Synthesis,computational molecular docking analysis and effectiveness on tyrosinase inhibition of kojic acid derivatives

Tyrosinase inhibitors have become increasingly important as whitening agents and for the treatment of pigmentary disorders. In this study, the synthesis of kojic acid derivatives having 2-substituted-3-hydroxy-6-hyroxymethyl/chloromethyl/methyl/morpholinomethylpiperidinyl- methyl/pyrrolidinylmethyl-4H-pyran-4-one structure (compounds 1–30) with inhibitory effects on tyrosinase enzyme were described. One-pot Mannich reaction was carried out by using kojic acid/chlorokojic acid/allomaltol and substituted benzylpiperazine derivatives in presence of formaline. Subsequently, cyclic amine (morpholine, piperidine and pyrrolidine) derivatives of the 6th-position of chlorokojic acid were obtained with nucleophilic substitutions in basic medium. The structures of new compounds were identified by FT-IR, ¹H- and ¹³C NMR, ESI-MS and elemental analysis data. The potential mushroom tyrosinase inhibitory activity of the compounds were evaluated by the spectrophotometric method using l-DOPA as a substrate and kojic acid as the control agent. The potential inhibitory activity was also investigated in silico using molecular docking simulation method. Tyrosinase inhibitory action was significantly more efficacious for several compounds (IC₅₀: 86.2–362.1 µM) than kojic acid (IC₅₀: 418.2). Compound 3 bearing 3,4-dichlorobenzyl piperazine moiety was proven to have the highest inhibitory activity. The results of docking studies showed that according to the predicted conformation of compound 3 in the enzyme binding site, hydroxymethyl group provides a metal complex with copper ions and enzyme. Thus, this interaction explain the high inhibitory activities of the compounds 1, 3 and 4 possessing hydroxymethyl substituent supporting the mushroom assay results with docking studies. In accordance with the results, it is suggested that Mannich bases of kojic acid bearing substituted benzyl piperazine groups (compounds 1, 3, 4, 11, 13, 14, 23, 24, 28, and 29) could be promising antityrosinase agents. Additionally, considering the relationship between tyrosinase inhibitory activity results and molecular docking, a new tyrosinase inhibition mechanism can be proposed.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

Specificity of inhibition of calcium- and calmodulin-dependent protein kinase by alloxan

Studies were undertaken to determine whether the effect of alloxan to inactivate a membrane-bound calcium- and calmodulin-dependent protein kinase was specific for the pancreatic islets and whether inactivation of the kinase occurred also after injection of a diabetogenic dose of alloxan into rats. The effect of alloxan was also examined on similar particulate calcium- and calmodulin-dependent kinases present in two other secretory tissues, mammary acini and forebrain. Exposure of alloxan to cell-free preparations of all secretory tissues examined inhibited the calcium- and calmodulin-dependent kinase activities, suggesting that the specificity of alloxan action was not due to the presence in islets of a kinase uniquely sensitive to alloxan. To determine whether the selective effect of alloxan action was mediated at the cellular level, experiments were performed with alloxan presented to intact cells. Whereas alloxan exposure to viable cell preparations of islets and brain decreased the subsequently measured calcium- and calmodulin-dependent protein kinase activity, the activity measured in mammary acini exposed to these alloxan concentrations was unaffected. Injection (i.v.) of a diabetogenic dose of alloxan (50 mg/kg) produced an immediate (10 min) and selective inactivation of the calcium- and calmodulin-dependent protein kinase in pancreatic islests but had no effect on the similar kinases measured in brain and mammary acini. These results indicate that the unique sensitivity of islets to alloxan may result from the ability of alloxan to rapidly gain intracellular access and then inactivate this kinase activity. The selective effect of alloxan injection on this islet protein kinase is consistent with the hypothesis that inactivation of the kinase by alloxan is related to its diabetogenic effect in vivo.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

Profiling the changes in signaling pathways in ascorbic acid/β‐glycerophosphate‐induced osteoblastic differentiation

Despite numerous reports on the ability of ascorbic acid and β‐glycerophosphate (AA/β‐GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β‐GP‐induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β‐GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR‐1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood. J. Cell. Biochem. 112: 71–77, 2011. © 2010 Wiley‐Liss, Inc.     

Existence of Sites for Anions and Divalent Cations in the Solubilized γ-Aminobutyric Acid/Benzodiazepine Receptor Complex

This study evaluated the ability of gamma-aminobutyric acid (GABA), baclofen, monovalent anions, divalent cations, and various combinations thereof to protect solubilized benzodiazepine (BZ) receptors of types 1 and 2, when contained together on the complex, against heat inactivation. Neither anions, cations, nor GABA alone provided significant protection of solubilized BZ receptors against heat, but inclusion of monovalent anions or divalent cations together with 500 microM GABA did afford protection. Monovalent anions combined with GABA (500 microM) provided 50% to full protection. Divalent cations, such as CaCl2 (2.5 mM) or MgCl2 (2.5 mM) in the presence of GABA (500 microM) yielded 45% and 24% protection, respectively. Other divalent cations tested (Zn2+, Hg2+, Co2+, and Ni2+) were poor protectors, even when combined with GABA. Monovalent anions (200 mM NaCl) and divalent cations (5 mM CaCl2) when tested together provided no protection. Similarly, baclofen (the GABA-B agonist) provided no protection, either alone or together with anions or divalent cations. These results indicate that the independent but interacting recognition sites of GABA, BZ, anions, and divalent cations, previously detected in the membrane-bound state, are retained in the solubilized state.     

Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

A new tubulin-binding protein

A new brain protein is described which forms an insoluble complex with tubulin, with concomitant stoichiometric hydrolysis of GTP. The complex contains a maximum of one tubulin-binding protein (MW 52,500) per two tubulin dimers. The tubulin-binding protein (TBP) does not compete with colchicine, but in the presence of microtubule-associated proteins tubulin appeared less accessible to it. Proteins such as TBP might sequester tubulin and thereby function either to inhibit indiscriminate polymerization, or to promote ordered nucleation by maintaining high local concentrations.