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A phylogeographic split in Buxus balearica (Buxaceae) as evidenced by nuclear ribosomal markers: when ITS paralogues are welcome.

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict.

Base Sequence↗

Phylogenetic relationships among early-diverging eudicots based on four genes: were the eudicots ancestrally woody?

Based on analyses of combined data sets of three genes (18S rDNA, rbcL, and atpB), phylogenetic relationships among the early-diverging eudicot lineages (Ranunculales, Proteales, Trochodendraceae, Sabiaceae, and Buxaceae) remain unclear, as are relationships within Ranunculales, especially the placement of Eupteleaceae. To clarify relationships among these early-diverging eudicot lineages, we added entire sequences of 26S rDNA to the existing three-gene data set. In the combined analyses of four genes based on parsimony, ML, and Bayesian analysis, Ranunculales are strongly supported as a clade and are sister to other eudicots. Proteales appear as sister to the remaining eudicots, which are weakly (59%) supported as a clade. Relationships among Trochodendraceae, Buxaceae (including Didymeles), Sabiaceae, and Proteales remain unclear. Within Ranunculales, Eupteleaceae are sister to all other Ranunculales, with bootstrap support of 70% in parsimony analysis and with posterior probability of 1.00 in Bayesian analysis. Our character reconstructions indicate that the woody habit is ancestral, not only for the basal angiosperms, but also for the eudicots. Furthermore, Ranunculales may not be ancestrally herbaceous, as long maintained. The woody habit appears to have been ancestral for several major clades of eudicots, including Caryophyllales, and asterids.

Bayes Theorem↗

Antifeedant activity of some Sri Lankan plants.

Twenty nine solvent extracts from twenty Sri Lankan plants were examined for antifeedant activity against the fourth instar larvae of Mexican bean beetle, Epilachna varivestis Muls., Coccinellidae. Extracts of Sarcococca brevifolia (Buxaceae), Strychnus nux vomica (Loganiaceae), Diploclisia glaucescens, Coscinium fenestratum (Menispermaceae), Syzygium caryophyllatum (Myrtaceae), Pittosporium zeylanicum (Pittosporaceae), Lasianthus gardneri (Rubiaceae), Ferronia limonia (Rutaceae) and Allophylus cobbe (Sapindaceae) have shown strong antifeedant activity.

Animals↗

Phylogenetic relationships of the Santalales and relatives.

Determining relationships among parasitic angiosperms has often been difficult owing to frequent morphological reductions in floral and vegetative features. We report 18S (small-subunit) rRNA sequences for representative genera of three families within the Santalales (Olacaceae, Santalaceae, and Viscaceae) and six outgroup dicot families (Celastraceae, Cornaceae, Nyssaceae, Buxaceae, Apiaceae, and Araliaceae). Using Wagner parsimony analysis, one most parsimonious tree resulted that shows the Santalales to be a holophyletic taxon most closely related to Euonymus (Celastraceae). The santalalean taxa showed approximately 13% more transitional mutations than the group of seven other dicot species. This suggests a higher fixation rate for mutations in these organisms, possibly owing to a relaxation of selection pressures at the molecular level in parasitic vs nonparasitic plants. Outgroup relationships are generally in accord with current taxonomic classifications, such as the grouping of Nyssaceae and Cornaceae together (Cornales) and the grouping of Araliaceae with Apiaceae (Apiales). These data provide the first nucleotide sequences for any parasitic flowering plant and support the contention that rRNA sequence analysis can result in robust phylogenetic comparisons at the family level and above.

Base Sequence↗

Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants.

