Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 μM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 μM, respectively. The critical micellar concentration (CMC) of ODPC was 200 μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T_c) of 41.5 °C and an enthalpy (?H) variation of 7.3 kcal mol⁻¹. The presence of 25 μM ODPC decreased T_c and ?H to 39.3 °C and 4.7 kcal mol⁻¹, respectively. ODPC at 250 μM destabilized the liposomes (36.3 °C, 0.46 kcal mol⁻¹). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.     

In rat dipeptidyl peptidase III,His is essential for catalysis,and Glu or Glu stabilizes the coordination bond between His or His and zinc ion

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K_m values or in the substrate specificity. However, the k_cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k_cat values of the mutants E507A and E512A were 2.38 and 3.88 s^− 1 toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10³ to 2 × 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.     

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K₁ 2,3-epoxide (K₁>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis–Menten constants of 1.20 μM for K₁>O and 260 μM for the active neutral form of THPP. The K_m value for K₁>O is in agreement with the value that we previously obtained using DTT (1.24 μM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K₁ and its previously reported base-catalyzed conversion to K₁, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.     

Equilibrium and structural studies on Co(II), Ni(II), Cu(II) and Zn(II) complexes with N-(2-benzimidazolyl)methyliminodiacetic acid: crystal structures of Ni(II) and Cu(II) complexes

Combined pH-metric, UV-Vis, ¹H NMR and EPR spectral investigations on the complex formation of M(II) ions (M=Co, Ni, Cu and Zn) with N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter H₂L) in aqueous solution at a fixed ionic strength, I=10⁻¹ mol dm⁻³, at 25 ± 1 °C indicate the formation of M(L), M(H₋₁L)⁻ and M₂(H₋₁L)⁺ complexes. Proton-ligand and metal-ligand constants and the complex formation equilibria have been elucidated. Solid complexes, [M(L)(H₂O)₂] · nH₂O (n=1 for M = Co and Zn, n=2 for M = Ni) and {Cu (μ-L) · 4H₂O}_n, have been isolated and characterized by elemental analysis, spectral, conductance and magnetic measurements and thermal studies. Structures of [Ni(L)(H₂O)₂] · 2H₂O and {Cu(μ-L) · 4H₂O}_n have been determined by single crystal X-ray diffraction. The nickel(II) complex exists in a distorted octahedral environment in which the metal ion is coordinated by the two carboxylate O atoms, the amino-N atom of the iminodiacetate moiety and the pyridine type N-atom of the benzimidazole moiety. Two aqua O atoms function as fifth and sixth donor atoms. The copper(II) complex is made up of interpenetrating polymeric chains of antiferromagnetically coupled Cu(II) ions linked by carboxylato bridges in syn-anti (apical-equatorial) bonding mode and stabilized via interchain hydrogen bonds and π-π stacking interactions.     

Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions

The hexahistidine (His₆)/nickel(II)-nitrilotriacetic acid (Ni²⁺-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His₆ proteins. Here we explore several applications of the NTA derivative, (Ni²⁺-NTA)₂-Cy3. This molecule binds our two model His₆ proteins, N-ethylmaleimide sensitive factor (NSF) and O⁶-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 10⁶ M⁻¹) and no effect on their activity. Its high specificity makes (Ni²⁺-NTA)₂-Cy3 ideal for detecting His₆ proteins in complex mixtures of other proteins, allowing (Ni²⁺-NTA)₂-Cy3 to be used as a probe in crude cell extracts and as a His₆-specific gel stain. (Ni²⁺-NTA)₂-Cy3 binding is reversible in 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (k_exchange ∼ 5 × 10⁻⁶ s⁻¹ with 0.2 μM labeled protein in the presence of 1 μM His₆ peptide). Labeling with (Ni²⁺-NTA)₂-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni²⁺-NTA)₂-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

Sulfated polysaccharides from Laminaria angustata: Structural features and in vitro antiviral activities

Sulfated polysaccharides potently inhibit the infectivity of herpes simplex virus (HSV) in cultured cells. In this study, we have analyzed sulfated xylogalactofucan and alginic acid containing fractions generated from Laminaria angustata, a marine alga. The xylogalactofucan that has apparent molecular mass of 56 ± 5 kDa and unusually low sulfate content contains, inter alia, 1,3-, 1,4- and 1,2-linked fucopyranosyl residues. The algin (molecular mass: 32 ± 5 kDa) contains gulo- (55.5%) and mannuronic (44.5%) acid residues. Introduction of sulfate groups enhanced the macromolecules capability to inhibit the infection of cells by HSV-1. The 50% inhibitory concentration (IC₅₀) values of these macromolecules against HSV-1 were in the range of 0.2-25 μg ml⁻¹ and they lacked cytotoxicity at concentrations up to 1000 μg ml⁻¹. The sulfate content appeared to be an important hallmark of anti-HSV-1 activity. Our results suggest the feasibility of inhibiting HSV attachment to cells by direct interaction of polysaccharides with viral particles.     

