Phosphatase-inhibitory activity and activation of murine macrophages by new 5'-nucleotidase inhibitors, NPF-86IA, NPF-86IB, NPF-86IIA and NPF-86IIB

New 5'-nucleotidase-inhibitory polyphenols named NPF-86IA, NPF-86IB, NPF-86IIA and NPF-86IIB were isolated from the seeds of Areca catechu L. The ability of the inhibitors to precipitate gelatin was investigated by microturbidimetry. These inhibitors produced weak turbidity. As 5'-nucleotidase is a kind of phosphatase, we examined the effects of these inhibitors on alkaline and acidic phosphatases. While they showed moderate inhibitory effects on the activity of acidic phosphatases, they did not have any significant effect on the activity of alkaline phosphatase. Therefore, they showed a higher inhibitory effect on the 5'-nucleotidase than the other phosphatases, Murine macrophages were directly stimulated by the 5'-nucleotidase inhibitors.     

The growth inhibition of Streptococcus mutans by 5'-nucleotidase inhibitors from Areca catechu L

New 5'-nucleotidase inhibitors named NF-86I, NF-86II were recently isolated from the seeds of Areca catechu L. NF-86I and NF86II showed inhibitory effects on the growth of Streptococcus mutans MT8148(c) and Streptococcus mutans MT6715(g), respectively. In addition, these inhibitors could inhibit insoluble glucan formation from sucrose. NF-86I and NF-86II were found to be polyphenolic substances. Some polyphenols such as tannic acid bind non-specifically to proteins (tannic activity). The 5'-nucleotidase inhibitors that we isolated did not show any such activity. However, the growth inhibitory activity and the inhibitory effect on water-insoluble glucan production were equal to tannic acid. It is therefore considered that these inhibitors bind specifically to the bacterial cell surface. Our findings suggest that the 5'-nucleotidase inhibitors NF-86I and NF-86II may be useful anti-plaque preventing agents.     

Application of solid-phase extraction on anion-exchange cartridges to quantify 5'-nucleotidase activity.

The enzyme 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) catalyzes a critical reaction in intermediary metabolism, the phosphohydrolysis of nucleoside 5'-monophosphates to their corresponding nucleosides. We have evaluated solid-phase extraction on pre-packed anion-exchange cartridges as a chromatographic technique with which 5'-nucleotidase activity may be detected and quantified. Chromatographic conditions were established whereby substrate nucleotide was rapidly and completely separated from its corresponding nucleoside by solid-phase extraction. Both analytes were recovered quantitatively, without loss or degradation. This chromatographic system was integrated into a discontinuous radiochemical assay for 5'-nucleotidase which enabled both substrate utilization and product formation to be assessed simultaneously. Enzyme reaction samples could be analyzed directly for 5'-nucleotidase activity without any pre-chromatography preparation. The high capacity of the solid-phase cartridges and the inability of 5'-nucleotidase to enter the packing bed during analyte elution facilitated termination of the enzyme reaction by applying the entire reaction mixture to the cartridge. Loaded cartridges could then be stored at 4 degrees C prior to chromatography and subsequently batch-eluted. The excellent resolution between substrate and product in solid-phase extraction and the sensitivity of radioisotopic counting enabled detection/quantification of low tissue levels of 5'-nucleotidase in conjunction with ancillary assays for secondary enzyme reactions with the potential to elicit the artifactual loss of 5'-nucleotidase substrate/product when crude biological preparations are examined for 5'-nucleotidase activity. Our results demonstrate that solid-phase extraction on anion-exchange cartridges with elution solvents of appropriate pH offers several unique advantages for 5'-nucleotidase determination.     

High-performance liquid chromatographic assay for acid and alkaline phosphatase in serum.

High-performance liquid chromatography was used to assay serum acid and alkaline phosphatase. Samples were incubated with adenosine-5'-monophosphoric acid (AMP) in a buffer of required pH, 5'-nucleotidase was inhibited with Ni2+ ions, and the phosphatase activity was determined by measuring the concentration of the reaction product, adenosine. The analysis time, after the incubation is terminated, is short (7 min), and the assay is quantitative and reproducible. Complete separation of the reaction product from the substrate and the naturally occurring serum constituents and the high sensitivity of the ultraviolet detection system eliminate some of the problems commonly encountered in spectrophotometric assays.     

Tracking antiangiogenic components from Glycyrrhiza uralensis Fisch. based on zebrafish assays using high-speed countercurrent chromatography

Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.     

