Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase

We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. K(m) for L-asparagine is 17 ± 0.9 μM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. colil-asparaginase (EcA) as the reference substance at similar conditions.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Recombinant intracellular <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> L-asparaginase with low L-glutaminase activity and antiproliferative effect

The recombinant producer strain expressing Rhodospirillum rubrum L-asparaginase (RrA) has been obtained and a purification procedure of RrA has been developed. The purified enzyme, RrA, has the following biochemical and catalytic characteristics: K_m for L-Asn of 0.22 mM, pH optimum at 9.2; temperature optimum at 54°C, pI = 5.1. RrA exhibited a significant cytotoxic effect towards the following cell lines: K562 (IC₅₀ = 1.80 U/mL), DU145 (IC₅₀ = 9.19 U/mL), and MDA-MB-231 (IC₅₀ = 34.62 U/mL). Comparative analysis employing E. coli L-asparaginase II type (EcA) and Erwinia carotovora L-asparaginase (EwA) has shown that the enzyme cytotoxicity towards these cell lines decreased in the following order: EcA > RrA > EwA. Daily administration of RrA (4000 U/kg) to L5178y bearing mice for 10 days (total dose of 40000 U/kg) showed T/C = 172. Data obtained suggest that RrA may be referred to intracellular L-asparaginases with low L-glutaminase activity and marked antiproliferative effect.     

Production,purification, characterization,antioxidant and antiproliferative activities of extracellular L-asparaginase produced by Fusarium equiseti AHMF4

L-Asparaginase is an antileukemic agent that depletes L-asparagine “an important nutrient for cancer cells” through the hydrolysis of L-asparagine into L-aspartic acid and ammonia leading to leukemia cell starvation and apoptosis in susceptible leukemic cell populations. Moreover currently, bacterial L-asparaginase has been limited by problems of lower productivity, stability, selectivity and a number of toxicities along with the resistance towards bacterial L-asparaginase. Then the current work aimed to provide pure L-asparaginase with in-vitro efficacy against various human carcinomas without adverse effects related to current L-asparaginase formulations. Submerged fermentation (SMF) bioprocess was applied and improved to maximize L-asparaginase production from Fusarium equiseti AHMF4 as alternative sources of bacteria. The enzyme production in SMF was maximized to reach 40.78 U mL⁻¹ at the 7th day of fermentation with initial pH 7.0, incubation temperature 30 °C, 1.0% glucose as carbon source, 0.2% asparagine as nitrogen source, 0.1% alanine as amino acid supplement and 0.1% KH₂PO₄. The purification of AHMF4 L-asparaginase yielded 2.67-fold purification and 48% recovery with final specific activity of 488.1 U mg⁻¹ of protein. Purified L-asparaginase was characterized as serine protease enzyme with molecular weight of 45.7 kDa beside stability at neutral pH and between 20 and 40 °C. Interestingly, purified L-asparaginase showed promising DPPH radical scavenging activity (IC₅₀ 69.12 μg mL⁻¹) and anti-proliferative activity against cervical epitheloid carcinoma (Hela), epidermoid larynx carcinoma (Hep-2), hepatocellular carcinoma (HepG-2), Colorectal carcinoma (HCT-116), and breast adenocarcinoma (MCF-7) with IC₅₀ equal to 2.0, 5.0, 12.40, 8.26 and 22.8 μg mL⁻¹, respectively. The enzyme showed higher activity, selectivity and anti-proliferative activity against cancerous cells along with tiny cytotoxicity toward normal cells (WI-38) which indicates that it has selective toxicity and it could be applied as a less toxic alternative to the current formulations.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Synthesis of new thiazolo-pyrrolidine–(spirooxindole) tethered to 3-acylindole as anticancer agents