Influence of ammonium sulphate feeding time on fed-batch Arthrospira (Spirulina) platensis cultivation and biomass composition with and without pH control

Previous work demonstrated that a mixture of NH₄Cl and KNO₃ as nitrogen source was beneficial to fed-batch Arthrospira (Spirulina) platensis cultivation, in terms of either lower costs or higher cell concentration. On the basis of those results, this study focused on the use of a cheaper nitrogen source mixture, namely (NH₄)₂SO₄ plus NaNO₃, varying the ammonium feeding time (T = 7-15 days), either controlling the pH by CO₂ addition or not. A. platensis was cultivated in mini-tanks at 30 °C, 156 μmol photons m⁻² s⁻¹, and starting cell concentration of 400 mg L⁻¹, on a modified Schlösser medium. T = 13 days under pH control were selected as optimum conditions, ensuring the best results in terms of biomass production (maximum cell concentration of 2911 mg L⁻¹, cell productivity of 179 mg L⁻¹ d⁻¹ and specific growth rate of 0.77 d⁻¹) and satisfactory protein and lipid contents (around 30% each).     

α-Glucosidase inhibition and antihyperglycemic activity of prenylated xanthones from Garcinia mangostana

An ethanol extract of the fruit case of Garcinia mangostan, whose most abundant chemical species are xanthones, showed potent α-glucosidase inhibitory activity (IC₅₀ = 3.2 μg/ml). A series of isolated xanthones (1-16) demonstrated modest to high inhibition of α-glucosidase with IC₅₀ values of 1.5-63.5 μM. In particular, one hitherto unknown xanthone 16 has a very rare 2-oxoethyl group on C-8. Kinetic enzymatic assays with a p-nitrophenyl glucopyranoside indicated that one of them, compound (9) exhibited the highest activity (K_i = 1.4 μM) and mixed inhibition. Using, a physiologically relevant substrate, maltose, as substrate, many compounds (6, 9, 14, and 15) also showed potent inhibition which ranged between 17.5 and 53.5 μM and thus compared favorably with deoxynojirimycin (IC₅₀ = 68.8 μM). Finally, the actual pharmacological potential of the ethanol extract was demonstrated by showing that it could elicit reduction of postprandial blood glucose levels. Furthermore, the most active α-glucosidase inhibitors (6, 9, and 14) were proven to be present in high quantities in the native seedcase by a HPLC chromatogram.     

The role of ammonium in photoprotection against high irradiance in the red alga Grateloupia lanceola

Combined and/or interactive effects of inorganic nitrogen (as ammonium) and irradiance on the accumulation of nitrogenous compounds, like UV-absorbing mycosporine-like amino acids (MAAs), chlorophyll a and phycobiliproteins, were examined in the red alga Grateloupia lanceola (J. Agardh) J. Agardh in a high irradiance laboratory exposure and a subsequent recovery period under low light. Also, photosynthetic activity as in vivo chlorophyll fluorescence of photosystem II, i.e. optimum quantum yield (F_v/F_m), electron transport rate (ETR) and quantum efficiency, were examined. Photosynthetic activity, phycobiliproteins and internal nitrogen content declined during the 3-day PAR (photosynthetically active radiation; 600 μmol s⁻¹ m⁻²) and PAR + UVR (ultraviolet radiation; UVB 280–315 nm 0.8 W m⁻², UVA 315–400 nm 16 W m⁻²) exposure. Ammonium supplied in the culture medium (0, 100 and 300 μM NH₄Cl) modified the responses of the alga to high irradiance exposures in a concentration dependent manner, mainly with respect to recovery, as the highest recovery during a 10-day low light period was produced under elevated concentration of ammonium (300 μM). The recovery of photosynthetic activity and phycobiliproteins was enhanced in the algae previously incubated under PAR + UVR as compared to exposure to only PAR, suggesting a beneficial effect of UVR on recovery or photoprotective processes under enriched nitrogen conditions. However, the content of MAAs did not follow the same pattern and thus it could not be concluded as the cause of observed enhanced recovery.     

Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes

Two Expressed Sequence Tagged (EST) clones were identified from the Arabidopsis database as encoding putative cytidine deaminases. Sequence analysis determined that the two clones overlapped and encoded a single cDNA. This cytidine deaminase corresponds to theArabidopsis thaliana gene,cda1. The deduced amino acid sequence was more closely related to prokaryotic cytidine deaminases than to eukaryotic enzymes. The cDNA shares 44% amino acid identity with theEscherichia coli cytidine deaminase but only 26 and 27% identity with human and yeast enzymes. A unique zinc-binding domain of the Ecoli enzyme forms the active site. A similar putative zinc-binding domain was identified in the Arabidopsis enzyme based upon primary sequence similarities. These similarities permitted us to model the active site of the Arabidopsis enzyme upon that of the Ecoli enzyme. In this model, the active site zinc is coordinated by His⁷³, Cys¹⁰³, Cys¹⁰⁷, and an active site hydroxyl. Additional residues that participate in catalysis, Asn⁶⁴, Glu⁶⁶, Ala⁷⁸, Glu⁷⁹, and Pro¹⁰², are conserved between the Arabidopsis and Ecoli enzymes suggesting that the Arabidopsis enzyme has a catalytic mechanism similar to the Ecoli enzyme. The two overlapping ESTs were used to prepare a single, full-length clone corresponding to theA thaliana cda1 cDNA. This cDNA was subcloned into pProExHtb and expressed as a fusion protein with an N-terminal His₆ tag. Following purification on a Ni-NTA-Agarose column, the protein was analyzed for its kinetic properties. The enzyme utilizes both cytidine (K_m = 226 μand 2’-deoxycytidine (K_m= 49 μM) as substrates. The enzyme was unable to deaminate cytosine, CMP or dCMP. journal Paper Number J-18324 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa Project No. 3340.     

Structure–activity relationships of analogs of 3,4,5-trimethylfuran-2(5H)-one with germination inhibitory activities

Smoke-derived butenolide compounds have, in recent years, been shown to be important germination signaling molecules, which also affect seedling growth. The butenolide 3,4,5-trimethylfuran-2(5H)-one was previously isolated from plant-derived smoke and was found to significantly reduce the effect on germination by the highly active promotor karrikinolide (KAR₁, 3-methyl-2H-furo[2,3-c]pyran-2-one), another smoke-derived compound. In this study, 11 analogs of 3,4,5-trimethylfuran-2(5H)-one were synthesized and their effect on the germination of light-sensitive ‘Grand Rapids’ lettuce seeds (Lactua sativa cv. ‘Grand Rapids’) were evaluated. A concentration series (1 mM–1 μM) of the analogs were tested alone, or in combination with 0.01 μM KAR₁. Only two compounds were found to reduce the germination promotory effect of 0.01 μM KAR₁ in a similar manner as observed with 3,4,5-trimethylfuran-2(5H)-one, with activity ranging from 1 mM to 10 μM. Four compounds were found to have inhibitory activity at 1 mM and 100 μM. The retention of activity by some of the analogs may be useful for designing novel compounds with improved activity. Furthermore, understanding the structure–activity relationships of these compounds may be helpful in synthesizing molecular probes that can be used to further investigate the mechanism of action of these compounds in regulating seed germination.     

Structural and equilibrium studies on mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-benzimidazolyl)methyliminodiacetic acid and typical N, N donor ligands

Mixed ligand complexes: [Co(L)(bipy)] · 3H₂O (1), [Ni(L)(phen)] · H₂O (2), [Cu(L)(phen)] · 3H₂O (3) and [Zn(L)(bipy)] · 3H₂O (4), where L²⁻ = two -COOH deprotonated dianion of N-(2-benzimidazolyl)methyliminodiacetic acid (H₂bzimida, hereafter, H₂L), bipy = 2,2′ bipyridine and phen = 1,10-phenanthroline have been isolated and characterized by elemental analysis, spectral and magnetic measurements and thermal studies. Single crystal X-ray diffraction studies show octahedral geometry for 1, 2 and 4 and square pyramidal geometry for 3. Equilibrium studies in aqueous solution (ionic strength I = 10⁻¹ mol dm⁻³ (NaNO₃), at 25 ± 1 °C) using different molar proportions of M(II):H₂L:B, where M = Co, Ni, Cu and Zn and B = phen, bipy and en (ethylene diamine), however, provides evidence of formation of mononuclear and binuclear binary and mixed ligand complexes: M(L), M(H₋₁L)⁻, M(B)²⁺, M(L)(B), M(H₋₁L)(B)⁻, M₂(H₋₁L)(OH), (B)M(H₋₁L)M(B)⁺, where H₋₁L³⁻ represents two -COOH and the benzimidazole N₁-H deprotonated quadridentate (O⁻, N, O⁻, N), or, quinquedentate (O⁻, N, O⁻, N, N⁻) function of the coordinated ligand H₂L. Binuclear mixed ligand complex formation equilibria: M(L)(B) + M(B)²⁺ ? (B)M(H₋₁L)M(B)⁺ + H⁺ is favoured with higher π-acidity of the B ligands. For Co(II), Ni(II) and Cu(II), these equilibria are accompanied by blue shift of the electronic absorption maxima of M(II) ions, as a negatively charged bridging benzimidazolate moiety provides stronger ligand field than a neutral one. Solution stability of the mixed ligand complexes are in the expected order: Co(II) < Ni(II) < Cu(II) > Zn(II). The Δ log K_M values are less negetive than their statistical values, indicating favoured formation of the mixed ligand complexes over the binary ones.     