DISTRIBUTION OF ENZYMES INVOLVED IN NUCLEOTIDE METABOLISM IN THE DISK AND OTHER ROD MEMBRANES

Abstract— Intact disks and inverted disks were prepared from bovine retinal rods and the distribution in the disk membrane of such enzymes as guanyl cyclase, cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase), 5'-nucleotidase and rhodopsin kinase was investigated. Guanyl cyclase was not detected in the disk; the enzyme activity was high in a membranous fraction containing the cilium or axoneme and the rod outer segment plasma membrane. Cyclic nucleotide phosphodiesterase, GTP binding protein (GTPase) and rhodopsin kinase were associated on the external surface of disk in the presence of 2 m M Mg²⁺. The enzymes dissociated from the membrane when Mg²⁺ was depleted. Thus, magnesium ion seems to regulate the state of these enzymes in the outer segment. 5'-Nucleotidase activity was low in intact disks but was significantly enhanced after inversion of the disk. The catalytic site of the enzyme, therefore, must be located on the internal (intradiscal) surface. Since the disks are known to be formed by invagination of the plasma membrane, 5'-nucleotidase, by inference, would have its catalytic site exposed on the external surface of the plasma membrane. Preliminary experiments showed that the capability of light-activated rhodopsin to activate cyclic nucleotide phosphodiesterase was inhibited by phosphorylation of the pigment. This supports the idea that rhodopsin kinase, cyclic nucleotide phosphodiesterase and GTPase exist as a functional complex on rod membranes.     

Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.     

Dictyodendrins A-E,the first telomerase-inhibitory marine natural products from the sponge Dictyodendrilla verongiformis

Five new alkaloids, dictyodendrins A-E (1-5), were isolated from the Japanese marine sponge Dictyodendrilla verongiformis as telomerase inhibitors. Their structures were elucidated by spectroscopic and chemical methods. Dictyodendrins are tyramine-based pyrrolocarbazole derivatives containing three or four p-hydroxybenzene groups. They inhibited telomerase completely at a concentration of 50 microg/mL.     

The yeast <Emphasis Type="Italic">ISN1</Emphasis> (<Emphasis Type="Italic">YOR155c</Emphasis>) gene encodes a new type of IMP-specific 5'-nucleotidase

Background  

The purine salvage enzyme inosine 5'-monophosphate (IMP)-specific 5'-nucleotidase catalyzes degradation of IMP to inosine. Although this enzymatic activity has been purified and characterized in Saccharomyces cerevisiae, the gene encoding IMP 5'-nucleotidase had not been identified.     

Characterization of aldose reductase and aldehyde reductase from the medulla of rat kidney

Aldose reductase and aldehyde reductase from the medulla of the rat kidney have been purified to homogeneity by using affinity chromatography, gel filtration and chromatofocusing. The molecular weights of aldose reductase and aldehyde reductase by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were found to be 37000 and 39000, respectively. The isoelectric points of aldose reductase and aldehyde reductase were found to be 5.4 and 6.2 by chromatofocusing, respectively. The major differences of amino acid compositions between both enzymes were found in serine, alanine and aspartic acid. Substrate specificity studies showed that aldose reductase utilized aldo-sugars such as D-glucose and D-galactose, but aldehyde reductase did not use them. The Km values of aldose reductase for various substrates were lower than those of aldehyde reductase. Aldose reductase utilized both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide adenine dinucleotide (NADH) as coenzymes, whereas aldehyde reductase utilized only NADPH. The presence of the sulfate ion resulted in a dramatic activation of aldose reductase whereas it did not affect aldehyde reductase activity. These enzymes were strongly inhibited by the known aldose reductase inhibitors. However, aldose reductase was more susceptible than aldehyde reductase to inhibition by the aldose reductase inhibitors.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Periandradulcins A, B and C: phosphodiesterase inhibitors from Periandra dulcis Mart

During the course of our screening of bioactive natural products, three new saponins named periandradulcins A (1), B (2) and C (3) were isolated as phosphodiesterase (PDE, EC 3.1.4.17) inhibitors from 80% MeOH extract of the roots of Periandra dulcis Mart. (Leguminosae) by a combination of column chromatography and reversed- and normal-phase high-performance liquid chromatography (HPLC). On the basis of 1H-, 13C- and two-dimensional nuclear magnetic resonance (NMR) spectral data and chemical evidence, their chemical structures were characterized as 3-O-beta-[alpha-L-rhamnopyranosyl(1----2)-beta-D-xylopyranosyl(1----2)-b eta-D- glucuronopyranosyl]-30-hydroxyl-25-formylolean-18-ene-22 beta-O-syringate, 3-O-beta-[alpha-L-rhamnopyranosyl(1----2)-beta-D- xylopyranosyl(1----2)-beta-D-glucuronopyranosyl]-22 beta-hydroxyl-25- formylolean-12-ene and 3-O-beta-[alpha-L-rhamnopyranosyl(1----2)-beta-D- glucopyranosyl(1----2)-beta-D-glucuronopyranosyl]-22 beta-hydroxyl-25-formylolean-18-ene, respectively. The concentrations of periandradulcins A, B and C required to give 50% inhibition (IC50 values) of PDE from bovine heart, were 0.033, 7.6 and 7.7 microM, respectively. Compound 1 was the most potent among the known PDE inhibitors; it inhibited PDE-I (IC50:0.0022 microM) twenty and forty times more effectively than PDE-II and -III, respectively.     

