Design,synthesis, MTT assay,DNA interaction studies of platinum(II) complexes

The square planar Pt(II) complexes of the type [Pt(Lⁿ)(Cl₂)] (where Lⁿ = L^1?3 = thiophene-2-carboxamide derivatives and L^4?6 = thiophene-2-carbothioamide derivatives) have been synthesized and characterized by physicochemical and various spectroscopic studies. MIC method was employed to inference the antibacterial potency of complexes in reference to free ligands and metal salt. Characteristic binding constant (K_b) and binding mode of complexes with calf thymus DNA (CT-DNA) were determined using absorption titration (0.76–1.61 × 10⁵ M^?1), hydrodynamic chain length assay and fluorescence quenching analysis, deducing the partial intercalative mode of binding. Molecular docking calculation displayed free energy of binding in the range of –260.06 to –219.63 kJmol^?1. The nuclease profile of complexes towards pUC19 DNA shows that the complexes cleave DNA more efficiently compared to their respective metal salt. Cytotoxicity profile of the complexes on the brine shrimp shows that all the complex exhibit noteworthy cytotoxic activity with LC₅₀ values ranging from 7.87 to 15.94 μg/mL. The complexes have been evaluated for cell proliferation potential in human colon carcinoma cells (HCT 116) and IC₅₀ value of complexes by MTT assay (IC₅₀ = 125–1000 μg/mL).     

Molecular dynamics and quantum chemical studies of solvent effects on cyclo glycylglycine and glycylalanine dipeptides

Hydrogen bond (H-bond) interactions between the two cyclo dipeptides, cyclo(glycyl-glycine) (CGG) and cyclo(glycyl-alanine) (CGA), and water have been studied using molecular dynamics (MD) and quantum chemical methods. The MD studies have been carried out on CGG and CGA in water using fixed charge force field (AMBER ff03) for over 10 ns with a MD time step of 2 fs. The results of this study show that the solvation pattern influences the conformations of the cyclo dipeptides. Following molecular simulations, post Hartree–Fock and density functional theory methods have been used to explore the molecular properties of the cyclo dipeptides in gaseous and aqueous phase environments. The self-consistent reaction field theory has been used to optimise the cyclopeptides in diethyl ether (? = 4.3) and water (? = 78.5), and the solvent effects have been analysed. A cluster of eight water molecules leads to the formation of first solvation shell of CGG and CGA and the strong H-bonding mainly contributes to the interaction energies. The H-bond interactions have been analysed by the calculation of electron density ρ(r) and its Laplacian ▽²ρ(r) at bond critical points using atoms in molecules theory. The natural bond orbital analysis was carried out to reveal the nature of H-bond interactions. In the solvated complexes, the keto carbons registered the maximum NMR chemical shifts.     

Cation Modulation of Hemoglobin Interaction with Sodium n-Dodecyl Sulfate (SDS). III: Calcium Interaction with R- and Mixed Spin States of Hemoglobin S at pH 5.0: The Musical Chair Paradox

We investigate the interaction of Ca²⁺ (0–500 µM) and a membrane mimic (0.60 mM SDS) with both the R- and mixed spin states hemoglobin S (HbS) as a function of time. These interactions were carried out at pH 5.0. We aim at ascertaining if there is or are differences in the UV–Visible spectra of such interactions to account for the dynamics of calcium ion concentrations [Ca²⁺] in initiating structures which may ultimately suggest HbS polymerization and or resistance to Plasmodium attack. From our results, we conclude that (a) simultaneous interaction of 40 µM Ca²⁺ and 0.60 mM SDS with the R state protein would promote structural formations that can “lock up” the protein for nucleation on the membranes and or become cytotoxic to the parasite; (b) simultaneous R state HbS-SDS or R state HbS-Ca²⁺ would lead to enhanced hemin formation and less deoxyHb species. This condition is unlikely to precipitate polymerization in the HbS but the resulting hemin would poison the parasite; (c) the mixed spin state HbS-SDS and 40 µM Ca²⁺ interaction yields more toxic products to that of the interaction of the mixed spin HbS-SDS with 500 µM Ca²⁺ thus suggesting why the 40 µM Ca²⁺ is important in parasite Hb proteolysis; and (d) pronounced structural changes on interaction with SDS and Ca²⁺ are more in the R state to the mixed spin state.     

Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways

A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150–250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9–10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.     

Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106–75, 75–25, ?25 μm fines), were used to confirm decreasing performance with decreasing particle size (D ₅₀ <40 μm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79–74.01 m² L^?1) and Fe²⁺ concentration (0.1–2.8 g?L^?1) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe²⁺ (0.78 mM). No ROS was detected from solutions containing only Fe³⁺ under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5–2.5 mM) showed significant decrease in both cell growth and Fe²⁺ oxidation rates within the concentration range 1.5–2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe²⁺, can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.     

Rational Improvement of Peptide Affinity to Human Pregnancy-Related Serine Protease HtrA3 PDZ Domain by Introducing a Halogen Bond to the Domain–Peptide Complex Interface

The pregnancy-related serine protease HtrA3 plays an important role in human placental development and has recently been recognized as a potential therapeutic target in the treatment of cancer. Previously, a C-terminal pentapeptide FGRWV^–COOH was identified to bind at the PDZ domain of HtrA3 with a moderate affinity. Here, based on the high-resolution complex crystal structure of HtrA3 PDZ domain with the pentapeptide ligand we successfully introduced a rationally designed halogen bond to the complex interface by substituting R₄-hydrogen atom of the indole moiety of peptide Trp_-1 residue with a halogen atom. High-level theoretical calculations suggested that bromine is the best choice that can render strong interaction energy for the halogen bond and can confer high affinity to the PDZ–peptide complex. Fluorescence spectroscopy characterizations revealed that the resulting R₄-brominated peptide (K _d = 0.15 ± 0.03 μM) exhibited 12-fold affinity improvement relative to its nonhalogenated counterpart (K _d = 1.8 ± 0.4 μM). In contrast, the PDZ-binding affinity of R₆-brominated peptide (K _d = 1.2 ± 0.1 μM), a negative control that was unable to form the halogen bond according to theoretical investigations, did not change substantially as compared to the nonhalogenated peptide.     

Structure‐activity relationship of Trp‐containing analogs of the antimicrobial peptide gomesin

Gomesin (Gm) has a broad antimicrobial activity making it of great interest for development of drugs. In this study, we analyzed three Gm analogs, [Trp¹]‐Gm, [Trp⁷]‐Gm, and [Trp⁹]‐Gm, in an attempt to gain insight into the contributions of different regions of the peptide sequence to its activity. The incorporation of the tryptophan residue in different positions has no effect on the antimicrobial and hemolytic activities of the Gm analogs in relation to Gm. Spectroscopic studies (circular dichroism, fluorescence and absorbance) of Gm and its analogs were performed in the presence of SDS, below and above its critical micelle concentration (CMC) (~8 mM), in order to monitor structural changes induced by the interaction with this anionic surfactant (0–15 mM). Interestingly, we found that the analogs interact more strongly with SDS at low concentrations (0.3‐6.0 mM) than close to or above its CMC. This suggests that SDS monomers are able to cover the whole peptide, forming large detergent‐peptide aggregates. On the other hand, the peptides interact differently with SDS micelles, inserting partially into the micelle core. Among the peptides, Trp in position 1 becomes more motionally‐restricted in the presence of SDS, probably because this residue is located at the N‐terminal region, which presents higher conformational freedom to interact stronger with SDS molecules. Trp residues in positions 7 and 9, close to and in the region of the turn of the molecule, respectively, induced a more constrained structure and the compounds cannot insert deeper into the micelle core or be completely buried by SDS monomers. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chiral Separation of Four Stereoisomers of Ketoconazole Drugs Using Capillary Electrophoresis

This work aimed to develop a chiral separation method of ketoconazole enantiomers using electrokinetic chromatography. The separation was achieved using heptakis (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin (TMβCD), a commonly used chiral selector (CS), as it is relatively inexpensive and has a low UV absorbance in addition to an anionic surfactant, sodium dodecyl sulfate (SDS). The influence of TMβCD concentration, phosphate buffer concentration, SDS concentration, buffer pH, and applied voltage were investigated. The optimum conditions for chiral separation of ketoconazole was achieved using 10 mM phosphate buffer at pH 2.5 containing 20 mM TMβCD, 5 mM SDS, and 1.0% (v/v) methanol with an applied voltage of 25 kV at 25 °C with a 5‐s injection time (hydrodynamic injection). The four ketoconazole stereoisomers were successfully resolved for the first time within 17 min (total analysis time was 28 min including capillary conditioning). The migration time precision of this method was examined to give repeatability and reproducibility with RSDs ≤5.80% (n =3) and RSDs ≤8.88% (n =9), respectively. Chirality 27:223–227, 2015. © 2014 Wiley Periodicals, Inc.     

Purification, molecular cloning, and antimicrobial activity of peptides from the skin secretion of the black-spotted frog, Rana nigromaculata

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13–8.3 μM against Gram-positive bacteria, 12.5–25.0 μM against Gram-negative bacteria, and 6.25–12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC₅₀: 100–150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58–72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.     