Anticancer therapeutics with profiles of high potency, low toxicity, and low resistance is of considerable interest. A new series of functionalized spirooxindole linked with 3-acylindole scaffold is reported, starting from chalcones derived from 3-acetyl indole with isatin, and l-4-thiazolidinecarboxylic acid. The reactions proceeded regioselectivity, stereoselectivity, without side products in high yield (71–89%). The new spirooxindole hybrids have been evaluated in vitro for their antiproliferative effects against colon cancer (HCT-116), hepatocellular carcinoma (HepG2) and prostate cancer (PC-3). The selectivity of their activity was evaluated. Some of the synthesized compounds showed considerable anticancer activities. Compound 4k proved to retain a high cytotoxic activity and selectivity against colon cancer cells HCT-116 (IC₅₀ = 7 ± 0.27 µM, SI: 3.7), and HepG2 (IC₅₀ = 5.5 ± 0.2 µM, SI: 4.7) in comparison to (IC₅₀ = 12.6 ± 0.5, SI: 0.4 and 5.5 ± 0.3 µM, SI: 0.9, respectively). Compound 4k was less active (IC₅₀ = 6 ± 0.3 µM, SI: 4.3) than cisplatin (IC₅₀ = 5 ± 0.56 µM, SI: 1.0) but showed greater selectivity towards prostate cancer cells PC-3 in comparison to cisplatin. The details of the binding mode of the active compounds were clarified by molecular docking. Ligand Efficiency (LE) and Ligand Lipophilic Efficiency (LLE) were evaluated and revealed that compound 4k had acceptable value.     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

Isolation and characterization of trichalasin-producing endophytic fungus from <Emphasis Type="BoldItalic">Taxus baccata</Emphasis>

An endophytic fungus (strain T1) isolated from Taxus baccata was studied for the production of metabolites with anticancer and antioxidant activities. This fungus was identified as Diaporthe sp. based on rDNA-internal transcribed spacer (ITS) sequence analysis. The crude extract showed cytotoxic activity against MCF-7 and HeLa cancer cell lines, with IC₅₀ (concentration inhibiting 50% of growth rate) values of 1058?±?44 and 1257?±?80 μg ml^?1, respectively. The scavenging activity of fungal extract increased significantly with increasing concentration [IC₅₀ (concentration required to scavenge 50% of free radicals) 482?±?9 μg ml^?1]. Ultra-high-performance liquid chromatography-quadrupole-time of flight analysis revealed the presence of three trichalasins (trichalasin E, F and H) in the crude extract of T1 which are known to have antitumour and antioxidant activities. These results suggest that Diaporthe sp. has the potential to be used for therapeutic purposes because of its antiproliferative and antioxidant potential and also for the production of cytochalasins.     

Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process

Abstract

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K₂HPO₄ 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µ_m 0.104 h^?1, X_m 6g/L P₀ 1.7U/mL P_m 8.2 U/mL Y_X/S 4 g-cell/g-glucose µP_m 0.35 h^?1 q_p 5.46 U/g/h Y_P/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.     

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin.The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties.Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [¹⁴C]choline uptake inhibition (IC₅₀ = 236.1 ± 1.28 μmol/L, and 217.4 ± 1.33 μmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ± 0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC₅₀ value i.e. inhibiting the growth of 50% of the cells (IC₅₀ = 0.72 ± 1.31 μmol/L).Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.     

CHEMICAL MODIFICATION OF L-ASPARAGINASE FROM Cladosporium sp. FOR IMPROVED ACTIVITY AND THERMAL STABILITY

L-Asparaginase (ASNase), an antileukemia enzyme, is facing problems with antigenicity in the blood. Modification of L-asparaginase from Cladosporium sp. was tried to obtain improved stability and improved functionality. In our experiment, modification of the enzyme was tried with bovine serum albumin, ovalbumin by crosslinking using glutaraldehyde, N-bromosuccinimide, and mono-methoxy polyethylene glycol. Modified enzymes were studied for activity, temperature stability, rate constants (k_d), and protection to proteolytic digestion. Modification with ovalbumin resulted in improved enzyme activity that was 10-fold higher compared to native enzyme, while modification with bovine serum albumin through glutaraldehyde cross-linking resulted in high stability of L-asparaginase that was 8.5- and 7.62-fold more compared to native enzyme at 28°C and 37°C by the end of 24 hr. These effects were dependent on the quantity of conjugate formed. Modification also markedly prolonged L-asparaginase half-life and serum stability. N-Bromosuccinimide-modified ASNase presented greater stability with prolonged in vitro half-life of 144 hr to proteolytic digestion relative to unmodified enzyme (93 h). The present work could be seen as producing a modified L-asparaginase with improved activity and stability and can be a potential source for developing therapeutic agents for cancer treatment.     

