8-Oxoerythrinine: An alkaloid from Erythrina brucei

The flowers of Erythrina brucei afforded, in addition to known compounds, a new erythrina alkaloid, 8-oxoerythrinine. Its structure was elucidated by spectroscopic investigations as well as by chemical transformations.     

Comparison of Mineral Balances in Germfree and Conventional Mice When Sodium Phytate is Added to Purified Diet

A strain of Alcaligenes isolated from soil was a good producer of β-glucuronidase, and the enzyme was purified from the cell-free extract by sequential column chromatography on DEAE-Toyopearl, Toyopearl HW-55F, and Phenyl-Sepharose CL-4B. By these procedures, two β-glucuronidases designated as β-glucuronidases I and II were purified 240- and 508-fold, respectively. β-Glucuronidase I, with a molecular weight of 75,000, had an optimum pH at 7.5 and the enzyme II, with a molecular weight of 300,000, had maximum activity at pH 6.0. Both enzymes were strongly inhibited by saccharo-1,4-lactone, glucaro-δ-lactam, p-chloromercuribenzoate, Hg²⁺, and N-bromosuccinimide. β-Glucuronidase I was active toward estrogen-3-β-glucuronides and inert toward β-glucuronide conjugates of menthol, estrogen-17β-, estrogen-16α-, androsterone-3α-, testosterone-17β-, cortisol-17α-. β-Glucuronidase II hydrolyzed all of these substrates. β-Glucuronidase I was inhibited by phenolphthalein and its glucuronide.     

Characteristics of Scopolamine-releasing Hairy Root Clones of Duboisia leichhardtii

A good scopolamine-releasing hairy root clone, DL47-1, was developed from Duboisia leichhardtii. Hyoscyamine 6β-hydroxylase activity of the clone DL47-1 in the medium for alkaloid release was 2-fold higher than that in the medium for hairy root growth. Other high scopolamine-producing clones also tended to have a high enzyme activity.     

Aporphine Alkaloids from Ocotea puberula with Anti-Trypanosoma Cruzi Potential – Activity of Dicentrine-β-N-Oxide in the Plasma Membrane Electric Potentials

One new aporphine, dicentrine-β-N-oxide ( 1 ), together with five related known alkaloids dehydrodicentrine ( 2 ), predicentrine ( 3 ), N-methyllaurotetanine ( 4 ), cassythicine ( 5 ), and dicentrine ( 6 ) were isolated from the leaves of Ocotea puberula (Lauraceae). Antiprotozoal activity of the isolated compounds was evaluated in vitro against trypomastigote forms of Trypanosoma cruzi. Among the tested compounds, alkaloid 1 exhibited higher potential with EC₅₀ value of 18.2 μM and reduced toxicity against NCTC cells (CC₅₀>200 μM – SI>11.0), similar to positive control benznidazole (EC₅₀ of 17.7 μM and SI=10.7). Considering the promising results of dicentrine-β-N-oxide ( 1 ) against trypomastigotes, the mechanism of parasite death caused by this alkaloid was investigated. As observed, this compound reached the plasma membrane electric potential directly after 2 h of incubation and triggered mitochondrial depolarization, which probably leads to trypomastigote death. Therefore, dicentrine-β-N-oxide ( 1 ), reported for the first time in this work, can contribute to future works for the development of new trypanocidal agents.     

Diversity of Methylxanthine Content inIlex cassine L. andIlex vomitoria Ait.: Assessing Sources of the North American Stimulant Cassina

Indigenous people of southeastern North America drank cassina, a stimulant and emetic decoction that the colonial British termed “black drink.” Though most authors citeIlex vomitoria Ait. as the botanical source of cassina, confusion persists because some researchers identify the source asI. cassine L. To clarify the link between plant and product, the methylxanthine alkaloid contents ofI. vomitoria andI. cassine were compared. Since methylxanthines (i.e., caffeine, theobromine, and theophylline) have pharmacological properties congruent with the recorded effects of cassina consumption, the alkaloids provide a chemical basis for the evaluation of both taxa as sources of the beverage. Methylxanthine levels are higher inI. vomitoria than inI. cassine, and the principal alkaloid of the former is caffeine. Based on its alkaloid content,I. vomitoria is the best-supported candidate source of cassina.     

Analysis of Phosphorylation of Wheat Elongation Factor 1β and β′ by Casein Kinase II

The purified casein kinase II (CK II) from Arabidopsis thaliana phosphorylates wheat elongation factor Iβ (EF- Iβ), but not elongation factor Iβ’ (EF-lβ’), which lacks a serine residue in the conserved phosphorylation site. Both EF-1β and1β’ subunits, with similar functions, seem to undergo different regulation despite the partial amino acid sequence of EF-lβ being similar to that of EF-1β’.     

