In Silico and Ex Vivo Studies on the Spasmolytic Activities of Fenchone Using Isolated Guinea Pig Trachea

Fenchone is a bicyclic monoterpene found in a variety of aromatic plants, including Foeniculum vulgare and Peumus boldus, and is used in the management of airways disorders. This study aimed to explore the bronchodilator effect of fenchone using guinea pig tracheal muscles as an ex vivo model and in silico studies. A concentration-mediated tracheal relaxant effect of fenchone was evaluated using isolated guinea pig trachea mounted in an organ bath provided with physiological conditions. Sustained contractions were achieved using low K⁺ (25 mM), high K⁺ (80 mM), and carbamylcholine (CCh; 1 µM), and fenchone inhibitory concentration–response curves (CRCs) were obtained against these contractions. Fenchone selectively inhibited with higher potency contractions evoked by low K⁺ compared to high K⁺ with resultant EC₅₀ values of 0.62 mg/mL (0.58–0.72; n = 5) and 6.44 mg/mL (5.86–7.32; n = 5), respectively. Verapamil (VRP) inhibited both low and high K⁺ contractions at similar concentrations. Pre-incubation of the tracheal tissues with K⁺ channel blockers such as glibenclamide (Gb), 4-aminopyridine (4-AP), and tetraethylammonium (TEA) significantly shifted the inhibitory CRCs of fenchone to the right towards higher doses. Fenchone also inhibited CCh-mediated contractions at comparable potency to its effect against high K⁺ [6.28 mg/mL (5.88–6.42, n = 4); CCh] and [6.44 mg/mL (5.86–7.32; n = 5); high K⁺]. A similar pattern was obtained with papaverine (PPV), a phosphodiesterase (PDE), and Ca²⁺ inhibitor which inhibited both CCh and high K⁺ at similar concentrations [10.46 µM (9.82–11.22, n = 4); CCh] and [10.28 µM (9.18–11.36; n = 5); high K⁺]. However, verapamil, a standard Ca²⁺ channel blocker, showed selectively higher potency against high K⁺ compared to CCh-mediated contractions with respective EC₅₀ values of 0.84 mg/mL (0.82–0.96; n = 5) 14.46 mg/mL (12.24–16.38, n = 4). The PDE-inhibitory action of fenchone was further confirmed when its pre-incubation at 3 and 5 mg/mL potentiated and shifted the isoprenaline inhibitory CRCs towards the left, similar to papaverine, whereas the Ca²⁺ inhibitory-like action of fenchone pretreated tracheal tissues were authenticated by the rightward shift of Ca²⁺ CRCs with suppression of maximum response, similar to verapamil, a standard Ca²⁺ channel blocker. Fenchone showed a spasmolytic effect in isolated trachea mediated predominantly by K⁺ channel activation followed by dual inhibition of PDE and Ca²⁺ channels. Further in silico molecular docking studies provided the insight for binding of fenchone with Ca²⁺ channel (−5.3 kcal/mol) and K⁺ channel (−5.7), which also endorsed the idea of dual inhibition.     

New sandwich-type polymeric potassium-dicyanoargentate(I) complex: synthesis,characterization and enzymatic activity

Coordination compounds containing dicyanoargentate(I) have remarkable biological potential due to their therapeutic antibacterial, antifungal, antibiofilm, and anticancer properties. In this study, a new dicyanoargentate(I)-based complex was synthesized and characterized by various procedures (elemental, thermal, FT-IR for complex) involving crystal analysis of the complex. In addition, the biological activity of this new compound on the acetylcholinesterase (AChE) enzyme, an important enzyme for the nervous system, was investigated. When the infrared (IR) spectrum of the complex is examined, the OH vibration peak resulting from H₂O molecules in the structure at 3948-3337 cm⁻¹ and at 2138 cm⁻¹, along with a CN peak coordinated to Ag, can be seen, indicating that the mass remaining in the thermal degradation of the complex at 1000 ◦ C is the weight corresponding to the metal mixture consisting of K+Ag (calc.: 68.06). The crystal method revealed that the complex has a sandwich-like, polymeric chemical structure with layers formed by K⁺ cations and [Ag(CN)₂H₂O]⁻ anions. Therefore, the AChE enzyme has potential therapeutic uses in improving ACh levels in brain cells, in reducing various side effects, and in improving cognitive impairment, especially in advanced Alzheimer’s disease patients. In this study, the activity of this newly synthesized complex on AChE was also investigated. As a result of this research, [Ag(CN)₂(H₂O)K] had 0.0282 ± 0.010 μM Ki values against AChE. The compound was therefore a good inhibitor for the AChE enzyme. This type of compound can be used for the development of novel anticholinesterase drugs.     