PHOTOCHEMISTRY OF 5-BROMOURIDINE AND 5-BROMO-2'-DEOXYURIDINE IN ICE AND IN "PUDDLES"*

Abstract. 5,5'-Diuridinyl and 5,5'-dideoxyuridinyl have been isolated from UV irradiation (254nm) in a frozen state of 5-bromouridine and 5-bromodeoxyuridine, respectively. These compounds were characterized by UV, IR, NMR, and mass spectra. They are formed in relatively high quantum yields which are comparable to thymidine dimerization and were found to be unaffected upon further irradiation. This photostability suggests that these 5,5'-coupled products, if formed, may accumulate in biological systems. Experiments carried out in "puddles" indicate, by negative inference, that the main reaction pathway is probably the recombination of two radicals to yield the coupled products in a frozen state. Therefore, the coupled products formation should be explored as a possible mechanism for the increased radiation and photosensitivity of biological systems when thymine in DNA is replaced with 5-halouracil. In addition, these compounds may be synthesized chemically, though, in very low yields, ˜7%.     

红葱中的新化学成分

通过正相和反相硅胶柱色以及高效液相色谱,从红葱60%乙醇提取物中分离得到3个新化合物,利用物理化学和光谱学方法鉴定了其结构,分别为9,9'-二羟基-8,8'-二甲氧基-1,1'-二甲基-1H,1H-['4,4']双[萘并(2,3-c)呋喃]-3,3'-二酮(1),6,8-二羟基-3,4-二甲氧基-1-甲基-蒽醌-2-羧酸甲酯(2),2-乙酰基-3,6,8-三羟基-1-甲基蒽醌(3).这3个化合物均能不同程度地诱导稻瘟霉菌丝变形,其最小变形质量浓度(Mini-mummorphologicaldeformationmassconcentration,MMDC)分别为145.8,50.2和124.8μg/mL.利用MTT法测定了化合物抑制人类红白血细胞株K562细胞生长的活性,化合物2的IC₅₀为49.1μg/mL.     

Study on the isolation, purification and physicochemical properties of polysaccharides from Indocalamus tesselatus

Polysaccharides prepared from Chinese traditional medicine "Ruoye" (Indocalamus tesselatus), a kind of bamboo leaves, were studied. Eight crude polysaccharides were isolated from the dried leaves by successive extraction with 85% ethanol (FE), 0.9% sodium chloride (FS), hot water (FI), 1% ammonium oxalate (FII), 5% NaOH (FIII-a, FIII-b) and 20% NaOH (FIV-a, FIV-b). They were decolorized by hydrogen peroxide treatment and the protein was removed with Savag's method. The polysaccharides were precipitated by three volumes of absolute ethanol. After further purification by gel filtration chromatography on a Sephadex G-200 column, these polysaccharides were lyophilized. Their physicochemical properties and structural feature were elucidated by modern analytical technique such as UV, elemental analysis and high performance gel permeation chromatography. From the results of partial hydrolysis, FE and FIII-b have a main chain comprised solely of glucuronic acid and xylose residues, respectively.     

Purification and characterization of a nuclease (3'-nucleotidase) from a Penicillium sp

A nuclease (3'-nucleotidase) similar to P1 nuclease from Penicillium citrinum was purified from a commercial digestive from a Penicillium sp. The activity of the nuclease (PA) was separated to three fractions by diethylaminoethyl-Toyopearl 650M column chromatography, in total yield of 10%. The apparent molecular weight of these three nucleases, PA1, PA2 and PA3 was 35000, 33000, and 32000, respectively. All of them were homogeneous so far as checked by sodium dodecyl sulfate slab gel electrophoresis. The three nucleases differed in carbohydrate content, but their amino acid composition was practically the same, and very similar to that of P1 nuclease. The molecular weight of nuclease PA3, the major component of nuclease PA, was approximately 27000 after digestion by endoglycosidase F. The N-terminal and C-terminal amino acid sequences of nuclease PA3 were determined by Edman degradation and carboxypeptidase(s) digestion, respectively. The nuclease PA3 was inactivated in the presence of 10 mM ethylenediamine tetraacetic acid (EDTA) and 65% of its native enzyme activity restored by the addition of 20 mM ZnCl2. The pH-dependent photooxidative inactivation of nuclease PA3 was accelerated by removal of Zn ion by EDTA or trishydroxymethyl aminomethane, indicating the possible chelation of Zn2+ with some histidine residues.     