A Highly Efficient NADH-dependent Butanol Dehydrogenase from High-butanol-producing Clostridium sp. BOH3

Butanol has been considered as a better alternative fuel and it can be produced from anaerobic Clostridial fermentation. Though several enzymes are involved in the biosynthesis of butanol in Clostridia, butanol dehydrogenase (BDH) is understood to play a major role, which catalyzes the conversion of butyraldehyde into butanol at the expenditure of a cofactor NAD(P)H. Recently, the strain Clostridium sp. BOH3 is reported to generate high level of butanol from monosugars. To investigate the BDH activity at various stages of fermentation, BOH3 was cultured in reinforced Clostridial medium with 30 g/l of glucose at 35 °C and the cells were harvested periodically from acid production and solvent production phases. During acid production, NADPH-dependent BDH activity is higher than NADH dependent BDH. Conversely, NADH-BDH activity is predominant during solvent production phase. The optimum pHs for NADH and NADPH-BDH are estimated as pH?6 and 8, respectively. By employing three steps of purification, NADH-BDH is purified to 102-fold with 36 % yield. Subsequent characterization reveals that NADH-BDH is a dimer composed of two subunits depicting the molecular weight of 44 kDa. The peptide finger printing analysis (MS/MS) suggests that the purified protein has higher homology with bifunctional acetaldehyde-CoA and alcohol dehydrogenase of Clostridium acetobutylicum. The extensive kinetic studies show that NADH-BDH follows an ordered sequential bi bi mechanism. The calculated values of K _{butyraldehyde} and K _NADH are 8.35?±?0.25 and 0.076?±?0.02 mM, respectively, whereas V _max is 4.02?±?0.07 μmol/(mg protein. min). The purified NADH-BDH retains 70 % of its initial activity after 7 days at 4 °C.     

Synthesis,biological evaluation,and molecular modelling of new naphthalene-chalcone derivatives as potential anticancer agents on MCF-7 breast cancer cells by targeting tubulin colchicine binding site

Abstract

A series of naphthalene-chalcone derivatives (3a–3t) were prepared and evaluated as tubulin polymerisation inhibitor for the treatment of breast cancer. All compounds were evaluated for their antiproliferative activity against MCF-7 cell line. The most of compounds displayed potent antiproliferative activity. Among them, compound 3a displayed the most potent antiproliferative activity with an IC₅₀ value of 1.42?±?0.15?µM, as compared to cisplatin (IC₅₀?=?15.24?±?1.27?µM). Additionally, the promising compound 3a demonstrated relatively lower cytotoxicity on normal cell line (HEK293) compared to tumour cell line. Furthermore, compound 3a was found to induce significant cell cycle arrest at the G₂/M phase and cell apoptosis. Compound 3a displayed potent tubulin polymerisation inhibitory activity with an IC₅₀ value of 8.4?µM, which was slightly more active than the reference compound colchicine (IC₅₀?=?10.6?µM). Molecular docking analysis suggested that 3a interact and bind at the colchicine binding site of the tubulin.     

The effect of calcium on the thermotropic properties of bovine blood coagulation factors IX and X and their activation intermediates and products

The thermotropic properties of bovine blood coagulation Factors IX and X, as well as the activation intermediates and products of these proteins, have been investigated by differential scanning microcalorimetry in the presence and absence of Ca²⁺. Bovine Factor IX displays a single thermal-denaturation transition characterized by a temperature midpoint (T_M) of 54.5 ± 0.5 °C and a calorimetric enthalpy (ΔH_c) of 105 ± 15 kcal/mol, in the absence of Ca²⁺. In the presence of Ca²⁺ concentrations sufficient to saturate its sites on Factor IX, the T_m value is increased to 57.0 ± 0.5 °C and the ΔH_c is virtually unchanged. When the activation intermediate, Factor IXα, is similarly analyzed in the absence of Ca²⁺, a broad, diffuse thermogram was obtained which did not lend itself to calculation of thermodynamic parameters. In the presence of Ca²⁺, Factor IXα displayed thermograms characterized by a T_M of 51.0 ± 0.5 °C and a ΔH_c of 109 ± 10 kcal/mol. The activated product, Factor IXaα, in the absence of Ca²⁺ (the values in the presence of saturating Ca²⁺ are given in parentheses), undergoes thermal denaturation with a T_M of 54.5 ± 0.5 °C (57.0 ± 0.5 °C) and a ΔH_c of 158 ±10 kcal/mol (156 ± 10 kcal/mol). Similarly, the terminal-activation product, Factor IXaβ, displays a T_M of 51.5 ± 0.5 °C (54.0 ± 0.5 °C) and a ΔH_c of 85 ± 5 kcal/mol (126 ± 10 kcal/mol). Bovine blood coagulation Factor X has been analyzed in this same fashion, and shows very similar thermal properties to Factor IX. The thermal denaturation of Factor X is represented by a T_M of 54.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 102 ± 10 kcal/mol (118 ± 10 kcal/mol), whereas its activated form, Factor Xaβ, possesses a T_M of 55.0 ± 0.5 °C (55.0 ± 0.5 °C) and a ΔH_c of 92.0 ± 5 kcal/mol (136 ± 10 kcal/mol). These studies indicate that, for many of these proteins, Ca²⁺ induces a conformational alteration to a more thermally stable form, which also requires the absorption of greater amounts of heat for thermal denaturation.     