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22–25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9–11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87–Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.     

Evaluation of role of the peptide transport system in absorption of dipeptides in the rat small intestine in chronic experiments <Emphasis Type="Italic">in vivo</Emphasis>

Hydrolysis and absorption of glycylglycine and glycyl-L-leucine as well as absorption of glycine and leucine were studied in chronic experiments on rats with their isolated small intestine loop. Values of the “true” kinetic constants (with taking into account effect of the preepithelial layer) were determined to be as follows: (1) K _t = 46.7 ± 4.0 and 2.15 ± 0.59 mM, J _max = 0.74 ± 0.15 and 0.16 ± 0.03 μmol min^?1 cm^?1 (for transport of free glycine and leucine, respectively); (2) K _t = 4.4 ± 0.6 and 4.8 ± 0.9 mM, J _max = 0.24 ± 0.02 and 0.23 ± 0.02 μmol min^?1 cm^?1 (for transport of glycylglycine and glycyl-L-leucine, respectively); (3) K _M = 5.4 ± 1.0 and 38.2 ± 4.4 mM, V _max = 0.09 ± 0.02 and 0.24 ± 0.07 μmol min^?1 cm^?1 (for membrane hydrolysis of these dipeptides, respectively). According to our calculations, in the wide range of the initial glycylglycine concentrations (2.5–40 mM) a part of the peptide component in its total absorption accounts for 0.77–0.80. In the case of glycyl-L-leucine a part of the peptide component in the total glycine absorption decreases from 0.89 to 0.84, while in the total leucine absorption—from 0.86 to 0.71, the initial dipeptide concentration rising from 5 to 40 mM. The obtained results show that the peptide component prevails in absorption of the studied dipeptides in the rat small intestine, but its role is much lesser than what many authors believe. In the case of glycyl-L-leucine, the peptide component can achieve saturation in the range of high substrate concentrations, its part decreasing essentially to become compared with absorption of free amino acids formed as a result of the dipeptide membrane hydrolysis.     

Zinc and Zinc Chelators Modify Taurine Transport in Rat Retinal Cells

Zinc regulates Na⁺/Cl^?-dependent transporters, similar to taurine one, such as those for dopamine, serotonin and norepinephrine. This study examined the ex vivo effect of zinc (ZnSO₄), N,N,N,N-tetraquis-(2-piridilmetil)etilendiamino (TPEN) and diethylenetriaminepenta-acetic acid (DTPA), intracellular and extracellular zinc chelators, respectively, on rat retina [³H]taurine transport. Isolated cells were incubated in Locke solution with 100 nM of [³H]taurine for 25 s. Different concentrations of ZnSO₄ (0.5–200 μM) were used. Low concentrations of ZnSO₄ (30 and 40 μM) increased the transport, while higher concentrations (100, 150 and 200 μM) decreased it. Various concentrations of TPEN (1–200 μM) were added. Intermediate concentrations of TPEN (10–60 μM) significantly decreased [³H]taurine transport. The presence of TPEN, 20 μM, plus ZnSO₄ reversed the effect of TPEN alone. Several concentrations of DTPA (1–500 μM) were also investigated. Reduction of transport took place at high concentrations of the chelator (100, 250 and 500 μM). DTPA, 500 μM, plus ZnSO₄, did not modify the effect of it. These results indicate that zinc modulates taurine transport in a concentration-dependent manner, directly acting on the transporter or by forming taurine–zinc complexes in cell membranes.     

Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.     

Salicylate Degradation by the Fungal Plant Pathogen Sclerotinia sclerotiorum

The fungal plant pathogen Sclerotinia sclerotiorum was studied to determine its ability to degrade salicylate, an important defense-signaling molecule in plants. S. sclerotiorum D-E7 was grown at 25 °C in an undefined medium (50 ml) containing minerals, 0.1 % soytone, 50 mM MES buffer (pH 6.5), 25 mM glucose, and 1 mM salicylate. Glucose, oxalate, and salicylate concentrations were monitored by HPLC. S. sclerotiorum D-E7 was found to be active in salicylate degradation. However, salicylate alone was not growth supportive and, at higher levels (10 mM), inhibited glucose-dependent growth. Biomass formation (130 mg [dry wt] of mycelium per 50 ml of undefined medium), oxalate concentrations (~10 mM), and culture acidification (final culture pH approximated 5) were essentially the same in cultures grown with or without salicylate (1 mM). Time-course analyses revealed that salicylate degradation and glucose consumption were complete after 7 days of incubation and was concomitant with growth. Trace amounts of catechol, a known intermediate of salicylate metabolism, were detected during salicylate degradation. Overall, these results indicated that S. sclerotiorum has the ability to degrade salicylate and that the presence of low levels of salicylate did not affect growth or oxalate production by S. sclerotiorum.     