Rapid and Sensitive Electrochemical Detection of Carbaryl Based on Enzyme Inhibition and Thiocholine Oxidation Mediated by a Ruthenium(III) Complex

In the electrochemical detection method for pesticides that measures their inhibitory effects on acetylcholinesterases (AChEs), the direct electrooxidation of the enzyme product (thiocholine, SCh) is slow at conventional electrodes. To overcome this limitation, an electron mediator is required to lower the applied potential and facilitate the transfer of electrons between the enzyme product and electrode. In this study, [Ru(NH₃)₅py]³⁺ is introduced as an electron mediator in inhibition‐based pesticide detection. To obtain a better signal‐to‐background ratio, [Ru(NH₃)₅py]³⁺, which undergoes a fast outer‐sphere reaction, is combined with low‐electrocatalytic indium‐tin‐oxide (ITO) electrodes at which many interfering species undergo slow redox reactions. AChE is immobilized onto an avidin‐modified ITO electrode via the direct adsorption of avidin onto ITO followed by the biospecific binding of biotinylated AChE to the avidin. SCh is generated from acetylthiocholine by AChE. Subsequently, SCh converts [Ru(NH₃)₅py]³⁺ to [Ru(NH₃)₅py]²⁺, which is then oxidized at the ITO electrode. This procedure allows the sensitive detection of carbaryl at a low applied potential of 0.15 V vs Ag/AgCl. The calculated detection limit for carbaryl is approximately 0.3 pM. This simple and sensitive pesticide sensor is thus very promising and should be extendable to the onsite environmental monitoring of other pesticides.     

Screening of Carbonic Anhydrase,Acetylcholinesterase, Butyrylcholinesterase,and α-Glycosidase Enzyme Inhibition Effects and Antioxidant Activity of Coumestrol

Coumestrol (3,9-dihydroxy-6-benzofuran [3,2-c] chromenone) as a phytoestrogen and polyphenolic compound is a member of the Coumestans family and is quite common in plants. In this study, antiglaucoma, antidiabetic, anticholinergic, and antioxidant effects of Coumestrol were evaluated and compared with standards. To determine the antioxidant activity of coumestrol, several methods—namely N,N-dimethyl-p-phenylenediamine dihydrochloride radical (DMPD^•+)-scavenging activity, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical (ABTS^•+)-scavenging activity, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^•)-scavenging activity, potassium ferric cyanide reduction ability, and cupric ion (Cu²⁺)-reducing activity—were performed. Butylated hydroxyanisole (BHA), Trolox, α-Tocopherol, and butylated hydroxytoluene (BHT) were used as the reference antioxidants for comparison. Coumestrol scavenged the DPPH radical with an IC₅₀ value of 25.95 μg/mL (r²: 0.9005) while BHA, BHT, Trolox, and α-Tocopherol demonstrated IC₅₀ values of 10.10, 25.95, 7.059, and 11.31 μg/mL, respectively. When these results evaluated, Coumestrol had similar DPPH^•-scavenging effect to BHT and lower better than Trolox, BHA and α-tocopherol. In addition, the inhibition effects of Coumestrol were tested against the metabolic enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BChE), carbonic anhydrase II (CA II), and α-glycosidase, which are associated with some global diseases such as Alzheimer’s disease (AD), glaucoma, and diabetes. Coumestrol exhibited K_i values of 10.25 ± 1.94, 5.99 ± 1.79, 25.41 ± 1.10, and 30.56 ± 3.36 nM towards these enzymes, respectively.     