Ammonium uptake kinetics in root and leaf cells of Zostera marina L.

Ammonium uptake rates and the mechanism for ammonium transport into the cells have been analysed in Zostera marina L. In the cells of this species, a proton pump is present in the plasmalemma, which maintains the membrane potential. However, this seagrass shows a high-affinity transport mechanism both for nitrate and phosphate which is dependent on sodium and is unique among angiosperms. We have then analysed if the transport of another N form, ammonium, is also dependent of sodium. First, we have studied ammonium transport at the cellular level by measurements of membrane potentials, both in epidermal root cells and mesophyll cells. And second, we have monitored uptake rates in whole leaves and roots by depletion experiments. The results showed that ammonium is taken up by a high-affinity transport system both in root and leaf cells, although two different of kinetics could be discerned in mesophyll cells (with affinity constants of 2.2 ± 1.1 μM NH₄⁺, in the range 0.01-10 μM NH₄⁺, and 23.2 ± 7.1 μM NH₄⁺, at concentrations between 10 and 500 μM NH₄⁺). However, only one kinetic could be observed in epidermal root cells, which showed a K_m = 11.2 ± 1.0 μM NH₄⁺, considering the whole ammonium concentration range assayed (0.01-500 μM NH₄⁺). The higher affinity of leaf cells for ammonium was consistent with the higher uptake rates observed in leaves, with respect to roots, in depletion experiments at 10 μM NH₄⁺ initial concentration. However, when an initial concentration of 100 μM was assayed, the difference between uptake rates was reduced, but still being higher in leaves. Variations in proton or sodium-electrochemical gradient did not affect ammonium uptake, suggesting that the transport of this nutrient is not driven by these ions and that the ammonium transport mechanism could be different to the transport of nitrate and phosphate in this species.     

New bis(thiosemicarbazonate) gold(III) complexes inhibit HIV replication at cytostatic concentrations: potential for incorporation into virostatic cocktails

Four bis(thiosemicarbazonate)gold(III) complexes (1-4) with a general formula [Au(L)]Cl {L = L1, glyoxal-bis(N⁴-methylthiosemicarbazone); L2, glyoxal-bis(N⁴-ethylthiosemicarbazone); L3, diacetyl-bis(N⁴-methylthiosemicarbazone); L4, diacetyl-bis(N⁴-ethylthiosemicarbazone)} were synthesised and screened for activity against the human immunodeficiency virus (HIV). Complexes 1-4 were characterised using ¹H-NMR and IR spectroscopy; and their purity established by micronanalysis. Complex 3 inhibited viral infection of TZM-bl cells by 98% (IC₅₀ = 6.8 ± 0.6 μM) at a non toxic concentration of 12.5 μM while complex 4 inhibited infection of these cells by 72 and 98% (IC₅₀ = 5.3 ± 0.4 μM) at concentrations of 6.25 and 12.5 μM respectively. The mechanism of inhibition of infection in TZM-bl cells is presumably as a result of the cytostatic or anti-proliferative activity that was observed for complex 4 in real time cell electronic sensing (RT-CES) and carboxyflourescein succinimidyl ester (CFSE) analysis. Treatment of T lymphocytes from HIV infected individuals with 4 decreased CD4+ T cell expression (p = 0.0049) as demonstrated by multi-parametric flow cytometry without suppressing cytokine production. None of the ligands (L1-L4) demonstrated anti-viral activity, supporting the importance of metal (gold) complexation in these potential drugs. Complexes 3 and 4 were shown to have ideal lipophilicity values that were similar when shake flask (0.97 ± 0.5 and 2.42 ± 0.6) and in silico prediction (0.8 and 1.5) methods were compared. The activity and drug-like properties of complexes 3 and 4 suggests that these novel metal-based compounds could be combined with virus inhibitory drugs to work as cytostatic agents in the emerging class of anti-HIV drugs known as virostatics.     

Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (K_m, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the “threonine dehydrogenase and related Zn-dependent dehydrogenases” (COG1063). Metal analysis confirmed the presence of 2 Zn²⁺ atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys₃₇, His₇₀, and Glu₇₁, while the structural zinc site is probably composed of Cys₁₀₀, Cys₁₀₃, Cys₁₀₆, and Cys₁₁₄. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.