica, decreased the incorporation of [³H] leucine and also caused a significant decrease in the overall protein content of the flukes. The effect of DAMD was similar to that of cycloheximide (1 × 10⁻³M), a potent inhibitor of protein synthesis, which also caused a significant decrease in the incorporation of [³H] leucine by the fluke and a decrease in the overall protein content of the fluke. Cordycepin (100 μg ml⁻¹) caused a significant decrease in the protein content of the fluke, but had no effect on the uptake or incorporation of [³H]uridine. 5-Fluorouracil (1 × 10⁻⁴ ) did not affect the uptake or incorporation of [³H]thymidine, nor did it decrease the protein content of the fluke. The results indicate that DAMD inhibits protein synthesis by F. hepatica, possibly by inhibiting RNA synthesis. The results are also consistent with previous morphological investigations involving DAMD.     

β-Lactam degradation catalysed by Cd ion in methanol

Kinetic schemes are established for degradation catalysed by Cd²⁺ ions in methanolic medium for penicillin G, penicillin V and cephalothin, a cephalosporin. Methanolysis of penicillin V and cephalothin occurs with the formation of a single substrate-metal ion intermediate complex, SM, while degradation of penicillin G occurs with the initial formation of two complexes with different stoichiometry, SM and S₂M. In each case, degradation is of first order with respect to SM with rate constant values equal to 0.079 min⁻¹, 0.120 min⁻¹ and 0.166 min⁻¹at 20, 25 and 30°C, respectively, for penicillin G; 0.061 min⁻¹ at 20°C for penicillin V; and 2.0×10⁻³ min⁻¹ at 20°C for cephalothin. Activation energy for the decomposition process of the SM intermediate for penicillin G was calculated to be about 5.5×10⁴ J/mol. Equilibrium constant values between SM compound and S₂M at 20°C (77.1 l/mol), 25°C (45.3 l/mol) and at 30°C (25.7 l/mol) were also calculated as well as the normal enthalpy of this equilibrium. With respect to the reaction products there is evidence that Cd²⁺ becomes part of their structure, forming complexes between Cd²⁺ and the product resulting from antibiotic methanolysis (L). Some characteristics of these complexes are discussed.     

Binding and the nature of Cu(II) ion interaction with nucleosomes

The energetics of Cu (II) ion binding to mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells was determined from their binding isotherms by equilibrium dialysis and pulse high frequency inductively coupled plasma atomic emission spectroscopy. Anticooperative binding of copper ions with normal and tumor mononucleosomes were observed under various NaCl concentrations (0.002; 0.02; 0.2 M). The binding constants of Cu(II) ion with normal mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.10×10⁴,5.22×10⁴,4.31×10⁴ respectively. The binding constants of Cu(II) ion with tumor mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.68×10⁴,6.12×10⁴,4.82×10⁴ respectively.     

Slippage of η-allenyl/propargyl to an η structure and tautomerization of η-propargyl to η-allenyl in ligand addition and substitution reactions of platinum(II) complexes

Reactions of [(PPh₃)₂Pt(η³-CH₂CCPh)]OTf with each of PMe₃, CO and Br⁻ result in the addition of these species to the metal and a change in hapticity of the η³-CH₂CCPh to η¹-CH₂CCPh or η¹-C(Ph)=C=CH₂. Thus, PMe₃ affords [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺, CO gives both [trans-(PPh₃)₂Pt(CO)(η¹-CH₂CCPh)]⁺ and [trans-(PPh₃)₂Pt(CO)(η¹-C(Ph)=C=CH₂)]⁺, and LiBr yields cis-(PPh₃)₂PtBr(η¹-CH₂CCPh), which undergoes isomerization to trans-(PPh₃)₂PtBr(η¹-CH₂CCPh). Substitution reactions of cis- and trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) each lead to tautomerization of η¹-CH₂CCPh to η¹-C(Ph)=C=CH₂, with trans-(PPh₃)₂PtBr(η¹-CH₂CCPh) affording [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]⁺ at ambient temperature and the slower reacting cis isomer giving [trans-(PPh₃)(PMe₃)₂Pt(η¹-C(Ph)=C=CH₂)]⁺ at 54 °C . All new complexes were characterized by a combination of elemental analysis, FAB mas spectrometry and IR and NMR (¹H, ¹³C{¹H} and ³¹P{¹H}) spectroscopy. The structure of [(PMe₃)₃Pt(η¹-C(Ph)=C=CH₂)]BPh₄·0.5MeOH was determined by single-crystal X-ray diffraction analysis.     

Topical delivery of retinyl ascorbate: 4. Comparative anti-oxidant activity towards DPPH

The free radical scavenging properties of retinyl ascorbate (RA-AsA) were determined by monitoring the decomposition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a function of time and in comparison with ascorbic acid (AsA), ascorbic acid palmitate (AsA-Pal), retinoic acid (RA), retinol (ROL) and retinol palmitate (Rol-Pal). The rate constant of RA-AsA (mean₃±SD) was 4.9±0.3 M^-1 s^-1, and indicated greater potency as an antioxidant compared to the rest of the test compounds (AsA 3.4±0.4 M^-1 s^-1, AsA-Pal, 2.9±0.2 M^-1 s^-1, RA 1.4±0.3 M^-1 s^-1, ROL 1.3±0.1 M^-1 s^-1, Rol-Pal exhibited insignificant activity). The decomposition rate constant of DPPH, 5±0.6 × 10^-8 M^-1 s^-1, in ethanol and BHA, 154±3 M^-1 s^-1 were both used as control. The compound RA-2-carboxy-2-hydroxy-ethanoate was isolated by prep-TLC and was identified, by ¹³C and ¹HNMR spectroscopy, as the major by-product from the reaction of RA-AsA with DPPH, which was also found to be potent antioxidant, 2.1±0.2 M^-1 s^-1. This suggests that oxidation of AsA moiety did not lead to the production of erythrulose species, which could cause deleterious modifications of cellular proteins.