ATP Regulates Sodium Channel Kinetics in Pancreatic Islet Beta Cells

Pancreatic beta cells act as glucose sensors, in which intracellular ATP ([ATP]_i) are altered with glucose concentration change. The characterization of voltage-gated sodium channels under different [ATP]_i remains unclear. Here, we demonstrated that increasing [ATP]_i within a certain range of concentrations (2–8 mM) significantly enhanced the voltage-gated sodium channel currents, compared with 2 mM cytosolic ATP. This enhancement was attenuated by even high intracellular ATP (12 mM). Furthermore, elevated ATP modulated the sodium channel kinetics in a dose-dependent manner. Increased [ATP]_i shifted both the current–voltage curve and the voltage-dependent inactivation curve of sodium channel to the right. Finally, the sodium channel recovery from inactivation was significantly faster when the intracellular ATP level was increased, especially in 8 mM [ATP]_i, which is an attainable concentration by the high glucose stimulation. In summary, our data suggested that elevated cytosolic ATP enhanced the activity of Na⁺ channels, which may play essential roles in modulating β cell excitability and insulin release when blood glucose concentration increases.     

Selective Cleavage of SNAREs in Sensory Neurons Unveils Protein Complexes Mediating Peptide Exocytosis Triggered by Different Stimuli

Oligomerisation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes is required for synaptic vesicle fusion and neurotransmitter release. How these regulate the release of pain peptides elicited by different stimuli from sensory neurons has not been established. Herein, K⁺ depolarization was found to induce multiple sodium dodecyl sulfate (SDS)-resistant SNARE complexes in sensory neurons exposed to botulinum neurotoxins (BoNTs), with molecular weights ranging from 104–288 k (large) to 38–104 k (small). Isoform 1 of vesicle-associated membrane protein 1 (VAMP 1) assembled into stable complexes upon depolarisation and was required for the participation of intact synaptosome-associated protein of relative molecular mass 25 k (SNAP-25) or BoNT/A-truncated form (SNAP-25_A) in the large functional and small inactive SDS-resistant SNARE complexes. Cleaving VAMP 1 decreased SNAP-25_A in the functional complexes to a much greater extent than the remaining intact SNAP-25. Syntaxin 1 proved essential for the incorporation of intact and SNAP-25_A into the large complexes. Truncation of syntaxin 1 by BoNT/C1 caused /A- and/or /C1-truncated SNAP-25 to appear in non-functional complexes and blocked the release of calcitonin gene-related peptide (CGRP) elicited by capsaicin, ionomycin, thapsigargin or K⁺ depolarization. Only the latter two were susceptible to /A. Inhibition of CGRP release by BoNT/A was reversed by capsaicin and/or ionomycin, an effect overcome by BoNT/C1. Unlike BoNT/B, BoNT/D cleaved VAMP 1 in addition to 2 and 3 in rat sensory neurons and blocked both CGRP and substance P release. Thus, unlike SNAP-25, syntaxin 1 and VAMP 1 are more suitable targets to abolish functional SNARE complexes and pain peptide release evoked by any stimuli.     

Sites, pathways, and mechanism of absorption of Cu-EDDS complex in primary roots of maize (Zea Mays L.): anatomical, chemical and histochemical analysis

To study the mechanism of chelant-metal complexes to be absorbed into plant roots in the presence of different concentration chelating agents, the sites, pathways, and mechanism of absorption of Cu-EDDS complex ([S, S’]-ethylene diamine disuccinic acid) in maize (Zea mays L.) primary roots were systematically studied. The results showed that, at low concentrations of the Cu-EDDS complex (<200 μmol L^?1) in hydroponic culture, the complex was passively absorbed mainly from the apoplastic spaces where lateral roots penetrate the endodermis and the cortical region into the root xylem, the lateral root zone were the main absorption sites. At higher concentrations (<3,000 μmol L^?1), under hydroponic culture and soil culture conditions, the passage cells, which form a physiological barrier controlling ion absorption, were either injured or killed, and the complex could enter the root xylem. Injury to the physiological barrier was a key factor in the complex being absorbed by roots in substantially larger quantities. In addition, the histochemical analysis of rubeanic acid can also be used for other researches involving Cu, and the negative–pressure measuring device provides a new research tool for studying the apoplastic absorption of other metal–chelating complexes, molecules, and ions.     