Discovery of Guanidine Derivatives from Buthus martensii Karsch with Metal-Binding and Cholinesterase Inhibition Properties

Two rare guanidine-type alkaloids, Buthutin A (1) and Buthutin B (2), along with two other compounds (3, 4), were isolated from Buthus martensii Karsch, and determined using extensive spectroscopic data analysis and high resolution-mass spectrometry. Compound 1 showed the most potent inhibition on AChE and BChE with IC₅₀ values of 7.83 ± 0.06 and 47.44 ± 0.95 μM, respectively. Kinetic characterization of compound 1 confirmed a mixed-type of AChE inhibition mechanism in accordance with the docking results, which shows its interaction with both catalytic active (CAS) and peripheral anionic (PAS) sites. The specific binding of compound 1 to PAS domain of AChE was also confirmed experimentally. Moreover, compounds 1 and 3 exhibited satisfactory biometal binding abilities toward Cu²⁺, Fe²⁺, Zn²⁺ and Al³⁺ ions. These results provide a new evidence for further development and utilization of B. martensii in health and pharmaceutical products.     

Synthesis,Biological Evaluation,and Molecular Modeling of Aza-Crown Ethers

Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and ε-aminocaproyl substituents and analyzed their biological effects in vitro. Ten of the compounds (8, 10–17, and 21) increased intracellular calcium ([Ca²⁺]_i) in human neutrophils, with the most potent being compound 15 (N,N’-bis[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could alter normal neutrophil [Ca²⁺]_i flux. Indeed, a number of these compounds (i.e., 8, 10–17, and 21) inhibited [Ca²⁺]_i flux in human neutrophils activated by N-formyl peptide (fMLF). Some of these compounds also inhibited chemotactic peptide-induced [Ca²⁺]_i flux in HL60 cells transfected with N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate (PMA) and neutrophil chemotaxis toward fMLF, as both of these processes are highly dependent on regulated [Ca²⁺]_i flux. Quantum chemical calculations were performed on five structure-related diaza-crown ethers and their complexes with Ca²⁺, Na⁺, and K⁺ to obtain a set of molecular electronic properties and to correlate these properties with biological activity. According to density-functional theory (DFT) modeling, Ca²⁺ ions were more effectively bound by these compounds versus Na⁺ and K⁺. The DFT-optimized structures of the ligand-Ca²⁺ complexes and quantitative structure-activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the N,N’-diacylated diaza-crown ethers participated in cation binding and could play an important role in Ca²⁺ transfer. Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore mechanism of biological action of aza-crown ethers.     

Effects of lanthanum on calcium-activated K+ currents and its kinetics in MC3T3 cells

Using the whole cell patch-clamp technique,we studied the effects of La3+ on calcium-activated K+ currents and its kinetics of activation and inactivation in non-excitable MC3T3 cells.Our results showed that the calcium-activated outward K+ currents were promoted with increasing concentration of Ca2+ in the pipette solution and a voltage- and Ca2+-dependent manner.La3+ in the bath solution inhibited the currents in a concentration-dependent manner and the inhibition EC50 was 8.23 ± 1.45 μmol/L.At the concentration of 50 μmol/L,La3+ significantly changed the Vh of the activation curve and shifted the activation curve to more positive potentials,but shifted the inactivation curve to more negative potentials.It had no effect on the slope factor k of the activation and inactivation curves.Potassium currents inhibition could induce a series of physiological and molecular biological functions,presumably because of its ability to depolarize the plasma membrane and enhance cell excitability,resulting in increasing Ca2+ influx and cytoplast Ca2+ concentration.This process may be one of the molecular mechanisms by which La3+ affects the cell growth and function of MC3T3 cells.     

