Phylogeny and biogeography of Tiliacoreae (Menispermaceae), a tribe restricted to tropical rainforests.

BACKGROUND AND AIMS: Modern tropical rainforests house the highest biodiversity of Earth's terrestrial biomes and are distributed in three low-latitude areas. However, the biogeographical patterns and processes underlying the distribution of biodiversity among these three areas are still poorly known. Here, we used Tiliacoreae, a tribe of pantropical lianas with a high level of regional endemism, to provide new insights into the biogeographical relationships of tropical rainforests among different continents. METHODS: Based on seven plastid and two nuclear DNA regions, we reconstructed a phylogeny for Tiliacoreae with the most comprehensive sampling ever. Within the phylogenetic framework, we then estimated divergence times and investigated the spatiotemporal evolution of the tribe. KEY RESULTS: The monophyletic Tiliacoreae contain three major clades, which correspond to Neotropical, Afrotropical and Indo-Malesian/Australasian areas, respectively. Both Albertisia and Anisocycla are not monophyletic. The most recent common ancestor of Tiliacoreae occurred in Indo-Malesia, the Afrotropics and Neotropics in the early Eocene, then rapidly diverged into three major clades between 48 and 46 Ma. Three dispersals from Indo-Malesia to Australasia were inferred, one in the middle Eocene and two in the late Oligocene-late Miocene, and two dispersals from the Afrotropics to Indo-Malesia occurred in the late Eocene-Oligocene. CONCLUSIONS: The three main clades of Anisocycla correspond to three distinct genera [i.e. Anisocycla sensu stricto and two new genera (Georgesia and Macrophragma)]. Epinetrum is a member of Albertisia. Our findings highlight that sea-level fluctuations and climate changes in the Cenozoic have played important roles in shaping the current distribution and endemism of Tiliacoreae, hence contributing to the knowledge on the historical biogeography of tropical rainforests on a global scale.

Clerodane Furanoditerpenoids from Tinospora bakis (A.Rich.) Miers (Menispermaceae).

Tinospora bakis (A.Rich.) Miers (Menispermaceae) has traditionally been used to alleviate headaches, rheumatism, mycetoma, and diabetes, among others. Despite its extensive use, the active components of the plant have never been investigated. In this work, a series of furanoditerpenoids (1-18) and five compounds from other classes (19-23) were isolated from T. bakis. Notably, two new compounds were discovered and named: tinobakisin (1) and tinobakiside (10). Their molecular structures were elucidated with NMR, MS, UV, IR, and ECD spectra. Additionally, known compounds (2-9 and 11-23) were corroboratively identified through spectral comparisons with previously reported data, while highlighting and addressing some inaccuracies in the prior literature. Remarkably, compounds 6, 7, 13, and 17 exhibited a superior anti-glycation effect, outperforming established agents like rutin and quercetin in a lab model of protein glycation with glucose. The overall findings suggest that furanoditerpenoids play a crucial role in the antidiabetic properties of T. bakis. This research marks the first comprehensive phytochemical investigation of T. bakis, opening the door for further investigation into furanoditerpenoids and their biological mechanisms.

A Review of Fibraurea tinctoria and Its Component, Berberine, as an Antidiabetic and Antioxidant.

Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia caused by resistance to insulin action, inadequate insulin secretion, or excessive glucagon production. Numerous studies have linked diabetes mellitus and oxidative stress. People with diabetes usually exhibit high oxidative stress due to persistent and chronic hyperglycemia, which impairs the activity of the antioxidant defense system and promotes the formation of free radicals. Recently, several studies have focused on exploring natural antioxidants to improve diabetes mellitus. Fibraurea tinctoria has long been known as the native Borneo used in traditional medicine to treat diabetes. Taxonomically, this plant is part of the Menispermaceae family, widely known for producing various alkaloids. Among them are protoberberine alkaloids such as berberine. Berberine is an isoquinoline alkaloid with many pharmacological activities. Berberine is receiving considerable interest because of its antidiabetic and antioxidant activities, which are based on many biochemical pathways. Therefore, this review explores the pharmacological effects of Fibraurea tinctoria and its active constituent, berberine, against oxidative stress and diabetes, emphasizing its mechanistic aspects. This review also summarizes the pharmacokinetics and toxicity of berberine and in silico studies of berberine in several diseases and its protein targets.