Inhibition of Cellobiohydrolases from Trichoderma reesei. Synthesis and Evaluation of Some Glucose-, Cellobiose-, and Cellotriose-Derived Hydroximolactams and Imidazoles

The lactam 16 , the hydroximolactams 8 , 20 , 23 , and 27 , and the imidazole 32 were prepared following known methods. They were tested together with the known tetrazole 35 and the hydroximolactams 2 and 36 as inhibitors of the cellobiohydrolases Cel7A and Cel6A from Trichoderma reesei. Cel7A is only weakly inhibited by these compounds. Comparing their inhibitory activity evidences the importance of occupying subsites +1 and +2. The results strongly suggest that the shape of none of the variants of the lactone-type inhibitor motif embodied by these inhibitors is complementary to the subsite −1, i. e., analogous to the transition state. Cel6A is rather strongly inhibited by the cellobiose analogues 20 , 23 , and 32 , and by the cellotriose analogue 27 . Their relative inhibitory activities evidence that binding at subsite −2 depends upon the shape of the moiety occupying subsite −1. There is only a small difference between the inhibition by the hydroximolactams 20 and 23 , which may be (partially) protonated by the catalytic acid of either anti- or syn-protonating glycosidases, and the imidazole 32 , which can only be protonated by anti-protonating glycosidases. The results strongly suggest that shape requirements must be met by glycosidase inhibitors before they can be used to characterize the proton trajectory of glycosidases.     

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.     

Development of a fast liquid chromatography/mass spectrometry screening method for angiotensin‐converting enzyme (ACE) inhibitors in complex natural mixtures like snake venom

A new robust high‐performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI‐MS)‐based screening method for angiotensin‐converting enzyme (ACE)‐inhibiting substances in crude samples is described. The ACE assay is carried out in a typical offline setup by incubation of the samples with ACE and angiotensin I (AI), followed by stopping the reaction with acetonitrile containing val⁵‐AI serving as internal standard (I.S.). AI and the product angiotensin II (AII) are extracted from the incubation mixture by turbulent‐flow chromatography (TFC) applied in backflush mode as online solid‐phase extraction and are directly quantified by ESI(+)‐MS. The presence of ACE inhibitors (ACEi) is detected by an increase in AI signal intensity and a corresponding decrease of AII signal, as compared to the blank assay. The overall time of analysis of the TFC/ESI‐MS method was 5 min, thus making the described setup suitable for a rapid screening method. The assay was validated using a known ACE inhibitor and the IC₅₀ values found were in good accordance with a common HPLC/UV method and literature data. The method was successfully applied for the screening of size‐exclusion chromatography fractions of the venom of the pitviper Bothrops moojeni. Three of 18 analyzed fractions inhibited ACE, due to peptides present as components of this snake venom. These compounds were extracted from the two most‐active fractions by means of TFC and isolated by means of HPLC. Three peptides with ACE inhibitory activity were characterized and their structures were elucidated with ESI‐MS/MS‐based de novo sequencing to be ZKWPPGKVPP, ZKWPRPGPEIPP and ZNWPRPGPEIPP, respectively (Z = pyroglutamic acid). Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis and Assessment of Two Malonyl Dihydrazide Derivatives as Corrosion Inhibitors for Carbon Steel in Acidic Media: Experimental and Theoretical Studies

Despite the extensive use of carbon steel in all industrial sectors, particularly in the petroleum industry, its low corrosion resistance is an ongoing problem for these industries. In the current work, two malonyl dihydrazide derivatives, namely 2,2’-malonylbis (N-phenylhydrazine-1-carbothiamide (MBC) and N’1, N’3-bis(-2-hydroxybenzylidene) malonohydrazide (HBM), were examined as inhibitors for the carbon steel corrosion in 1.0 M HCl. Both MBC and HBM were characterised using thin-layer chromatography, elemental analysis, infrared spectroscopy, and nuclear magnetic resonance techniques. The corrosion tests were performed using mass loss measurements, polarisation curves, and electrochemical impedance spectroscopy. It is obtained from the mass loss studies that the optimal concentration for both inhibitors is 2.0 × 10⁻⁵ mol/L, and the inhibition efficiencies reached up to 90.7% and 84.5% for MBC and HBM, respectively. Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarisation (PDP) indicate an increased impedance in the presence of both MBC and HBM and mixed-type inhibitors, respectively. Both inhibitors can mitigate corrosion in the range of 298–328 K. Values of free energy changes obtained from the Langmuir model suggest that the inhibitors suppress the corrosion process principally by chemisorption. The computational investigations were conducted to identify the factors connected with the anti-corrosive properties of the examined inhibitors.