The chloroform:medianol (1:1) extracts of a number of the plant species belonging to eight families, namely Corydalis solida (L.) Swartz subsp. solida and Glaucium corniculatum (L.) J. H. Rudolph (Papaveraceae), Rhododendron ponticum L. subsp. ponticum and Rhododendron luteum Sweet. (Ericaceae), Buxus sempervirens L. (Buxaceae), Vicia faba L. (Fabaceae), Robinia pseudoacacia L. (Caeselpiniaceae), Tribulus terrestris L. and Zygophyllum fabago L. (Zygophyllaceae), Lycopodium clavatum L. (Lycopodiaceae), Fumaria vaillantii Lois., Fumaria capreolata L., Fumaria kralikii Jordan, Fumaria asepala Boiss., Fumaria densiflora DC., Fumaria flabellata L., Fumaria petteri Reichb. subsp. thuretii (Boiss.) Pugsley, Fumaria macrocarpa Boiss. ex Hausskn., Fumaria cilicica Hauskkn., Fumaria parviflora Lam. and Fumaria judaica Boiss. (Fumariaceae) were screened for their anticholinesterase activity on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes by in vitro Ellman method at 10 microg/ml and 1 mg/ml concentrations. The extracts did not show any noticeable inhibitory activity against both of the enzymes at 10 microg/ml. The extracts of Rhododendron ponticum subsp. ponticum, Rhododendron luteum, Corydalis solida subsp. solida, Glaucium corniculatum, and Buxus sempervirens showed remarkable inhibitory activity above 50% inhibition rate on AChE at 1 mg/ml. Among them, Rhododendron ponticum subsp. ponticum, Corydalis solida subsp. solida and Buxus sempervirens were the most active extracts against BChE having 95.46 +/- 1.03%, 93.08 +/- 0.97%, and 93.45 +/- 0.88% inhibition rates, respectively. Among the extracts screened, all of the Fumaria extracts displayed highly potent inhibition against both of the enzymes at 1 mg/ml concentration compared to the standard.

Butyrylcholinesterase↗

Iso-N-formyl-5-en-chonemorphine, a steroidal alkaloid from Sarcococca zeylanica.

Chemical investigation of the non-quaternary alkaloidal fraction of the aerial parts of Sarcococca zeylanica of the family Buxaceae furnished a steroidal alkaloid iso-N-formyl-5-en-chonemorphine, which has not been previously reported as a natural product. The structure of this alkaloid was established on the basis of spectroscopic evidence.

Alkaloids↗

Nematicidal activity of some Sri Lankan plants.

Nineteen solvent extracts from seven Sri Lankan plants were examined for nematicidal activity against the root knot nematode Meloidogyne incognita. Extracts of Allophyhu cobbe, Lepisanthes tetraphylla (Sapindaceae), Sarcococca zeylanica (Buxaceae) and Hedyotis lawsoniae (Rubiaceae) have shown significant nematicidal activity.

Animals↗

A simplified explanation for the frameshift mutation that created a novel C-terminal motif in the APETALA3 gene lineage.

BACKGROUND: The evolution of type II MADS box genes has been extensively studied in angiosperms. One of the best-understood subfamilies is that of the Arabidopsis gene APETALA3 (AP3). Previous work has demonstrated that the ancestral paleoAP3 lineage was duplicated at some point within the basal eudicots to give rise to the paralogous TM6 and euAP3 lineages. This event was followed in euAP3 orthologs by the replacement of the C-terminal paleoAP3 motif with the derived euAP3 motif. It has been suggested that the new motif was created by an eight-nucleotide insertion that produced a translational frameshift. RESULTS: The addition of 25 eudicot AP3 homologs to the existing dataset has allowed us to clarify the process by which the euAP3 motif evolved. Phylogenetic analysis indicates that the euAP3/TM6 duplication maps very close to the base of the core eudicots, associated with the families Trochodendraceae and Buxaceae. We demonstrate that although the transformation of paleoAP3 into euAP3 was due to a frameshift mutation, this was the result of a single nucleotide deletion. The use of ancestral character state reconstructions has allowed us to demonstrate that the frameshift was accompanied by few other nucleotide changes. We further confirm that the sequence is evolving as coding region. CONCLUSION: This study demonstrates that the simplest of genetic changes can result in the remodeling of protein sequence to produce a kind of molecular 'hopeful monster.' Moreover, such a novel protein motif can become conserved almost immediately on the basis of what appears to be a rapidly generated new function. Given that the existing data on the function of such C-terminal motifs are somewhat disparate and contradictory, we have sought to synthesize previous findings within the context of the current analysis and thereby highlight specific hypotheses that require further investigation before the significance of the euAP3 frameshift event can be fully understood.

Amino Acid Sequence↗

Cytotoxic alkaloids of Pachysandra terminalis.