In Vitro and In Silico Evaluation of Cholinesterase Inhibition by Alkaloids Obtained from Branches of Abuta panurensis Eichler.

Alkaloids are natural products known as ethnobotanicals that have attracted increasing attention due to a wide range of their pharmacological properties. In this study, cholinesterase inhibitors were obtained from branches of Abuta panurensis Eichler (Menispermaceae), an endemic species from the Amazonian rainforest. Five alkaloids were isolated, and their structure was elucidated by a combination of 1D and 2D 1H and 13C NMR spectroscopy, HPLC-MS, and high-resolution MS: Lindoldhamine isomer m/z 569.2674 (1), stepharine m/z 298.1461 (2), palmatine m/z 352.1616 (3), 5-N-methylmaytenine m/z 420.2669 (4) and the N-trans-feruloyltyramine m/z 314.1404 (5). The compounds 1, 3, and 5 were isolated from A. panurensis for the first time. Interaction of the above-mentioned alkaloids with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes was investigated in silico by molecular docking and molecular dynamics. The molecules under investigation were able to bind effectively with the active sites of the AChE and BChE enzymes. The compounds 1-4 demonstrated in vitro an inhibitory effect on acetylcholinesterase with IC50 values in the range of 19.55 µM to 61.24 µM. The data obtained in silico corroborate the results of AChE enzyme inhibition.

Antidepressant effects of total alkaloids of Fibraurea recisa on improving corticosterone-induced apoptosis of HT-22 cells and chronic unpredictable mild stress-induced depressive-like behaviour in mice.

CONTEXT: Fibraurea recisa Pierre. (Menispermaceae) (FR) is a traditional Chinese medicine known as "Huangteng." The total alkaloids of FR (AFR) are the main active ingredients. However, the pharmacological effects of AFR in the treatment of depression have not been reported. OBJECTIVES: This study investigates the antidepressant effects of AFR by network pharmacology and verification experiments. MATERIALS AND METHODS: Compound-Target-Pathway (C-P-T) network of FR and depression was constructed through network pharmacology. In vitro, HT-22 cells were treated with corticosterone (CORT) solution (0.35 mg/mL), then AFR (0.05 mg/mL) solution and inhibitor AZD6244 (14 µM/mL) or BAY11-7082 (10 µM/mL) were added, respectively. The cell viability was detected by CCK-8. In vivo, C57BL/6 mice were divided into 5 groups, namely the normal group, the CUMS group, the AFR (400 mg/kg) group, and the 2 groups that were simultaneously administered the inhibitory group AZD6244 (8 mg/kg) and BAY11-7082 (5 mg/kg). Western blotting was used to assess the expression level of the proteins. RESULTS: AFR could protect HT-22 cells from CORT-induced damage and increase the cell viability from 49.12 ± 3.4% to 87.26 ± 1.5%. Moreover, AFR significantly increased the levels of BDNF (1.3, 1.4-fold), p-ERK (1.4, 1.2-fold) and p-CERB (1.6, 1.3-fold), and decreased the levels of NLRP3 (11.3%, 31.6%), ASC (19.2%, 34.2%) and caspase-1 (18.0%, 27.6%) in HT-22 cells and the hippocampus, respectively. DISCUSSION AND CONCLUSIONS: AFR can improve depressive-like behaviours and can develop drugs for depression treatment. Further studies are needed to validate its potential in clinical medicine.

Complete chloroplast genome of Stephania tetrandra (Menispermaceae) from Zhejiang Province: insights into molecular structures, comparative genome analysis, mutational hotspots and phylogenetic relationships.