Detection of DNA polymorphism in Papaver somniferum genotypes differing in straw morphinan alkaloid content

Abstract

Thirty‐two distinct accessions of Papaver somniferum were screened for morphinan alkaloid content in the straw. The combined content of major morphinan alkaloids (morphine+codeine+thebaine) was found to vary in the range 0.2260–0.0683%. Two genotypes each, were selected as prototypes for low [I‐48 (0.0683%) and I‐344 (0.0878%)] and high [Pps‐1 (0.2260%) and N‐3 (0.2074%)] morphinan alkaloid content for studying DNA polymorphism. RAPD analysis of these four genotypes using 80 primers could not detect the polymorphism. However, AFLP analysis of these genotypes with 12 EcoRI/MseI primer pairs could distinctly group the high‐ and low‐morphinan alkaloid genotypes separately. Furthermore, 50 AFLP fragments, specific to high‐straw morphinan alkaloid genotypes (Pps‐1 and N‐3) and 28 DNA fragments specific to low‐straw morphinan alkaloid genotypes (I‐48 and I‐344) could be identified. This investigation is the first report on the polymorphism identified in the genotypes differing in their straw morphinan alkaloid content. This DNA polymorphism could be exploited for defining chemotypes at an early seedling stage in poppy breeding programmes.     

Further constituents of Galianthe thalictroides (Rubiaceae) and inhibition of DNA topoisomerases I and IIα by its cytotoxic β-carboline alkaloids

A new cytotoxic β-carboline alkaloid, 1-methyl-3-(2-hydroxypropan-2-yl)-2-(5-methoxy-9H-β-carbolin-1-yl)-cyclopentanol (1), was isolated from roots of Galianthe thalictroides, together with the alkaloid 1-(hydroxymethyl)-3-(2-hydroxypropan-2-yl)-2-(5-methoxy-9H-β-carbolin-1-yl)-cyclopentanol (2), the anthraquinones 1-methyl-alizarin and morindaparvin-A, the coumarin scopoletin, homovanillic alcohol, (−)-epicatechin, and the steroids stigmast-4-en-3-one, 4,22-stigmastadien-3-one, campest-4-en-3-one, stigmast-4-en-3,6-dione, 6-β-hydroxy-stigmast-4-en-3-one, stigmasterol, campesterol, β-sitosterol, and β-sitosterol-3-O-β-d-glucopyranoside. Among the previously known compounds, homovanillic alcohol is a novel finding in Rubiaceae, while 1-methyl-alizarin, morindaparvin-A, scopoletin, stigmast-4-en-3-one, 4,22-stigmastadien-3-one, campest-4-en-3-one, stigmast-4-en-3,6-dione, and 6-β-hydroxy-stigmast-4-en-3-one is reported for the first time in the genus Galianthe. The cytotoxic β-carboline alkaloids 1 and 2 exhibited potent antitopoisomerase I and IIα activities and strong evidence is provided for their action as topoisomerase IIα poisons and redox-independent inhibitors.     

Stemospironine,a New Insecticidal Alkaloid of Stemona japonica Miq. Isolation,Structural Determination and Activity

A new alkaloid named stemospironine (I) was isolated together with stemofoline (II) as the main insecticidal constituents of leaves and stems of Stemona japonica Miq. The absolute stereostructure of I has been determined by X-ray crystallographic analysis. Stemofoline (II) showed a much stronger activity than I against silkworm larvae (Bombyx mori L.) by oral administration.     

A Novel Tropoloisoquinoline Alkaloid,Neotatarine, from Acorus calamus L.

A novel tropoloisoquinoline alkaloid, neotatarine ( 1 ), was isolated from the 95% ethanol extract of the rhizome parts of Acorus calamus L. The chemical structure was unambiguously elucidated by spectroscopic and single‐crystal X‐ray diffraction analysis. Neotatarine ( 1 ) exhibited significantly inhibitory activity against Aβ_{25 – 35} induced PC12 cell death with 2, 4 and 8 μm comparing with the assay control (P < 0.01).     

Alkaloids and triterpenes from the african toddalioideae

Stem and root barks of Araliopsis tabouensis, Diphasia klaineana and Teclea verdoorniana have been investigated. A. tabouensis yielded flindissol (I), (+)-N-methylplatydesminium ion (III) and a second furoquinoline alkaloid: D. klaineana yielded lupeol (IX). evoxanthine (XI), arborinine (X) and skimmianine (V). T. verdoorniana gave lupeol (IX) and evoxanthine (XI). A further alkaloid, designated DK/1, was isolated from both the Diphasia and Teclea species. The value, as chemotaxonomic indicators, of the alkaloids and other constituents known to occur in six African genera of the Toddalioideae has been examined and possible affinities with other rutaceous taxa are suggested.     