Changes in bacterial CO2 fixation with depth in agricultural soils

Soils were incubated continuously in an atmosphere of ¹⁴CO₂ and the distribution of labeled C into soil organic carbon (¹⁴C-SOC) was determined at 0–1, 1–5, and 5–17 cm down the profile. Significant amounts of ¹⁴C-SOC were measured in paddy soils with a mean of 1,180.6?±?105.2 mg kg^–1 at 0–1 cm and 135.3?±?47.1 mg kg^?1 at 1–5 cm. This accounted for 5.9?±?0.7 % and 0.7?±?0.2 %, respectively, of the total soil organic carbon at these depths. In the upland soils, the mean ¹⁴C-SOC concentrations were 43 times (0–1 cm) and 11 times (1–5 cm) lower, respectively, than those in the paddy soils. The amounts of ¹⁴C incorporated into the microbial biomass (MBC) were also much lower in upland soils (5.0?±?3.6 % and 2.9?±?1.9 % at 0–1 and 1–5 cm, respectively) than in paddy soils (34.1?±?12.4 % and 10.2?±?2.1 % at 0–1 and 1–5 cm, respectively). Similarly, the amount of ¹⁴C incorporated into the dissolved organic carbon (DOC) was considerably higher in paddy soils (26.1?±?6.9 % and 6.9?±?1.3 % at 0–1 and 1–5 cm, respectively) than in upland soils (6.0?±?2.7 % and 4.3?±?2.2 %, respectively). The observation that the majority of the fixed ¹⁴C-SOC, RubisCO activity and cbbL gene abundance were concentrated at 0–1 cm depth and the fact that light is restricted to the top few millimeters of the soil profiles highlighted the importance of phototrophs in CO₂ fixation in surface soils. Phylogenetic analysis of the cbbL genes showed that the potential for CO₂ fixation was evident throughout the profile and distributed between both photoautotrophic and chemoautotrophic bacteria such as Rhodopseudomonas palustris, Bradyrhizobium japonicum, Rubrivivax gelatinosus and Ralstonia eutropha.     

Semi-synthesis and anti-tumor activity of novel 25-OCH3-PPD derivatives incorporating aromatic moiety

Previously we have reported that 25-OCH₃-PPD could suppress the reproduction of cancer cells and cause apoptosis without obvious toxicity. Herein, we aimed to enhance its bioactivity by introducing aromatic groups to its dammarane-type skeleton. These synthesized derivatives were tested for their inhibitory activities against five cancer cell lines. Of them, compounds 3a, 14a and 18a had the strongest antiproliferative activities against tumor cells (IC₅₀?<?15?µM, 5-fold to 10-fold increases than 25-OCH₃-PPD). Especially compound 14a displayed the most potent activity against DU145, MCF-7 and HepG2 cells (IC₅₀?=?6.7?±?0.8, 4.3?±?0.8 and 5.8?±?0.6?µM, respectively). Structure-activity relationships demonstrated that having aromatic ester at the C3 position could improve the bioactivity. The data provided new insights into exploring novel antiproliferative lead compounds.     