BACKGROUND: The Stephania tetrandra S. Moore (S. tetrandra) is a medicinal plant belonging to the family Menispermaceae that has high medicinal value and is well worth doing further exploration. The wild resources of S. tetrandra were widely distributed in tropical and subtropical regions of China, generating potential genetic diversity and unique population structures. The geographical origin of S. tetrandra is an important factor influencing its quality and price in the market. In addition, the species relationship within Stephania genus still remains uncertain due to high morphological similarity and low support values of molecular analysis approach. The complete chloroplast (cp) genome data has become a promising strategy to determine geographical origin and understand species evolution for closely related plant species. Herein, we sequenced the complete cp genome of S. tetrandra from Zhejiang Province and conducted a comparative analysis within Stephania plants to reveal the structural variations, informative markers and phylogenetic relationship of Stephania species. RESULTS: The cp genome of S. tetrandra voucher ZJ was 157,725 bp, consisting of a large single copy region (89,468 bp), a small single copy region (19,685 bp) and a pair of inverted repeat regions (24,286 bp each). A total of 134 genes were identified in the cp genome of S. tetrandra, including 87 protein-coding genes, 8 rRNA genes, 37 tRNA genes and 2 pseudogene copies (ycf1 and rps19). The gene order and GC content were highly consistent in the Stephania species according to the comparative analysis results, with the highest RSCU value in arginine (1.79) and lowest RSCU value in serine of S. tetrandra, respectively. A total of 90 SSRs have been identified in the cp genome of S. tetrandra, where repeats that consisting of A or T bases were much higher than that of G or C bases. In addition, 92 potential RNA editing sites were identified in 25 protein-coding genes, with the most predicted RNA editing sites in ndhB gene. The variations on length and expansion extent to the junction of ycf1 gene were observed between S. tetrandra vouchers from different regions, indicating potential markers for further geographical origin discrimination. Moreover, the values of transition to transversion ratio (Ts/Tv) in the Stephania species were significantly higher than 1 using Pericampylus glaucus as reference. Comparative analysis of the Stephania cp genomes revealed 5 highly variable regions, including 3 intergenic regions (trnH-psbA, trnD-trnY, trnP) and two protein coding genes (rps16 and ndhA). The identified mutational hotspots of Stephania plants exhibited multiple SNP sites and Gaps, as well as different Ka/Ks ratio values. In addition, five pairs of specific primers targeting the divergence regions were accordingly designed, which could be utilized as potential molecular markers for species identification, population genetic and phylogenetic analysis in Stephania species. Phylogenetic tree analysis based on the conserved chloroplast protein coding genes indicated a sister relationship between S. tetrandra and the monophyletic group of S. japonica and S. kwangsiensis with high support values, suggesting a close genetic relationship within Stephania plants. However, two S. tetrandra vouches from different regions failed to cluster into one clade, confirming the occurrences of genetic diversities and requiring further investigation for geographical tracing strategy. CONCLUSIONS: Overall, we provided comprehensive and detailed information on the complete chloroplast genome and identified nucleotide diversity hotspots of Stephania species. The obtained genetic resource of S. tetrandra from Zhejiang Province would facilitate future studies in DNA barcode, species discrimination, the intraspecific and interspecific variability and the phylogenetic relationships of Stephania plants.

Triterpenoid saponins from the leaves and stems of Pericampylus glaucus and their insulin mimetic activities.

During an attempt to discover insulin mimetics, thirteen new triterpenoid saponins (1-13), including three phytolaccagenic acids (1, 2, and 12) and ten serjanic acids (3-11 and 13), as aglycones were isolated from a 70% ethanol extract of leaves and stems from Pericampylus glaucus. The chemical structures of compounds 1-13 were determined through spectroscopic data analysis, including NMR, IR, and HRESIMS. All isolated compounds (1-13) were evaluated using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG) as a fluorescent-tagged glucose probe to determine their stimulatory effects on glucose uptake in differentiated 3 T3-L1 adipocyte cells. Consequently, four compounds (4, 7, 11, and 12) exhibited stimulatory effects on glucose uptake.

Tiliacora triandra (Colebr.) Diels Leaf Aqueous Extract Inhibits Hepatic Glucose Production in HepG2 Cells and Type 2 Diabetic Rats.

This study investigated the effects of Tiliacora triandra (Colebr.) Diels aqueous extract (TTE) on hepatic glucose production in hepatocellular carcinoma (HepG2) cells and type 2 diabetic (T2DM) conditions. HepG2 cells were pretreated with TTE and its major constituents found in TTE, epicatechin (EC) and quercetin (QC). The hepatic glucose production was determined. The in vitro data were confirmed in T2DM rats, which were supplemented daily with 1000 mg/kg body weight (BW) TTE, 30 mg/kg BW metformin or TTE combined with metformin for 12 weeks. Results demonstrate that TTE induced copper-zinc superoxide dismutase, glutathione peroxidase and catalase genes, similarly to EC and QC. TTE decreased hepatic glucose production by downregulating phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) and increasing protein kinase B and AMP-activated protein kinase phosphorylation in HepG2 cells. These results correlated with the antihyperglycemic, antitriglyceridemic, anti-insulin resistance, and antioxidant activities of TTE in T2DM rats, similar to the metformin and combination treatments. Consistently, impairment of hepatic gluconeogenesis in T2DM rats was restored after single and combined treatments by reducing PEPCK and G6Pase genes. Collectively, TTE could potentially be developed as a nutraceutical product to prevent glucose overproduction in patients with obesity, insulin resistance, and diabetes who are being treated with antidiabetic drugs.

