Purification of a non-specific nucleoside hydrolase from Alaska pea seeds.

A non-specific nucleoside hydrolase has been isolated from germinated Alaska pea seeds. The enzyme catalyzes the hydrolysis of both purines and pyrimidines along with ribo- and deoxyribonucleosides. A purification scheme utilized ammonium sulfate precipitation, ion exchange chromatography and size exclusion chromatography, resulted in 103-fold purification with a recovery of 2.8%. The purified protein has a specific activity of 0.308 µmol/min•mg. The subunit molecular weight was 26103 Da and the enzyme exists as a dimer. The enzyme retains a significant amount of activity over a wide pH range with the maximum activity occurring at a pH of 6.0. The maximum activity was observed with adenosine as the substrate followed by inosine and guanosine, respectively. The Km for adenosine was 184 ±â€¯34 µM and for inosine 283 ±â€¯88 µM. In addition to the nucleoside hydrolase activity, adenosine deaminase activity was seen in the initial extract. Using adenosine as the substrate with the initial extract from the germinated seeds, the products adenine, inosine, and hypoxanthine were identified based on their retention times during reverse phase HPLC.

The classification of Cannabis hemp cultivars by thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) with chemometrics.

Cannabis has been cultivated as a source of food, fiber, and medicine globally, so the classification of Cannabis cultivars based on their chemical fingerprints is important to standardize and control the quality of Cannabis, ensure that patients receive a full and consistent spectrum of therapeutic benefits, and promote the further implementation of Cannabis-based products in clinical uses. In this study, a high-throughput analytical method, thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS), was employed to classify various Cannabis hemp cultivars with multivariate analysis. Cannabis plant materials from four cultivars were analyzed directly by TD-DART-MS without solvent extraction. The total run time was 15 min including 8 min for data acquisition and 7 min for cooling down the thermal stage. Data preprocessing strategy such as data transformation was evaluated on the TD-DART-MS data set and cubic root transform has shown significant improvement to the classification. TD-DART-MS data was then processed by principal component analysis (PCA) and the results were compared with those from liquid chromatography-mass spectrometry (LC-MS) data. The samples were clustered based on cultivars by PCA, and the validation samples collected 2 months later were also grouped together with the original samples by cultivars after mean-centering the data sets. Partial least squares discriminant analysis (PLS-DA) models were constructed with the TD-DART-MS data sets and a 99.3 ± 0.3% classification accuracy was obtained from 100 independent bootstrapped Latin partition evaluations. Our results indicate that TD-DART-MS may be used as a screening tool for the classification of Cannabis cultivars. Graphical abstract.

Stability of pentobarbital in soil.

Intravenous injection of barbiturates, particularly pentobarbital (5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,5-trione), is a widely used method to euthanize large animals such as horses. However, one concern with this method is the fate of pentobarbital after the disposal of the carcass. As tissues decompose, pentobarbital may leach into the soil and from there migrate to groundwater. A method using methanol extraction, solid phase concentration, and liquid chromatography (LC/MS) has been developed to measure pentobarbital in soils. Recovery of pentobarbital from soil averaged approximately 85% from different soil types including topsoil, potting soil, sand, stall sweepings, and loam. The method was capable of detecting pentobarbital levels of 0.1 ppm. A calibration curve was constructed with a linear range of 1 ppm to 100 ppm. The limit of quantification was 0.5 ppm. The rate of degradation of pentobarbital in sand, topsoil, and potting soil was measured over a 17-week period. At the end of week 17, approximately 17% of the pentobarbital remained in the sand, 19% remained in the topsoil, and 10% remained in the potting soil. While there was a significant decrease in the pentobarbital recovered from the soil, there were still detectable amounts of pentobarbital present in the soil after 17 weeks. To determine the importance of bacterial degradation, the three soil types were autoclaved before addition of pentobarbital. After autoclaving, no degradation of pentobarbital was observed in sand and one topsoil sample, while there was no difference in the degradation of pentobarbital in autoclaved potting soil versus potting soil that had not undergone autoclaving.

1-(beta-D-Erythrofuranosyl)adenosine.

The title compound, also known as beta-erythroadenosine, C(9)H(11)N(5)O(3), (I), a derivative of beta-adenosine, (II), that lacks the C5' exocyclic hydroxymethyl (-CH(2)OH) substituent, crystallizes from hot ethanol with two independent molecules having different conformations, denoted (IA) and (IB). In (IA), the furanose conformation is (O)T(1)-E(1) (C1'-exo, east), with pseudorotational parameters P and tau(m) of 114.4 and 42 degrees, respectively. In contrast, the P and tau(m) values are 170.1 and 46 degrees, respectively, in (IB), consistent with a (2)E-(2)T(3) (C2'-endo, south) conformation. The N-glycoside conformation is syn (+sc) in (IA) and anti (-ac) in (IB). The crystal structure, determined to a resolution of 2.0 A, of a cocrystal of (I) bound to the enzyme 5'-fluorodeoxyadenosine synthase from Streptomyces cattleya shows the furanose ring in a near-ideal (O)E (east) conformation (P = 90 degrees and tau(m) = 42 degrees) and the base in an anti (-ac) conformation.