Molecular dynamics simulation exploration of cooperative migration mechanism of calcium ions in sarcoplasmic reticulum Ca2+‐ATPase

Calcium ATPase is a member of the P‐type ATPase, and it pumps calcium ions from the cytoplasm into the reticulum against a concentration gradient. Several X‐ray structures of different conformations have been solved in recent years, providing basis for elucidating the active transport mechanism of Ca²⁺ ions. In this work, molecular dynamics (MD) simulations were performed at atomic level to investigate the dynamical process of calcium ions moving from the outer mouth of the protein to their binding sites. Five initial locations of Ca²⁺ ions were considered, and the simulations lasted for 2 or 6 ns, respectively. Specific pathways leading to the binding sites and large structural rearrangements around binding sites caused by uptake of calcium ions were identified. A cooperative binding mechanism was observed from our simulation. Firstly, the first Ca²⁺ ion binds to site I , and then, the second Ca²⁺ ion approaches. The interactions between the second Ca²⁺ and the residues around site I disturb the binding state of site I and weaken its binding ability for the first bound Ca²⁺. Because of the electrostatic repulsion of the second Ca²⁺ and the electrostatic attraction of site II , the first bound Ca²⁺ shifts from site I to site II . Concertedly, the second Ca²⁺ binds to site I , forming a binding state with two Ca²⁺ ions, one at site I and the other at site II . Both of Glu908 and Asp800 coordinate with the two Ca²⁺ ions simultaneously during the concerted binding process, which is believed to be the hinge to achieve the concerted binding. In our simulations, four amino acid residues that serve as the channel to link the outer mouth and the binding sites during the binding process were recognized, namely Tyr837, Tyr763, Asn911, and Ser767. The analyses regarding the activity of the proteins via mutations of some key residues also supported our cooperative mechanism. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Peak parking technique for the simultaneous determination of anions and cations

An ion chromatography method is described for the simultaneous determination of anions (Cl^–, NO₃^–, and SO₄^2–) and cations (Na⁺, NH₄⁺, K⁺, Mg²⁺, and Ca²⁺) using a single pump, a single eluent and a single detector. An anion-exchange column modified with chondroitin sulfate C facilitated the elution of the above three anions using 5 mM tartaric acid as the eluent in isocratic mode, whereas the same eluent facilitated the separation of the above five cations on a commercially-available cation-exchange column. The separation columns were connected in series via two six-port switching valves, so the required cation-exchange or anion-exchange separation could be carried out by selecting the appropriate positions for the switching valves. The separations were completed in 30 min.     

Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305

l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na⁺, K⁺) and phosphate ion activated the enzyme activity, while divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cu²⁺) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K _m, V _max, K _cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?10² M^?1s^?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC₅₀ value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).     

Protective effect of total aralosides of Aralia elata (Miq) Seem (TASAES) against diabetic cardiomyopathy in rats during the early stage, and possible mechanisms

Total aralosides of Aralia elata (Miq) Seem (TASAES) from Chinese traditional herb Longya Aralia chinensis L was found to improve cardiac function. The present study was to determine the protective effects of TASAES on diabetic cardiomyopathy, and the possible mechanisms. Therefore, a single dose of streptozotocin was used to induce diabetes in Wister rats. Diabetic rats were immediately treated with low, medium and high doses of TASAES at 4.9, 9.8 mg/kg and 19.6 mg/kg body weight by gavage, respectively, for eight weeks. Cardiac function was evaluated by in situ hemodynamic measurements, and patch clamp for the L-type Ca²⁺ channel current (I_Ca²⁺-L) and transient outward K⁺ channel current (I_to). Histopathological changes were observed under light and electron microscope. The expression of pro-fibrotic factor, connective tissue growth factor (CTGF) was monitored using immunohistochemistry staining. Compared with diabetic group, medium and high doses, but not low dose, of TASAES showed a significant protection against diabetes-induced cardiac dysfunction, shown by increased absolute value of left ventricular systolic pressure (LVSP) and maximum rates of pressure development (±dp/dt_max), and enhanced amplitude of I_Ca²⁺-L (P < 0.05). Histological staining indicated a significant inhibition of diabetes-caused pathological changes and up-regulation of CTGF expression (P < 0.05). The results suggest that TASAES prevents diabetes-induced cardiac dysfunction and pathological damage through up-regulating I_Ca²⁺-L in cardiac cells and decreasing CTGF expression.     