Tiliacora triandra extract and its major constituent attenuates diabetic kidney and testicular impairment by modulating redox imbalance and pro-inflammatory responses in rats.

BACKGROUND: Literature has demonstrated that diabetes is associated with renal complication and testicular dysfunctions. The current study explored the potential of Tiliacora triandra extract and its major component against diabetic kidney and testicular damages in rats. METHODS: Diabetes was induced by high fat diet/streptozotocin (HFD/STZ) and treated orally with Tiliacora triandra extract (TTE, 100 and 400 mg kg-1 body weight) and its major component, 5,7-dihydroxy-6-oxoheptadecanoic acid (DHA, 25 mg kg-1 body weight) for 30 consecutive days. Testicular activities of testicular enzymes, serum levels of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), sperm parameters and urinalysis for protein and albumin levels were evaluated. Renal and testicular biomarkers of oxidative stress and pro-inflammation were analysed along with histology. RESULTS: The experimental diabetes induced significant alterations in the levels and activities of indices evaluated compared to non-diabetic normal rats. The 28-day treatment of diabetic rats with TTE and DHA markedly improved activities of testicular enzymes, restored levels of testosterone, LH and FSH and sperm parameters compared to untreated diabetic rats. TTE and DHA abrogated proteinuria and reversed urine albumin level. Testicular and renal oxidative stress and pro-inflammation were attenuated in diabetic rats treated with TTE and DHA. The diabetes-mediated histopathological damage was alleviated in the kidney and testis. CONCLUSION: The protective effect of TTE and DHA against diabetes induced kidney and testicular damages may be related to its antioxidant and anti-inflammatory activities. © 2020 Society of Chemical Industry.

Phylogeny and biogeography of Pachygoneae (Menispermaceae), with consideration of the boreotropical flora hypothesis and resurrection of the genera Cebatha and Nephroia.

The tribe Pachygoneae consists of four genera with about 40 species, primarily distributed in tropical and subtropical Asia and America, also in Australasia and Africa. This tribe presents an ideal model to investigate the origin of the tropical and subtropical amphi-Pacific disjunction pattern. More specifically, it allows us to test whether the tropical lineages diverged earlier than the subtropical ones during the fragmentation of the boreotropical flora. In this study, we reconstructed the phylogeny of Pachygoneae using five plastid (rbcL, atpB, matK, ndhF, trnL-F) and one nuclear (26S rDNA) DNA regions. Our results indicate that Pachygoneae is not monophyletic unless Cocculus pendulus and Cocculus balfourii are excluded. We resurrected the genus Cebatha to include these two species and established a new tribe for this genus. Within Pachygoneae, the species of Cocculus are distributed in three different clades, among which two are recognized as two distinct genera, Cocculus s.str. and Nephroia resurrected, and one species is transferred into Pachygone. Our molecular dating and ancestral area reconstruction analyses suggest that Pachygoneae began to diversify in tropical Asia around the early-middle Eocene boundary (c. 48 Ma) and expanded into the New World by c. 44 Ma. In the New World, tropical Hyperbaena originated in the late Eocene (c. 40 Ma), whereas the subtropical Cocculus carolinus and Cocculus diversifolius originated later, in the early Oligocene (c. 32  Ma). These two timings correspond with the two climatic cooling intervals, which suggests that the formation and breakup of the boreotropical floral may have been responsible for the amphi-Pacific disjunct distribution within Pachygoneae. One overland migration event from Asia into Australasia appears to have occurred in the early to late Miocene.

The whole-genome sequence analysis of Enterobacter cloacae strain Ghats1: insights into endophytic lifestyle-associated genomic adaptations.