Purification and Some Properties of Urethane Hydrolase II from Lactobacillus casei ω ATCC 7469

The β-1,3-glucanase (1,3-β-d-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKUβG1 (8.2 kb), showed a high β-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the peripiasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-β-1,3-glucanase II and a nonlytic endo-β-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-β-1,3-glucanase I was also formed by a proteolytic digestion of endo-β-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-β-1,3-glucanase II was coded for in the cloned gene.     

Solanopubamine,a steroidal alkaloid from Solanum pubescens

Aerial parts of Solanum pubescens yielded a new steroidal alkaloid, solanopubamine, the structure of which was elucidated as 3β-amino-5α,22αH,25βH-solanidan-23β-ol by ¹³C NMR, ¹H NMR, IR, mass spectral analysis and chemical degradation methods.     

Isolation and Characterization of Two β-d-Glucosidases from Bifidobacterium breve 203

Two β-d-glucosidases were purified to homogeneity from Bifidobacterium breve 203: one ( β-d-glucosidase I; molecular weight, 96,000) showed reactivity toward p-nitrophenyl (p-NP) β-d-fucoside, 74% of that to p-NP β-d-glucoside, and the other ( β-dglucosidase II; molecular weight, 450,000) did not. They also differed in their thermal and pH stabilities. Laminaribiose, cellobiose and gentiobiose were hydrolyzed by β-d-glucosidase I, with 53%, 34% and 3% of the reactivity in the case of p-NP β-d-glucoside, and by β-dglucosidase II, with 53%, 6% and 107% of the reactivity. The reaction of β-dglucosidase I with p-NP β-dfucoside was enhanced by the addition of glucose and other monosaccharides to the reaction mixture, whereas that with p-NP β-dglucoside was not affected. The activity of β-dglucosidase II with p-NP β-dglucoside was inhibited by glucose.     

Polymorphisms of estrogen receptors alpha and beta in idiopathic, infertile Brazilian men: a case-control study

Estrogen plays an important role in the human reproductive system and its action is mediated mainly by two specific receptors: α (ERα) and β (ERβ). There are polymorphic variants in both ER genes, and studies showed their association with reproductive outcomes. We aimed to determine the distribution of ERα and ERβ gene polymorphisms in idiopathic, infertile Brazilian patients in a case–control study comprising 187 idiopathic, infertile Brazilian men with nonobstructive azoospermia (NOA, n = 78) or severe oligozoospermia (SO, n = 109) and 216 fertile men. Detection of ERα (PvuII and XbaI) and ERβ (AluI and RsaI) gene polymorphisms were performed using TaqMan PCR. The results were analyzed statically, and a P‐value < 0.05 was considered significant. Single‐marker analysis revealed that neither PvuII nor XbaI polymorphisms of the ERα gene were associated either with NOA group (P = 0.662 and P = 0.527, respectively) or SO group (P = 0.777 and P = 1.0, respectively). Regarding ERβ polymorphisms, no statistical difference was observed between the AluI polymorphism and NOA group compared to controls (P = 1.0) or between SO group and controls (P = 0.423). We found similar results with the RsaI polymorphism. Statistical analysis did not reveal a difference between NOA (P = 0.740) and SO (P = 0.920) groups compared to controls. Combined genotypes of ERα and ERβ polymorphisms did not identify a haplotype associated with idiopathic infertility. Thus, in the Brazilian population, genetic variations in both estrogen receptors alpha (PvuII and XbaI) and beta (AluI and RsaI) were not relevant to idiopathic infertility. Mol. Reprod. Dev. 78:665–672, 2011. © 2011 Wiley‐Liss, Inc.     

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of β‐2‐microglobulin by antibody BBM.1

Human β‐2‐microglobulin (β2m) is the light chain of human leucocyte antigen‐I (HLA‐I). It can disassociate from HLA‐I and accumulate to cause serious dialysis‐related amyloidosis (DRA) in long‐term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free‐form and HLA‐I associated β2m. It can be used for specific elimination of β2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv‐β2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu⁴⁴ and Arg⁴⁵ of β2m have direct interactions with BBM.1, while Asp³⁸ exerts its function mainly via stabilization of Arg⁴⁵. In addition, Arg⁸¹ of β2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA‐I heavy chain. These results provide insights into the molecular basis of the recognition of β2m by BBM.1 and explain why BBM.1 can bind both free‐form and HLA‐1 associated β2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other β2m‐targeting mAbs for therapeutic purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

Detailed Structure of an Acidic Polysaccharide in Soy Sauce,Confirmed by Use of Two Kinds of Purified Pectinases

The partially degraded polysaccharide obtained by means of mild acid hydrolysis of APS–I, an acidic polysaccharide in soy sauce, was incubated with an endo-polygalacturonase, and some acidic sugars liberated were fractionated and purified from the enzymatic hydrolyzate. They were identified as d-galacturonic acid (Gal A), its α-l,4-linked dimer and trimer, d-xylose (Xyl) β1→3 Gal A and Xyl β1→3 Gal A α1→4 Gal A, and an acidic polymer composed of the above-mentioned sugars through methylation analysis and other methods.

