Optimizing the formulation of a myoglobin molecularly imprinted thin-film polymer--formed using a micro-contact imprinting method

Thin-film myoglobin molecularly imprinted polymers have been fabricated using a micro-contact approach. By initially selecting the cross-linker on the basis of it having a minimal recognition for the template and using this as a starting point for functional monomer selection, we have produced myoglobin imprinted polymers with exceptionally high selectivities.

The affinity of the polymers, for myoglobin, when prepared with a variety of different cross-linkers and no functional monomer was evaluated. Of these, tetraethylene glycol dimethacrylate (TEGDMA) exhibited the lowest affinity for the template species. Methyl methacrylate (MMA) was chosen as the functional monomer as when it was used in conjunction with TEGDMA, it exhibited maximum selectivity for the template compared to polymers made with other functional monomers.

With a MMA to TEGDMA ratio of 1 to 3, the myoglobin molecularly imprinted polymer adsorbed 15.03 ± 0.89 × 10⁻¹¹ mole/cm² of template from a 5.68 × 10⁻⁷ M myoglobin solution, compared to 2.58 ± 0.02 × 10⁻¹¹ mole/cm² for a polymer of similar composition, but formed in the absence of a template. Various washing conditions, using alkaline media to remove the template, were investigated. An extraction solvent comprising 2 wt.% SDS and 0.6 wt.% NaOH used at 80 °C for 30 min was shown to give the highest imprinting factor i.e. 5.83 with 72.82% myoglobin removal.

The saturation kinetics of template binding to the thin-film MIP were examined and found to display a simple two-phase profile typical of non-cooperative binding. A Scatchard binding plot showed the dissociation constant (K_d) for the specific binding phase to be 3.4 × 10⁻⁷ M and the binding site capacity to be 7.24 × 10⁻¹¹ mole/cm². For the non-specific binding phase, K_d was found to be 1.355 × 10⁻⁵ M and the binding site capacity was determined as 9.62 × 10⁻¹⁰ mole/cm².

Selectivity experiments were carried out in both single protein and binary protein systems all using a total protein concentration of 5.68 × 10⁻⁷ M. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5, respectively. While, in binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was, respectively, 94.18, 98.21 and 61.09%. Rebinding in natural biological matrices, i.e. human serum or urine, showed the imprinted films to have significantly greater uptake than non-imprinted films. Re-binding in undiluted urine was found to be a facile process, with the imprinting factor, i.e. the ratio of MIP to NIP binding, being determined as 37.4.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

The presence of cortisol receptors in the human amnion

Cytosol extracts of human amnion tissue contained high affinity binding of cortisol (K_a = 2.48 ± 1.06 × 10⁹ M⁻¹; N = 30) and low capacity binding of cortisol (N_max = 279 ± 15.5 fmol mg⁻¹ protein). Kinetic studies of cortisol binding resulted in a similar value of K_a to that obtained by Scatchard analysis. Nuclear extracts of amnion tissue contained high affinity binding of cortisol (K_a = 5.8 ± 1.91 × 10⁷ M⁻¹) and low binding capacity (N_max = 91.4±21.4 fmol mg⁻¹ protein). K_a values were an order of magnitude higher in cytosol than in blood serum when amnion and blood were obtained from the same individuals. Differences in competitive ligand binding, especially dexamethasone, were observed between the amnion receptor and transcortin in serum. Gel permeation chromatography gave only one peak at 320 kDa for amnion receptor and only one peak at 48 kDa for transcortin from serum. When amnion tissue was incubated with or without cortisol, cytosol receptor activity was significantly lower in cortisol treated tissue than in control. The nuclear extracted receptor activity was significantly higher in cortisol treated tissue than control. The K_a values from cortisol treated tissue were significantly lower from control. Together the data support the presence of a specific cortisol receptor in the human amnion that is different from transcortin.     