Enterobacter cloacae is normally considered to be an opportunistic human pathogen. Here, we report on the whole-genome sequence of an endophytic E. cloacae, strain "Ghats1", isolated from leaves of the medicinal plant Coscinium fenestratum Gaertn. Functional analysis of the Ghats1 genome revealed an enrichment for genes involved in the uptake and exchange of nutrients, for chemotaxis and for plant colonization. Unexpectedly though, there were no ORFs belonging to the "virulence factors and antibiotic resistance". Moreover, the presence of hydrolytic enzymes and motility functions reveals the characteristics of an endophyte lifestyle of a bacterium that can colonize and adapt to plant environment. These results provide a better understanding of an endophytic lifestyle through plant-microbe interaction, which can be further exploited as a biocontrol agent.

GC-HRMS screening of bioactive compounds responsible for antimicrobial and antioxidant activities of blood fruit (Haematocarpus validus Bakh. F. Ex Forman) of North-East India.

The aims of this study are to evaluate phytochemical composition, antimicrobial and antioxidant activities of different solvent-assisted blood fruit pulp extracts. The extracts of blood fruit were prepared with various solvents namely, water, ethyl acetate, methanol and acetone. These extracts were divided into two groups, first group of extract were thermosonicated (treated) and the second group were not exposed to thermosonication (untreated). The sample extracts were then evaluated for their antimicrobial properties against various microorganisms. The results revealed that the antimicrobial activity of thermosonicated ethyl acetate extracts at 12 mg/mL concentration exhibited the highest inhibition zone of 19.4 mm against Staphylococcus aureus. The phenolic content of ethyl acetate extract was found to be the highest as compared to that of other solvent extracts and the values were 1508.08 and 1478.34 mg GAE/100 g for treated and untreated samples, respectively. The treated ethyl acetate extract antioxidative activity by DPPH, ORAC, ABTS, and FRAP assay were 1627.68, 2119.82, 938.11, and 2360.18 µmol TE/g, respectively. The minimum bactericidal concentration (MBC - 1.5 mg/mL) against the selected foodborne pathogens was obtained with thermosonicated ethyl acetate blood fruit extract. In the present study, it was observed that the thermosonicated ethyl acetate extract exhibits maximum antioxidant and antimicrobial activities than any other examined solvent extracts. The major bioactive volatile compounds found in treated ethyl acetate extracts were 2-bromotetradecane, tetracosane, heptadecane, eicosane and palmitic acid. These bioactive compounds can be utilizable for the development of functional, nutraceutical and pharmacological products.

Comparison of the antimalarial activity of a Colombian traditional Uitoto remedy with laboratory preparations.

BACKGROUND & OBJECTIVES: In Colombian Amazonia, Uitoto indigenous people use a preparation of Curarea toxicofera (Menispermaceae) to prevent and treat malaria. To open the way for the production of a standardized herbal remedy, we compared the activity of the traditional preparation with laboratory preparations. METHODS: People were interviewed on their mode of use and preparation of what is considered the best remedy against fevers in this area. The herbal remedy was prepared according to the healer's recommendations. The plant was also submitted to continuous distillation and percolation extraction. The preparations were then tested against Plasmodium falciparum, in vitro. Traditional preparation and extract obtained by percolation were tested on Plasmodium berghei infected mice. Chemical profiles were also explored by thin-layer chromatography. RESULTS: Yields of extraction were around 7% in the preparations (percolation was the most efficient). The phytochemical profile showed a mix of steroids, flavonoids and alkaloids qualitatively similar in all preparations. In vitro, the extracts showed inhibitory concentration 50 <10µg/mL: the traditional preparation was almost three times less active than laboratory preparations. In vivo, percolation was also more active than traditional preparation, inhibiting 78% of the parasite growth at 400mg/kg/day by oral route. INTERPRETATION & CONCLUSION: Pharmacological activities suggest that both the original remedy (prepared according to traditional pharmacopeia) and the extracts obtained by percolation extraction exhibit relevant antiparasitic activity. C. toxicofera should therefore be considered for the elaboration of an improved traditional medicine by implementing toxicological studies and carefully following quality control guidelines for its preparation.

A multi-locus phylogeny for the Neotropical Anomospermeae (Menispermaceae): Implications for taxonomy and biogeography.