Methyl-esterized APS–I was subjected to β-elimination with pectin lyase or by heat treatment. The high molecular fraction which was highly resistant to β-elimination, was concluded to be β-1,4-linked d-galactan of which reducing-end group was attached to d-galacturonic acid. On the basis of these findings and previous knowledges, the construction of APS–I was also discussed.     

Distinct solid and solution state self‐assembly pathways of RADA16‐I designer peptide

Solid state NMR measurements on selectively ¹³C‐labeled RADA16‐I peptide (COCH₃–RADARADARADARADA–NH₂) were used to obtain new molecular level information on the conversion of α‐helices to β‐sheets through self‐assembly in the solid state with increasing temperature. Isotopic labeling at the A4 C_β site enabled rapid detection of ¹³C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α‐helix and β‐strand content. These samples were then probed at room temperature for intermolecular ¹³C–¹³C nuclear dipolar couplings with the PITHIRDS‐CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X‐ray diffraction. Independence of PITHIRDS‐CT decay shapes on overall α‐helical and β‐strand content infers that β‐strands are not observed without association with β‐sheets, indicating that β‐sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS‐CT NMR data were compared with results of similar measurements on RADA16‐I nanofibers produced by self‐assembly in aqueous salt solution. We report that β‐sheets formed through self‐assembly in the solid state have a structure that differs from those formed through self‐assembly in the solution state. Specifically, solid state RADA16‐I self‐assembly produces in‐register parallel β‐sheets, whereas nanofibers are composed of stacked parallel β‐sheets with registry shifts between adjacent β‐strands in each β‐sheet. These results provide evidence for environment‐dependent self‐assembly mechanisms for RADA16‐I β‐sheets as well as new constraints on solid state self‐assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

(−)-3β-13α-dihydroxylupanine from cytisus scoparius

A new lupin alkaloid, (−)-3β,13α-dihydroxylupanine was isolated from Cytisus scoparius together with five known sparteine-type lupin alkaloids and tyramine. The absolute structure of the new alkaloid was confirmed by comparison of the natural product with the synthetic sample derived from (+)-13-hydroxylupanine. It was also shown that the alkaloid constituents of C. scoparius differed considerably in the aerial parts, flowers and seeds.     

Feeding behavior of aphids on narrow‐leafed lupin (Lupinus angustifolius) genotypes varying in the content of quinolizidine alkaloids

Since the beginning of breeding narrow‐leafed lupins [Lupinus angustifolius L. (Fabaceae)] with a low alkaloid content, susceptibility to several aphid species has increased. Therefore, the probing and feeding behavior of Aphis fabae Scopoli, Aphis craccivora Koch, Acyrthosiphon pisum (Harris), Myzus persicae (Sulzer), and the well‐adapted Macrosiphum albifrons Essig (all Hemiptera: Aphididae) was studied over 12 h on narrow‐leafed lupin genotypes containing varying amounts and compositions of alkaloids. We used the electrical penetration graph (EPG) technique to obtain information on the influence of alkaloid content and composition on the susceptibility to various aphid species. Results indicated that the total time of probing of A. fabae, A. craccivora, A. pisum, and M. persicae increased with a reduced alkaloid content, whereas the alkaloid content had no influence on M. albifrons. Almost all of the individuals (>93%) conducted sieve element phases on the highly susceptible genotype Bo083521AR (low alkaloid content). A reduced occurrence of phloem phases was observed during the 12‐h recording on the alkaloid‐rich cultivar Azuro, especially for A. pisum (37.5%) and A. fabae (55.0%). Furthermore, aphids feeding on genotypes with low alkaloid content had in most cases significantly longer sieve element phases than when feeding on resistant genotypes (Kalya: low alkaloid content, yet resistant; Azuro: high alkaloid content, resistant), whereas M. albifrons showed the longest phloem phase on the alkaloid‐rich cultivar Azuro. As most significant differences were found in phloem‐related parameters, it is likely that the most important plant factors influencing aphid probing and feeding behavior are localized in the sieve elements. The aphids’ feeding behavior on the cultivar Kalya, with a low alkaloid content but reduced susceptibility, indicates that not only the total alkaloid content influences the feeding behavior but additional plant factors have an impact.