An acidic polysaccharide (AGC3) isolated from North American ginseng (Panax quinquefolius) suspension culture as a potential immunomodulatory nutraceutical.

Polysaccharides isolated from Panax quinquefolius roots are widely used as nutraceuticals due to their immunomodulatory properties. Despite their popularity, several challenges exist in isolating ginseng root polysaccharides such as batch-to-batch structural inconsistencies and bacterial endotoxin contamination. A plant tissue culture-based platform offers a potential solution to isolate natural polysaccharide fractions with consistent chemical characteristics and reduced endotoxin content. In this study, an acidic polysaccharide fraction (AGC3) with immunomodulatory properties was isolated from Panax quinquefolius suspension cultures. The heterogeneous fraction (molecular weight: 4.81 and 32.14 kDa), purified by anion exchange chromatography, was predominantly composed of galactose (>60%) along with the presence of rhamnose, arabinose, glucose, glucuronic acid and galacturonic acid. The major glycosidic linkages were found to be t-Galp (47.7%), 4-Galp (15.6%), 2,4-Rhap (8.1%), 6-Galp (8.1%) and 4-GalAp (6.8%). Structural analyses indicated the presence of a pectic rhamnogalacturonan I polysaccharide in AGC3. AGC3 significantly (p < 0.05) stimulated RAW 264.7 murine macrophage cells and primary murine splenocytes by enhancing the production of several immunomodulatory mediators such as IL-6, TNF-α, GM-CSF and MCP-1. The results also indicated the putative roles of NF-κB (p65/RelA) and MAPK (p38) signaling pathways in the immunostimulatory response. Additionally, AGC3 induced murine splenocyte proliferation, another major indicator of immunostimulation. Overall, AGC3 has the potential to be used as an immunostimulatory nutraceutical.

Isolation, structure elucidation, and immunostimulatory activity of polysaccharide fractions from Boswellia carterii frankincense resin.

Frankincense has a long history in religious, cultural, and medicinal use. In this study polysaccharides were extracted from frankincense from Boswellia carterii. The polysaccharides were purified by anion exchange chromatography on a DEAE-Sepharose Fast Flow 16/10 FPLC column. Six fractions were obtained and the three most active immunomodulatory fractions were further purified by size exclusion chromatography on a Superdex-200 column. The composition showed the monosaccharides present were predominantly galactose, arabinose, and glucuronic acid along with small amounts of rhamnose and glucose. The monosaccharide composition and glycosyl linkage analysis revealed the polysaccharides belong to the type II arabinogalactans. Fourier-transform infrared spectroscopy and bicinchoninic acid assay showed that the amount of protein in the samples was <1 wt%. One-dimensional 1H NMR were consistent with high molecular weight compounds. The monosaccharides were primarily in the ß conformation. The three fractions exhibited an immunostimulatory effect on RAW 264.7 murine macrophage cells. The most active immunostimulatory fraction FA2, stimulated a range of pro-inflammatory mediators including iNOS, NO, TNF-α, and IL-6 in RAW 264.7 cells. The fractions were effective in proliferating primary murine splenocytes. The results indicate that the polysaccharides isolated from frankincense have the potential to be used as an immunological stimulant or nutraceutical.

Panax quinquefolius (North American ginseng) cell suspension culture as a source of bioactive polysaccharides: Immunostimulatory activity and characterization of a neutral polysaccharide AGC1.

In this study, we propose the use of a plant tissue culture-based system for the production of polysaccharides with consistent chemical characteristics and reduced endotoxin content. Polysaccharides were isolated from suspension cultures of Panax quinquefolius (American ginseng), a widely used medicinal herb. A neutral fraction, AGC1, purified by anion exchange and size exclusion chromatography, displayed immunostimulatory activity in vitro and ex vivo. AGC1 (average molecular weight: 5.2kDa) was predominantly composed of galactose (>60%) along with the presence of several other neutral sugars such as arabinose, xylose, glucose, mannose and rhamnose in minor amounts. The major glycosidic linkages were found to be 3-Galp (48.5%), 3,6-Galp (10.2%), t-Galp (5.2%), 6-Galp (4.4%), 4-Glcp (5.7%), 4-Arap/5-Araf (4.0%) and t-Araf (4.5%). AGC1 significantly (p<0.05) stimulated the expression of a range of proinflammatory mediators in RAW 264.7 murine macrophages such as IL-6, TNF-α, MCP-1 and GM-CSF. Additionally, AGC1 treatment of RAW 264.7 cells stimulated NOS2 gene expression, leading to increased levels of iNOS and downstream NO. Consistent with this, AGC1 was able to act as an immunostimulant in primary murine splenocytes, enhancing cell proliferation, as well as NO and TNF-α production. Our results also indicate the partial role of NF-κB pathway in the immunostimulatory response.