Neotropical rainforests cover about half of the world's tropical rainforests and house most of the biodiversity available on Earth. Australasia has been suggested as a potential source for Neotropical diversity. However, it remains unclear whether megathermal lineages could indeed have migrated to South America though Antarctica. The Neotropical Anomospermeae (Menispermaceae) consists of large, canopy lianas and is entirely restricted to tropical lowland rainforests. The sister relationship identified between this group and its Australasian ally represents an excellent model to test hypotheses regarding past connections between those landmasses. In this study, we used six chloroplast and two nuclear DNA markers to reconstruct phylogenetic relationships within the Neotropical Anomospermeae (Menispermaceae). The phylogeny of this group was then used as basis to reconstruct its biogeographical history. The phylogenetic framework reconstructed here strongly supports the monophyly of the Neotropical Anomospermeae and recovers the species of Anomospermum in three different clades: (i) Anomospermum sect. Anomospermum plus Orthomene; (ii) Anomospermum grandifolium and A. solimoesanum (Anomospermum sect. Elissarrhena); and (iii) Anomospermum bolivianum (Anomospermum sect. Elissarrhena). Each of these clades is recognized as a different genus and the necessary taxonomic changes are proposed. Furthermore, the Neotropical Anomospermeae seems to have split from its Australasian sister-group at c. 62 Ma. Ancestral area reconstructions support an Australasian origin for the Neotropical Anomospermeae, providing additional support for the hypothesis that Australasia is a source of Neotropical diversity, with megathermal lineages having dispersed via Antarctica. The Neotropical Anomospermeae differentiated in the late Eocene and subsequently diversified rapidly into seven lineages, suggesting that Neotropical lowland rainforests resembling modern rainforests physiognomically and structurally might not have developed until the late Eocene. The Neotropical Anomospermeae exemplifies the contributions of Australasian migration to Neotropical diversity.

Effect of tiliamosine, a bis, benzylisoquinoline alkaloid isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom on the immature stages of filarial mosquito Culex quinquefasciatus say (Diptera: Culicidae).

The present study was aimed to check the mosquitocidal activity of tiliamosine isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom against immature stages of Culex quinquefasciatus. Eggs and larvae of Cx. quinquefasciatus were exposed to different concentrations of tiliamosine - 0.5, 1.0, 1.5 and 2.0 ppm - prepared using DMSO. The compound tiliamosine showed good larvicidal activity with LC50 and LC90 values of 1.13 and 2.85 ppm respectively, against third-instar larvae of Cx. quinquefasciatus at 24 h. In control, the larvae exhibited normal movement. Tiliamosine exhibited 91% ovicidal activity at 2.0 ppm concentration after 120 h post-treatment. Lowest concentration of tiliamosine (0.5 ppm) showed 19% egg mortality. Histopathology study of the compound-treated larvae showed serious damage on the larval midgut cells. The treated larvae showed restless movement which was different from that of the control larvae. The larvae exhibited malformation in development. The compound tiliamosine was harmless to non-target organisms P. reticulata and Dragon fly nymph at tested concentrations. The compound was highly active and inhibited AChE in a concentration-dependent manner. Computational analysis of the tiliamosine had strong interaction with AChE1 of Cx. quinquefasciatus. This report clearly suggests that the isolated compound can be used as an insecticide to control mosquito population and thus prevent the spread of vector-borne diseases.

Fossil moonseeds from the Paleogene of West Gondwana (Patagonia, Argentina).

PREMISE OF THE STUDY: The fossil record is critical for testing biogeographic hypotheses. Menispermaceae (moonseeds) are a widespread family with a rich fossil record and alternative hypotheses related to their origin and diversification. The family is well-represented in Cenozoic deposits of the northern hemisphere, but the record in the southern hemisphere is sparse. Filling in the southern record of moonseeds will improve our ability to evaluate alternative biogeographic hypotheses. METHODS: Fossils were collected from the Salamanca (early Paleocene, Danian) and the Huitrera (early Eocene, Ypresian) formations in Chubut Province, Argentina. We photographed them using light microscopy, epifluorescence, and scanning electron microscopy and compared the fossils with similar extant and fossil Menispermaceae using herbarium specimens and published literature. KEY RESULTS: We describe fossil leaves and endocarps attributed to Menispermaceae from Argentinean Patagonia. The leaves are identified to the family, and the endocarps are further identified to the tribe Cissampelideae. The Salamancan endocarp is assigned to the extant genus Stephania. These fossils significantly expand the known range of Menispermaceae in South America, and they include the oldest (ca. 64 Ma) unequivocal evidence of the family worldwide. CONCLUSIONS: Our findings highlight the importance of West Gondwana in the evolution of Menispermaceae during the Paleogene. Currently, the fossil record does not discern between a Laurasian or Gondwanan origin; however, it does demonstrate that Menispermaceae grew well outside the tropics by the early Paleocene. The endocarps' affinity with Cissampelideae suggests that diversification of the family was well underway by the earliest Paleocene.