Four steroidal alkaloids, epipachysamines B (1) and E (2), pachystermine A (3) and pachysamine E (4), were isolated as cytotoxic principles from the MeOH extract of the stems of Pachysandra terminalis SIEB. et ZUCC. (Buxaceae). These alkaloids showed cytotoxic activity against P388 and P388/ADR leukemia cells in vitro. Three of the alkaloids (1-3) were previously isolated from this plant material, and this is the first report of their cytotoxic activity. Pachysamine E (4) is a new alkaloid.

Alkaloids↗

[Studies on the steroidal alkaloids from Sarcococca vagans].

The root of Sarcococca vagans Stapt (Buxaceae) is well-known Chinese folk drug called as "Jie Gu Mu", distributed in southern of China. They are used in treatment of traumatic injury. Four new steroidal alkaloids, named sarcovagine A, B, C and D, were isolated from the root of Sarcococca vagans. Their structures were established on the basis of spectral evidence.

Alkaloids↗

Studies on cardio-suppressant, vasodilator and tracheal relaxant effects of Sarcococca saligna.

Sarcococca saligna is a shrub that is traditionally used for its medicinal properties in Pakistan. In this study we report the cardio-suppressant, vasodilator and tracheal relaxant activities of the aqueous-methanolic extract (Ss.Cr) of the plant. Ss.Cr, that tested positive for the presence of saponins, flavonoids, tannins, phenols, and alkaloids, exhibited a dose-dependent (0.3-5 mg/mL) negative inotropic and chronotropic effect on the isolated guinea-pig atrium which was resistant to atropine (1 microM) and aminophylline (10 microM) pretreatment. In rabbit thoracic aorta, Ss.Cr dose-dependently (0.1-3 mg/mL) relaxed the high K+ (80 mM) and phenylephrine (PE, 1 microM)-induced contractions, indicating a possible Ca++ channel blocking (CCB) effect. When tested against PE (1 microM) control peaks in normal Ca++ and Ca++-free Kreb's solution, Ss.Cr exhibited dose-dependent (0.1-3 mg/mL) inhibition, being more potent in relaxing the PE responses in Ca++-free Kreb's solution, thus indicating specific blockade of Ca++ release from the intracellular stores. Ss.Cr also relaxed the agonist-induced contractions in: a) rat aorta irrespective of the presence of endothelium or nitric oxide synthase inhibitor L-NAME and b) rabbit and guinea-pig tracheal strips. The data shows that Ss.Cr possesses possible Ca++ channel blocking activity which might be responsible for its observed cardio-suppressant, vasodilator and tracheal relaxant effects though more tests are required to confirm this Ca++ channel blocking effect.

Aminophylline↗

3D-QSAR studies on natural acetylcholinesterase inhibitors of Sarcococca saligna by comparative molecular field analysis (CoMFA).

We have derived a comprehensive structure-activity relationship (SAR) picture for a new series of natural acetylcholinesterase inhibitors isolated from Sarcococca saligna. A set of 32 previously isolated and tested pregnane-type steroidal alkaloids inhibitors were investigated with respect to their IC(50) values (pIC(50)) against the AChE enzyme in order to derive CoMFA models using atom-based alignment. A highly significant CoMFA model was obtained with r(2) value of 0.974. The q(2) (cross validation r(2)) value also confirms the statistical significance of our model.

Acetylcholinesterase↗

Cholinesterase inhibitory and spasmolytic potential of steroidal alkaloids.

A new steroidal alkaloid, isosarcodine (1) along with four known bases, sarcorine (2), sarcodine (3), sarcocine (4) and alkaloid-C (5) were isolated from the MeOH extract of Sarcococca saligna. The structures of these alkaloids were identified by spectral data interpretation. These compounds were subjected to acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition studies, and were found to be noncompetitive inhibitors of AChE (Ki = 21.8, 90.3, 32.2, 16.0 and 50.0 microM, respectively) and uncompetitive or noncompetitive inhibitors of BChE (Ki = 8.3, 7.5, 15.6, 5.0 and 12.0 microM, respectively). The compounds (2-5) also showed dose-dependent spasmolytic activity in the rabbit jejunum intestinal preparations and also relaxed the high K+ (80 mM)-induced contraction, indicative of a calcium channel-blocking mechanism. Structure-activity relationship suggested that the nitrogen substituents at C-3 and/or C-20 of steroidal skeleton and the hydrophobic properties of the pregnane skeleton are the key structural features contributed to the inhibitory potency of these steroidal alkaloids against AChE and BChE.