A Thermodynamic Study Between 18-Crown-6 with Mg2+, Ca2+, Sr2+and Ba2+ Cations in Water–Methanol and Water–Ethanol Binary Mixtures Using The Conductometric Method

The complexation reactions between Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ cations with the macrocyclic ligand, 18-Crown-6 (l8C6) in water–methanol (MeOH) binary systems as well as the complexation reactions between Ca²⁺ and Sr²⁺ cations with 18C6 in water–ethanol (EtOH) binary mixtures have been studied at different temperatures using conductometric method. The conductance data show that the stoichiometry of all the complexes is 1:1. It was found that the stability of 18C6 complexes with Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺ cations is sensitive to solvent composition and in all cases, a non-linear behaviour was observed for the variation of log K _f of the complexes versus the composition of the mixed solvents. In some cases, the stability order is changed with changing the composition of the mixed solvents. The selectivity order of 18C6 for the metal cations in pure methanol is: Ba²⁺ > Sr²⁺ > Ca²⁺ > Mg²⁺. The values of thermodynamic parameters (Δ H _c ° and Δ S _c °) for formation of 18C6–Mg²⁺, 18C6–Ca²⁺, 18C6–Sr²⁺ and 18C6–Ba²⁺complexes were obtained from temperature dependence of the stability constants. The obtained results show that the values of (Δ H _c ° and Δ S _c °) for formation of these complexes are quite sensitive to the nature and composition of the mixed solvent, but they do not vary monotonically with the solvent composition.This revised version was published online in July 2005 with a corrected issue number.     

Field-evaporation mass spectroscopy applied to crown ether complexes with alkali and alkaline-earth metals

Field evaporation from solution has been used to detect crown-ether 15K5 and 18K6 coordination compounds formed with Na⁺, K⁺, Cs⁺, Ba²⁺ and Ca²⁺ in aqueous solution, both hydrated and unhydrated; the Ca²⁺·18K6·(H₂O)_n (n=0, 1, 2,...) compounds have been observed before. 18K6 present in the solution greatly increases the yields of 18K6 compounds with the dissociation products from Ba(NO₃)₂. The detection limits for 15K5 and 18K6 combined with Na⁺, K⁺, and Cs⁺ are approximately the same at 10^–7 g.Translated from Teoreticheskaya i Éksperimental'naya Khimiya, Vol. 24, No. 2, pp. 239–242, March–April 1988.     

Characterizing Enzyme Cooperativity with Imaging SAMDI-MS

This paper describes a method that combines a microfluidic device and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI) mass spectrometry to calculate the cooperativity in binding of calcium ions to peptidylarginine deiminase type 2 (PAD2). This example uses only 120 μL of enzyme solution and three fluidic inputs. This microfluidic device incorporates a self-assembled monolayer that is functionalized with a peptide substrate for PAD2. The enzyme and different concentrations of calcium ions are flowed through each of eight channels, where the position along the channel corresponds to reaction time and position across the channel corresponds to the concentration of Ca²⁺. Imaging SAMDI (iSAMDI) is then used to determine the yield for the enzyme reaction at each 200 μm pixel on the monolayer, providing a time course for the reactions. Analysis of the peptide conversion as a function of position and time gives the degree of cooperativity (n) and the concentration of ligand required for half maximal activity (K_0.5) for the Ca²⁺ – dependent activation of PAD2. This work establishes a high-throughput and label-free method for studying enzyme-ligand binding interactions and widens the applicability of microfluidics and matrix-assisted laser desorption/ionization mass spectrometry (MALDI) imaging mass spectrometry.     

Chalcone Scaffolds Exhibiting Acetylcholinesterase Enzyme Inhibition: Mechanistic and Computational Investigations

This study was aimed to perform the mechanistic investigations of chalcone scaffold as inhibitors of acetylcholinesterase (AChE) enzyme using molecular docking and molecular dynamics simulation tools. Basic chalcones (C1–C5) were synthesized and their in vitro AChE inhibition was tested. Binding interactions were studied using AutoDock and Surflex-Dock programs, whereas the molecular dynamics simulation studies were performed to check the stability of the ligand–protein complex. Good AChE inhibition (IC₅₀ = 22 ± 2.8 to 37.6 ± 0.75 μM) in correlation with the in silico results (binding energies = −8.55 to −8.14 Kcal/mol) were obtained. The mechanistic studies showed that all of the functionalities present in the chalcone scaffold were involved in binding with the amino acid residues at the binding site through hydrogen bonding, π–π, π–cation, π–sigma, and hydrophobic interactions. Molecular dynamics simulation studies showed the formation of stable complex between the AChE enzyme and C4 ligand.     