Synthesis of (aminoalkyl)cycleanine analogues: cytotoxicity, cellular uptake, and apoptosis induction in ovarian cancer cells.

Our previous studies demonstrated that cycleanine, a macrocyclic bisbenzylisoquinoline (BBIQ) alkaloid, showed potent anti-ovarian cancer activity via apoptosis induction. Here, we synthesized two novel (aminoalkyl)cycleanine analogues (2 and 3) through a simple and efficient two-step reaction starting from cycleanine isolated from Triclisia subcordata Oliv. These analogues showed greater potency than the unmodified cycleanine in three human ovarian cancer cell lines. Both 2 and 3 induced apoptosis in ovarian cancer cells by activations of caspases 3/7, cleavage of PARP, increase in subG1 cell cycle phase and in the percentage of apoptotic cells. Further confocal fluorescence microscopy analysis confirmed the cellular uptake of alkaloids in ovarian cancer cells by using the unique (alkynyl)cycleanine (3) via click chemistry reaction. Our results suggest that cycleanine could be a hit compound for the future development in attacking ovarian cancer.

Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition.

Protein folding/unfolding transitions between the native (N) and unfolded (U) states are usually describable as two-state, only because of the dominant presence of the N and/or U states, because of which high energy intermediates remain undetected. Delineation of the cooperativity underlying these transitions, and characterization of high energy intermediates that are populated sparsely, have been difficult challenges, especially under equilibrium conditions, and require the use of a sensitive probe that reports on both the structures and population distributions of the partially unfolded intermediates. In this study, the use of multisite time-resolved FRET to monitor structural change in five specific segments of the small protein monellin, has brought out local deviations from two-state behavior during unfolding. It is shown that in some segments of the protein structure, denaturant-induced unfolding proceeds first by gradual expansion of the N state, then by an all-or-none transition from the N state ensemble to the U state ensemble, followed finally by expansion of the U state. Segments encompassing the sole helix appear, however, to unfold completely through a gradual transition from the N to U states. Finally, it is shown that equilibrium unfolding of monellin is not only heterogeneous, but that the degree of non-cooperativity differs between the sole α-helix and different parts of the ß-sheet.

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

New insights into the phylogeny of Burasaieae (Menispermaceae) with the recognition of a new genus and emphasis on the southern Taiwanese and mainland Chinese disjunction.

Taiwan is a continental island lying at the boundary between the Eurasian and the Philippine tectonic plates and possesses high biodiversity. Southern Taiwan, viz. Hengchun Peninsula, is notably floristically different from northern Taiwan. The floristic origin and relationships of the Hengchun Peninsula have been rarely investigated in a phylogenetic context. In this study, data from six plastid and nuclear sequences were used to reconstruct phylogenetic relationships within Burasaieae (Menispermaceae), which mainly inhabits tropical rainforests. The tree-based comparisons indicate that the position of Tinospora sensu stricto conflicts significantly between the cpDNA and ITS trees. However, alternative hypothesis tests from the ITS data did not reject the result of the cpDNA data, which suggests that tree-based comparisons might sometimes generate an artificial incongruence, especially when markers with high homoplasy are used. Based on the combined cpDNA and ITS data, we present an inter-generic phylogenetic framework for Burasaieae. Sampled species of Tinospora are placed in three different clades, including Tinospora dentata from southern Taiwan and T. sagittata from mainland China in an unresolved position alongside six lineages of Burasaieae. By integrating lines of evidence from molecular phylogeny, divergence times, and morphology, we recognize the three Tinospora clades as three different genera, including Tinospora sensu stricto, a new genus (Paratinospora) for T. dentata and T. sagittata, and Hyalosepalum resurrected. Tinospora dentata, now endemic to the Hengchun Peninsula, originated from the Late Eocene (ca. 39Ma), greatly predating the formation of Taiwan. Our study suggests that the flora of the Hengchun Peninsula contains some ancient components that might have migrated from mainland China.