Acetamides↗

Cholinesterase inhibitory pregnane-type steroidal alkaloids from Sarcococca hookeriana.

The bioassay-guided phytochemical investigation on Sarcococca hookeriana have resulted in the isolation of four new pregnane-type steriodal alkaloids: hookerianamide-D [(2'E,20S)-20-(N,N-formyl(methyl)amino)-3beta-(3',4'-dimethyl-2'-pentenamido)-5alpha-pregnane] (1), hookerianamide-E [(2'E,20S)-20-(N,N-dimethylamino)-3beta-(senecioylamino)-5alpha-pregn-14-en-2beta-O-acetate] (2), hookerianamide-F [(2'E,20S)-20-(N-methylamino)-3beta-(tigloylamino)-5alpha-pregn-2,14-dien-4-one] (3), and hookerianamide-G [(20S)-20-(N,N-dimethylamino)-3beta-(N-methylbenzamido)-5alpha-pregn-4beta-O-acetate] (4), along with five known alkaloids 5-9. Their structures were determined by spectroscopic analysis. These steroidal alkaloids and chemically derived derivatives of compound 5 have displayed varying degree of inhibitory activities against acetylcholinesterase and butyrylcholinesterase enzymes in a concentration-dependent fashion, with the IC(50) values ranging from 1.5 to 148.2 and 0.6 to 100.2 microM, respectively.

Acetylcholinesterase↗

Molecular docking studies of natural cholinesterase-inhibiting steroidal alkaloids from Sarcococca saligna.

Alkaloids isolated from Sarcococca saligna significantly inhibit acetyl- and butyrylcholinesterase enzyme, suggesting discovery of inhibitors for nervous-system disorders. Studying interactions with the active site of the AChE enzyme from Torpedo californica, we have identified hydrophobic interactions inside the aromatic gorge area as the major stabilizing factor in enzyme-inhibitor complexes of these alkaloids. Molecular Dynamics simulation of a predicted complex indicates that ligand binding does not extensively alter enzyme structure, but reduces flexibility at the gorge.

Acetylcholinesterase↗

Presence of antispasmodic, antidiarrheal, antisecretory, calcium antagonist and acetylcholinesterase inhibitory steroidal alkaloids in Sarcococca saligna.

The aim of this investigation was to see if the crude extract of Sarcococca saligna (Ss.Cr) contains chemicals with gut function inhibitory activity by using in vitro and in vivo assays. Ss.Cr caused a dose-dependent (0.03 - 3 mg/mL) inhibitory effect on K+-induced contractions in rat stomach fundus, guinea-pig ileum and rabbit jejunum preparations. The calcium channel blocking(CCB) activity was confirmed when Ss.Cr caused a rightward shift in the Ca++ dose-response curves. It also potentiated, at lower do-ses (0.001 - 0.03 mg/mL), the contractile effect of a fixed dose of acetylcholine (ACh), similar to physostigmine, and suppressed the effect of ACh at higher doses (0.3 - 1.0 mg/mL). Both Ss.Cr and physostigmine inhibited acetylcholinesterase (AChE), in the in vitro assay, confirming the AChE inhibitory activity. In the in vivo studies, Ss.Cr exhibited antidiarrheal and antisecretory activities against castor oil-induced diarrhea and intestinal fluid accumulation in mice. Characteristic steroidal compounds of the plant (saracocine, saracodine, saracorine and alkaloid-C), exhibited a similar combination of AChE inhibitory and CCB activities. Thus this study provides a sound mechanistic base for some of the traditional uses of the plant in hyperactive gut states, in addition to providing the first evidence for verapamil to possess additional AChE inhibitory activity. Furthermore, these characteristic compounds with dual activity may be good candidates for further studies on their usefulness in Alzheimer's disease.

Alkaloids↗