Effect of melanin on netilmicin-induced inhibition of collagen biosynthesis in human skin fibroblasts

It is known that various drugs form complexes with melanins and that melanins are abundant constituents of the inner ear. In this study, we determined whether the aminoglycoside antibiotic, netilmicin, interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. The obtained results indicate that netilmicin forms stable complexes with melanin characterized by the association constants K₁  10⁶ M⁻¹ and K₂  10³ M⁻¹. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for aminoglycoside-induced inhibition of collagen biosynthesis. We found that netilmicin strongly induced inhibition of prolidase activity (IC₅₀ < 5 μM) and collagen biosynthesis (IC₅₀  10 μM). At 10 μM concentration of netilmicin, prolidase activity in human skin fibroblasts was inhibited by about 80% and DNA biosynthesis—only by about 25%. Melanin at 100 μg/mL produced about 30% inhibition of collagen biosynthesis and about 30% inhibition of prolidase activity in cultured fibroblasts. However, the addition of melanin (100 μg/mL) to netilmicin-treated cells (10 μM) restored the prolidase activity in fibroblasts to almost 100% of control values and partially reversed the inhibitory action of the drug on collagen and DNA biosynthesis. The data suggest that the ability of netilmicin to form stable complexes with melanin may prevent its toxicity on prolidase activity and collagen biosynthesis.     

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k_inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 °C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5–4.6)×10⁴ M⁻¹ s⁻¹) > BHT-OOH (0.7–1.9×10⁴ M⁻¹ s⁻¹) > BHT-CHO ((0.4–1.7)×10⁴ M⁻¹ s⁻¹) > BHT ((0.1–0.2)×10⁴ M⁻¹ s⁻¹). The k_inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.     

Purification and characterization of a Na/K dependent alginate lyase from turban shell gut Vibrio sp. YKW-34

An alginate lyase with high specific enzyme activity was purified from Vibrio sp. YKW-34, which was newly isolated from turban shell gut. The alginate lyase was purified by in order of ion exchange, hydrophobic and gel filtration chromatographies to homogeneity with a recovery of 7% and a fold of 25. This alginate lyase was composed of a single polypeptide chain with molecular mass of 60 kDa and isoelectric point of 5.5–5.7. The optimal pH and temperature for alginate lyase activity were pH 7.0 and 40 °C, respectively. The alginate lyase was stable over pH 7.0–10.0 and at temperature below 50 °C. The alginate lyase had substrate specificity for both poly-guluronate and poly-mannuronate units. The k_cat/K_m value for alginate (heterotype) was 1.7 × 10⁶ s⁻¹ M⁻¹. The enzyme activity was completely lost by dialysis and restored by addition of Na⁺ or K⁺. The optimal activity exhibited in 0.1 M of Na⁺ or K⁺. This enzyme was resistant to denaturing reagents (SDS and urea), reducing reagents (β-mercaptoethanol and DTT) and chelating reagents (EGTA and EDTA).     

Endogenous heptapeptide Met-enkephalin-Gly-Tyr binds differentially to duplicate delta opioid receptors from zebrafish

Met-enkephalin-Gly-Tyr (MEGY) is an endogenous peptide that binds to opioid sites in zebrafish and in rat brain homogenates. The aim of this work is to characterize the binding profile of this opioid ligand on two duplicate delta receptors from zebrafish, ZFOR1 and ZFOR4. Our results show that, while ZFOR1 presents one single binding site for [³H]-MEGY (K_D = 4.0 ± 0.4 nM), the experimental data from ZFOR4 fit better to the two-site binding model (K_D1 = 0.8 ± 0.2 nM and K_D2 = 30.2 ± 10.2 nM). Two other MEGY synthetic analogues, (D-Ala²)-MEGY and (D-Ala², Val⁵)-MEGY were also prepared and tested, together with the original peptide MEGY and other opioid ligands, in competition binding assays. While these peptides presented K_i values on the nanomolar range when using [³H]-MEGY as radioligand, these parameters were two orders higher in competition binding assays with the antagonist [³H]-diprenorphine. Functional [³⁵S]GTPγS stimulation analysis has revealed that these two receptors can be activated by several opioid agonists. Our results prove that although the MEGY peptide acts as an agonist on ZFOR1 and ZFOR4, there are subtle pharmacological differences between these two delta opioid receptors from zebrafish.     