Biochemical,Kinetic and Biological Properties of Group V Phospholipase A2 from Dromedary

Secretory group V phospholipase A2 (PLA₂-V) is known to be involved in inflammatory processes in cellular studies, nevertheless, the biochemical and the enzymatic characteristics of this important enzyme have been unclear yet. We reported, as a first step towards understanding the biochemical properties, catalytic characteristics, antimicrobial and cytotoxic effects of this PLA₂, the production of PLA₂-V from dromedary. The obtained DrPLA₂-V has an absolute requirement for Ca²⁺ and NaTDC for enzymatic activity with an optimum pH of 9 and temperature of 45 °C with phosphatidylethanolamine as a substrate. Kinetic parameters showed that K_cat/Km_app is 2.6 ± 0.02 mM⁻¹ s⁻¹. The enzyme was found to display potent Gram-positive bactericidal activity (with IC50 values of about 5 µg/mL) and antifungal activity (with IC50 values of about 25 µg/mL)in vitro. However, the purified enzyme did not display a cytotoxic effect against cancer cells.     

Synthesis of Small‐Molecule Fluorescent Probes for the In Vitro Imaging of Calcium‐Activated Potassium Channel KCa3.1

Small‐molecule probes for the in vitro imaging of K_Ca3.1 channel‐expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K_Ca3.1 channels, was chosen as the targeting component. BODIPY dyes 15 – 20 were synthesized and connected by a Cu^I‐catalyzed azide–alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8 , yielding fluorescently labeled ligands 21 – 26 . The dimethylpyrrole‐based imaging probes 25 and 26 allow staining of K_Ca3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre‐incubation with senicapoc. The density of K_Ca3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc‐targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.     

Purification and Characterization of Extracellular Inulinase from a Marine Yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and Inulin Hydrolysis by the Purified Inulinase

The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca²⁺, K⁺, Na⁺, Fe²⁺, and Zn²⁺. However, Mg²⁺, Hg²⁺, and Ag⁺ acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase activity.     

Kaempferol 3-Rhamnoside on Glutamate Release from Rat Cerebrocortical Nerve Terminals Involves P/Q-Type Ca2+ Channel and Ca2+/Calmodulin-Dependent Protein Kinase II-Dependent Pathway Suppression

Excess synaptic glutamate release has pathological consequences, and the inhibition of glutamate release is crucial for neuroprotection. Kaempferol 3-rhamnoside (KR) is a flavonoid isolated from Schima superba with neuroprotective properties, and its effecton the release of glutamate from rat cerebrocortical nerve terminals was investigated. KR produced a concentration-dependent inhibition of 4-aminopyridine (4-AP)-evoked glutamate release with half-maximal inhibitory concentration value of 17 µM. The inhibition of glutamate release by KR was completely abolished by the omission of external Ca²⁺ or the depletion of glutamate in synaptic vesicles, and it was unaffected by blocking carrier-mediated release. In addition, KR reduced the 4-AP-evoked increase in Ca²⁺ concentration, while it did not affect 4-AP-evoked membrane potential depolarization. The application of selective antagonists of voltage-dependent Ca²⁺ channels revealed that the KR-mediated inhibition of glutamate release involved the suppression of P/Q-type Ca²⁺ channel activity. Furthermore, the inhibition of release was abolished by the calmodulin antagonist, W7, and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN62, but not by the protein kinase A (PKA) inhibitor, H89, or the protein kinase C (PKC) inhibitor, GF109203X. We also found that KR reduced the 4-AP-induced increase in phosphorylation of CaMKII and its substrate synapsin I. Thus, the effect of KR on evoked glutamate release is likely linked to a decrease in P/Q-type Ca²⁺ channel activity, as well as to the consequent reduction in the CaMKII/synapsin I pathway.