Competitive substrate inhibition of amyloglucosidase from Rhizopus sp. by vanillin and curcumin

Kinetic studies of two glucosylation reactions catalyzed by an amyloglucosidase from Rhizopus sp. leading to the synthesis of vanillin-α/β-D-glucoside from D-glucose and vanillin and curcumin-bis-α-D-glucoside from D-glucose and curcumin were investigated in detail. Initial reaction rates were determined from kinetic runs involving different concentrations of D-glucose and vanillin (5?mM to 0.1?M) or D-glucose and curcumin (5?mM to 0.1?M). Graphical double reciprocal plots showed that the kinetics of the two enzyme catalyzed reactions exhibited Ping-Pong Bi-Bi mechanism where competitive substrate inhibition by vanillin/curcumin led to dead-end amyloglucosidase–vanillin/curcumin complexes at higher concentrations of vanillin/curcumin. An attempt to obtain the best fit of this kinetic model through computer simulation yielded in good approximation, the values of four important kinetic parameters, vanillin-α/β-D-glucoside: k_cat=35.0±3.2 10^?5M?h^?1·mg, K_i=10.5±1.1?mM, K_mD-glucose=60.0±6.2?mM, K_mvanillin=50.0±4.8?mM; curcumin-bis-α-D-glucoside: k_cat=6.07±0.58 10^?5M?h^?1·mg, K_i=3.0±0.28?mM, K_mD-glucose=10.0±0.9?mM, K_mcurcumin=4.6±0.5?mM.     

Design of p53-derived peptides with cytotoxicity on breast cancer

The tumor suppressor p53 plays essential role in conserving stability by preventing genome mutation, which is inactivated naturally by its negative regulator MDM2. Thus, targeting p53–MDM2 protein–protein interaction has been raised as a new cancer therapy in the medicinal community. In the current study, we report a successful application of an integrative protocol to design novel p53-derived peptides with cytotoxicity on human breast cancer cells. A quantitative structure–activity relationship-improved statistical potential was used to evaluate the binding potency of totally 24,054 single- and dual-point mutants of p53 peptide to MDM2 in a high-throughput manner, from which 46 peptide mutants with high predicted affinity and typical helical feature were involved in a rigorous modeling procedure that employed molecular dynamics simulations and post-binding energy analysis to systematically investigate the structural, energetic and dynamic aspects of peptide interactions with MDM2. Subsequently, a biological analysis was performed on a number of promising peptide candidates to determine their cytotoxic effects on human breast cancer cell line MDF-7. Six dual-point mutants were found to have moderate or high activities with their IC₅₀ values ranging from 16.3 to 137.0 μM, which are better than that of wild-type p53 peptide (IC₅₀ = 182.6 μM) and close to that of classical anticancer agent cis-platin (IC₅₀ = 4.3 μM). Further, the most active peptide ETFSDWWKLLAE was selected as parent to further derive new mutants on the basis of the structural and energetic profile of its complex with MDM2. Consequently, three triple-point mutants (LTFSDWWKLLAE, ESFSDWWKLLAE and ETFADWWKLLAE) were obtained, and their biological activities (IC₅₀ = 15.1, 27.0 and 8.7 μM, respectively) were determined to be comparable or better than the parent (IC₅₀ = 16.3 μM).     

Effect of Pectin Type on Association and pH Stability of Whey Protein—Pectin Complexes

The purpose of this study was to investigate the influence of pectin type on complex formation between whey protein isolate (WPI) and high methoxy pectins with varying degrees of esterification (DE), and their pH stability. The biopolymer particles with protein-to-polysaccharide mass ratio set to 2:1 were formed at pH 3–7 by heating at 85 °C for 20 min. The particle size, electrical charge, turbidity and microstructure of the biopolymer complexes were evaluated. The optimal conditions for forming WPI-pectin complexes were at the initial pH of 4.5–4.75, just below the isoelectric point of the WPI, where complex formation occurs. At this pH range, the smallest biopolymer complexes (d?=?225–300 nm) could be created. Pectins with 50, 55, 62 and 70 % DE formed relatively small and monomodal complexes with WPI, except for pectin with 71 % DE, which showed major aggregation. The pH stability against aggregation was best with the biopolymer complexes assembled from pectins with 50 % DE (stable at pH 3.5–6.0) and with 62 % DE (stable at pH 3.0–6.0). The results suggest that pectins with varying DE can be used to form small particles and therefore can offer new possibilities in designing novel hierarchical structures and delivery systems.