Modifying the N-terminus of polyamides: PyImPyIm has improved sequence specificity over f-ImPyIm

Seven N-terminus modified derivatives of a previously published minor-groove binding polyamide (f-ImPyIm, 1) were synthesized and the biochemical and biophysical chemistry evaluated. These compounds were synthesized with the aim of attaining a higher level of sequence selectivity over f-ImPyIm (1), a previously published strong minor-groove binder. Two compounds possessing a furan or a benzofuran moiety at the N-terminus showed a footprint of 0.5 μM at the cognate ACGCGT site (determined by DNase I footprinting); however, the specificity of these compounds was not improved. In contrast, PyImPyIm (4) produced a footprint of 0.5 μM but showed a superior specificity using the same technique. When evaluated by thermal melting experiments and circular dichroism using ACGCGT and the non-cognate AAATTT sequence, all compounds were shown to bind in the minor-groove of DNA and stabilize the cognate sequence much better than the non-cognate (except for the non-amido-compound that did not bind either sequence, as expected). PyImPyIm (4) was interesting as the ΔT_m for this compound was only 4 °C but the footprint was very selective. No binding was observed for this compound with a third DNA (non-cognate, ACCGGT). ITC studies on compound 4 showed exothermic binding with ACGCGT and no heat change was observed for titrating the compound to the other two DNA sequences. The heat capacity (ΔC_p) of the PIPI/ACGCGT complex calculated from the hydrophobic interactions and SASA calculations was comparable to the experimental value obtained from ITC (−146 cal mol⁻¹ K⁻¹). SPR results provided confirmation of the sequence specificity of PyImPyIm (4), with a K_eq value determined to be 7.1 × 10⁶ M⁻¹ for the cognate sequence and no observable binding to AAATTT and ACCGGT. Molecular dynamic simulations affirmed that PyImPyIm (4) binds as a dimer in an overlapped conformation, and it fits snugly in the minor-groove of the ACGCGT oligonucleotide. PyImPyIm (4) is an especially interesting molecule, because although the binding affinity is slightly reduced, the specificity with respect to f-ImPyIm (1) is significantly improved.     

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.     

Kinetic studies on 1:1 electron transfer reactions involving blue copper proteins : 8. Reactions of plastocyanin and azurin with cytochrome c and high potential iron-sulfur protein

Rate constants determined by the stopped-flow method for four protein-protein reactions at 25°C, pH's in the range 5.8–7.5. I = 0.10 M (NaCI), are as follows: cytochrome c(II) with plastocyanin, PCu(II). 1.5 × 10⁶ M⁻¹ sec⁻¹, pH 7.6; high-potential iron-sulfur protein (Hipip) with PCu(II), 3.7 × 10⁵ M⁻¹sec⁻¹. pH 5.8; cytochrome c(II) with azurin, ACu(ll). 6.4 × 10³ M⁻¹sec⁻¹, pH 6.1; Hipip with ACu(II), 2.2 × 10⁵ M⁻¹sec⁻¹, pH 5.8. Activation parameters have been determined for all four reactions; they indicate higher enthalpy requirements and less negative entropy requirements for the PCu(II) as opposed to ACu(II) reactions. Equilibrium constants K for association prior to electron transfer are < 150 M⁻¹ for the cytochrome c(II) reduction of PCu(II) (estimated charges 8 + and 9-,respectively), and < 300 M⁻¹ for the other reactions, indicating no favorable interactions. Rate constants have been analyzed in terms of the simple Marcus theory, which has previously given an excellent fit to thirteen protein-protein reactions considered by Wherland and Pecht. No similar correlation exists in the present studies, and calculated rate constants differ by orders of magnitude from experimentally determined values.     

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10⁴ M⁻¹. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H₂O₂.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

Kinetics of laccase-catalysed TEMPO oxidation

The oxidation of TEMPO (2,2,6,6-tetramethyl-piperidine-1-oxyl radical) has been studied in the presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL) in buffer solution pH 4.5–7.3 and at 25 °C. At pH 5.5 the oxidation constant calculated from the initial rate of TEMPO oxidation was 1.7 × 10⁴, 1.4 × 10³, 7.8 × 10² and 5.2 × 10² M⁻¹ s⁻¹ for rPpL, rRsL, rCcL and rMtL, respectively. The maximal activity of rPpL-catalysed TEMPO oxidation was at pH 5.0. The pK_a obtained in neutral pH range was 6.2. The reactivity of laccases is in a good agreement with laccases copper type I redox potential.