Substituted hippurates and hippurate analogs as substrates and inhibitors of peptidylglycine alpha-hydroxylating monooxygenase (PHM).

Peptidyl alpha-hydroxylating monooxygenase (PHM) functions in vivo towards the biosynthesis of alpha-amidated peptide hormones in mammals and insects. PHM is a potential target for the development of inhibitors as drugs for the treatment of human disease and as insecticides for the management of insect pests. We show here that relatively simple ground state analogs of the PHM substrate hippuric acid (C(6)H(5)-CO-NH-CH(2)-COOH) inhibit the enzyme with K(i) values as low as 0.5microM. Substitution of sulfur atom(s) into the hippuric acid analog increases the affinity of PHM for the inhibitor. Replacement of the acetylglycine moiety, -CO-NH-CH(2)-COOH with an S-(thioacetyl)thioglycolic acid moiety, -CS-S-CH(2)-COOH, yields compounds with the highest PHM affinity. Both S-(2-phenylthioacetyl)thioglycolate and S-(4-ethylthiobenzoyl)thioglycolic acid inhibit the proliferation of cultured human prostate cancer cells at concentrations >100-fold excess of their respective K(i) values. Comparison of K(i) values between mammalian PHM and insect PHM shows differences in potency suggesting that a PHM-based insecticide with limited human toxicity can be developed.

Nonfunctional Missense Mutants in Two Well Characterized Cytosolic Enzymes Reveal Important Information About Protein Structure and Function.

The isolation and characterization of 42 unique nonfunctional missense mutants in the bacterial cytosolic ß-galactosidase and catechol 2,3-dioxygenase enzymes allowed us to examine some of the basic general trends regarding protein structure and function. A total of 6 out of the 42, or 14.29% of the missense mutants were in α-helices, 17 out of the 42, or 40.48%, of the missense mutants were in ß-sheets and 19 out of the 42, or 45.24% of the missense mutants were in unstructured coil, turn or loop regions. While α-helices and ß-sheets are undeniably important in protein structure, our results clearly indicate that the unstructured regions are just as important. A total of 21 out of the 42, or 50.00% of the missense mutants caused either amino acids located on the surface of the protein to shift from hydrophilic to hydrophobic or buried amino acids to shift from hydrophobic to hydrophilic and resulted in drastic changes in hydropathy that would not be preferable. There was generally good consensus amongst the widely used algorithms, Chou-Fasman, GOR, Qian-Sejnowski, JPred, PSIPRED, Porter and SPIDER, in their ability to predict the presence of the secondary structures that were affected by the missense mutants and most of the algorithms predicted that the majority of the 42 inactive missense mutants would impact the α-helical and ß-sheet secondary structures or the unstructured coil, turn or loop regions that they altered.

Kinetic isotope effects of nucleoside hydrolase from Escherichia coli.

rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012+/-0.006, 1.027+/-0.005, 1.134+/-0.007, 1.122+/-0.008, and 1.002+/-0.004 for [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3'-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus).

The transition state of adenosine nucleosidase (EC 3.2.2.7) isolated from yellow lupin (Lupinus luteus) was determined based upon a series of heavy atom kinetic isotope effects. Adenosine labeled with 13C, 2H, and 15N was analyzed by liquid chromatography/electrospray mass spectrometry to determine kinetic isotope effects. Values of 1.024+/-0.004, 1.121+/-0.005, 1.093+/-0.004, 0.993+/-0.006, and 1.028+/-0.005 were found for [1'-13C], [1'-2H], [2'-2H], [5'-2H], and [9-15N] adenosine, respectively. Using a bond order bond energy vibrational analysis, a transition state consisting of a significantly broken C-N bond, formation of an oxocarbenium ion in the ribose ring, a conformation of C3-exo for the ribose ring, and protonation of the heterocyclic base was proposed. This transition state was found to be very similar to the transition state for nucleoside hydrolase, another purine metabolizing enzyme, isolated from Crithidia fasciculata.