The ruthenium complexes cis-(dichloro)tetramineruthenium(III) chloride and cis-tetraammine(oxalato)ruthenium(III) dithionate overcome resistance inducing apoptosis on human lung carcinoma cells (A549)

Lung cancer is one of the leading causes of death in the world, and non-small cell lung carcinoma accounts for approximately 75–85 % of all lung cancers. In the present work, we studied the antitumor activity of the compound cis-(dichloro)tetramineruthenium(III) chloride {cis-[RuCl₂(NH₃)₄]Cl} against human lung carcinoma tumor cell line A549. The present study aimed to investigate the relationship between the expression of MDR1 and CYP450 genes in human lung carcinoma cell lines A549 treated with cisCarboPt, cisCRu(III) and cisDRu(III). The ruthenium-based coordinated complexes presented low cytotoxic and antiproliferative activities, with high IC₅₀ values, 196 (±15.49), 472 (±20.29) and 175 (±1.41) for cisCarboPt, cisCRu(III) and cisDRu(III), respectively. The tested compounds induced apoptosis in A549 tumor cells as evidenced by caspase 3 activation, but only at high concentrations. Results also revealed that the amplification of P-gp gene is greater in A549 cells exposed to cisCarboPt and cisCRu(III) than cisDRu(III). Taken together all these results strongly demonstrate that MDR-1 over-expression in A549 cells could be associated to a MDR phenotype of these cells and moreover, it is also contributing to the platinum, and structurally-related compound, resistance in these cells. The identification and characterization of novel mechanisms of drug resistance will enable the development of a new generation of anti-cancer drugs that increase cancer sensitivity and/or represent more effective chemotherapeutic agents.     

Anti-inflammatory and antiproliferative prenylated carbazole alkaloids from Clausena vestita

Twelve prenylated carbazole alkaloids, containing a novel prenylated carbazole alkaloid, named as clausevestine (1), and 11 known prenylated carbazole alkaloids (2–12), were isolated and identified from the stems and leaves of Clausena vestita, which is a Chinese endemic plant. The chemical structure of 1 was established by means of comprehensive spectroscopic data analyses and the known compounds were determined via comparing their NMR and MS data as well as optical rotation values with those reported in literature. Especially, clausevestine (1) is an unusual prenylated carbazole alkaloid possessing an unprecedented carbon skeleton holding 20 carbon atoms. The anti-inflammatory effects and antiproliferative activities of those isolated prenylated carbazole alkaloids were tested. Prenylated carbazole alkaloids 1–12 displayed remarkable inhibitory effects on NO (nitric oxide) production with IC₅₀ values equivalent to that of the positive control (hydrocortisone). Meanwhile, prenylated carbazole alkaloids 1–12 exhibited remarkable antiproliferative activities against diverse human cancer cell lines in vitro holding the IC₅₀ values ranging from 0.32 ± 0.04 to 18.76 ± 0.18 µM. These findings indicate that these prenylated carbazole alkaloids possessing remarkable anti-inflammatory effects and antiproliferative activities could be meaningful to the discovery of new anti-inflammatory and anti-tumor candidate drugs.     

Brain temperature regulation of panting and non-panting pigeons exposed to extreme thermal conditions

• 1.1. Brain (hypothalamic), skin and body temperatures were measured in hand-reared acclimated (Acc, n = 5) and non-acclimated (NAcc, n =7) rock pigeons (Columba livia, mean body mass 237 g) exposed to increasing ambient temperatures (T_a) (30–60°C) and low humidities.
• 2.2. In non-panting Acc birds, brain temperature gradually increased from 40.1 ± 0.4°C at 30°C to 41.2 ± 0.4°C at 60°C T_a. A mean body temperature (T_b) of 41.2 ± 0.2°C was measured at T_a up to 50°C; an increase of 1.1°C was observed at 60°C (T_b 42.2 ±0.6°C).
• 3.3. In Acc panting birds exposed for 2 hr to 60°C, T_hy was 41.9 ± 0.8°C and T_s was somewhat (but insignificantly) higher, i.e., 42.2 ± 0.7°C. It looks as if both values were increased as a result of a slight hyperthermia that developed (T_b = 43.5 ± 0.9°C).
• 4.4. The significance of the present results for evaluating neuronal thermoresponsiveness of birds' hypothalamus is discussed.

     

1,3-β-d-Glucan synthase ofNeurospora crassa: Partial purification and characterization of solubilized enzyme activity

1,3-β-d-Glucan synthase activity ofNeurospora crassa was rendered soluble by treatment of crude protoplast lysates with 0.1% 3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 1 mM dithiothreitol, 200 mM inorganic phosphate, 10 μM GTP, and 600 mM glycerol. Approximately 50% of enzyme activity was solubilized; soluble enzyme activity was purified 5.6-fold with a net 38% recovery by sucrose gradient density sedimentation. Partially purified enzyme activity had a half-life of 60 ± 10 h at 4°C, aK_m,app of 0.75 ± 0.05 mM, and a V_max,app of 35 ± 1 enzyme units/mg protein.