TEMPO oxidation rate increased 541 times in the presence of 10-(3-propylsulfonate) phenoxazine (PSPX). The model of synergistic TEMPO and PSPX oxidation was proposed. Experimentally obtained rate constants for rPpL-catalysed PSPX oxidation were in a good agreement with those calculated from the synergistic model, therefore confirming the feasibility of the model. The acceleration of TEMPO oxidation with high reactive laccase substrates opens new possibilities for TEMPO application as a mediator.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Structure of Co2(CO)6(dppm) and Co2(CO)5(CHCO2Et)(dppm) (dppm = Ph2PCH2PPh2) and exchange reaction with CO: An experimental and computational study

Crystal structures of Co₂(CO)₆(dppm) (1) and Co₂(CO)₅(CHCO₂Et)(dppm) (2) (dppm = Ph₂PCH₂PPh₂) show asymmetry with respect to the orientation of the phenyl groups in 1 and owing to the bridging ethoxycarbonylcarbene ligand in 2. The effect of this asymmetry was recognized in the solid-state ³¹P NMR spectra of 1 and 2 and in the solid-state and solution ¹³C NMR spectra of 2 as well, but not in the solid-state and solution ¹³C NMR spectra of 1. In CH₂Cl₂ solution under an atmosphere of ¹³CO, the CO ligands of both complexes exchange with ¹³CO. The overall rate of ¹³CO exchange at 10 °C was found to be k_obs = 0.107 × 10⁻³ s⁻¹ for 1 and k_obs = 0.243 × 10⁻³ s⁻¹ for 2. Two-layered ONIOM(B3LYP/6-31G(d):LSDA/LANL2MB) studies revealed fluxional behavior of 1 with rather small barriers of activation of the rearrangements. Four possible isomers have been computed for 2, close to each other energetically.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

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.     

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.     

Study of the interaction of artemisinin with bovine serum albumin

The study on the interaction of artemisinin with bovine serum albumin (BSA) has been undertaken at three temperatures, 289, 296 and 303 K and investigated the effect of common ions and UV C (253.7 nm) irradiation on the binding of artemisinin with BSA. The binding mode, the binding constant and the protein structure changes in the presence of artemisinin in aqueous solution at pH 7.40 have been evaluated using fluorescence, UV–vis and Fourier transform infrared (FT-IR) spectroscopy. The quenching constant K_q, K_sv and the association constant K were calculated according to Stern–Volmer equation based on the quenching of the fluorescence of BSA. The thermodynamic parameters, the enthalpy (ΔH) and the entropy change (ΔS) were estimated to be −3.625 kJ mol⁻¹ and 107.419 J mol⁻¹ K⁻¹ using the van’t Hoff equation. The displacement experiment shows that artemisinin can bind to the subdomain IIA. The distance between the tryptophan residues in BSA and artemisinin bound to site I was estimated to be 2.22 nm using Föster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of enough common ions and UV C exposure, indicates that common ions and UV C irradiation have effect on artemisinin binding to BSA.     

Tetrahydrocortisol-apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

Tetrahydrocortisol stimulates DNA and protein biosynthesis in hepatocytes only when it enters the complex with apolipoprotein A-I. Tetrahydrocortisol–apolipoprotein A-I (THC–apoA-I) complex specifically interacts with eukaryotic DNA isolated from rat liver. In the process of interaction, rupture of hydrogen bonds between the pairs of nitrous bases occurs with the formation of single-stranded DNA structures. In such state DNA forms complexes with DNA-dependent RNA-polymerase. The most probable site of binding the tetrahydrocortisol–apolipoprotein A-I complex with DNA is the sequence of CC(GCC)_n type entering the structure of many genes, among them the structure of human apolipoprotein A-I gene. Oligonucleotide of this type has been synthesized. Association constant (K_ass) of it with tetrahydrocortisol–apolipoprotein A-I complex was shown to be 1.66×10⁶ M⁻¹. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. It was assumed that in the GC-pairs of the given sequence tetrahydrocortisol itself participates in the formation of hydrogen bonds with cytosine, favoring their rupture with complementary base—guanine.