Gnetum montanum extract induces apoptosis by inhibiting the activation of AKT in SW480 human colon cancer cells.

CONTEXT: Gnetum montanum Markgr. (Gnetaceae) is used to treat rheumatic arthralgia and bruises in the clinic. OBJECTIVE: To exam the activity and mechanism of G. montanum extract (GME) against colon cancer cells SW480. MATERIALS AND METHODS: The anti-proliferative activity of GME (0-120 µg/mL) on SW480 cells was determined using MTS assay at 24, 48, and 72 h. The in vitro activity of GME (0-120 µg/mL) on SW480 cells was investigated using flow cytometry and western blotting analysis. The in vivo activity of GME was evaluated using xenograft tumour model of zebrafish and nude mice. The chemical composition of GME was detected by using HPLC-MS/MS. RESULTS: The IC50 value SW480 cells viability by GME were 126.50, 78.25, and 50.77 µg/mL, respectively, for 24, 48, and 72 h. The experiments showed that apoptotic cells and G2/M phase cells increased from 20.81 to 61.53% (p < 0.01) and 25.76 to 34.93% with 120 µg/mL GME, respectively. GME also down-regulated the protein expression of P-AKT, P-GSK-3ß, P-PDK1, P-c-Raf, caspase-3, and Bcl-2, and up-regulated the expression cleaved caspase-3, cleaved PARP, and Bax. In vivo study found that GME can significantly inhibit the growth and migration of SW480 cells in xenograft zebrafish. GME reduced the nude mice tumour weight to approximately 32.19% at 28 mg/kg/day and to 53.17% (p < 0.01) at 56 mg/kg/day. Forty-two compounds were identified from the GME. DISCUSSION AND CONCLUSIONS: GME has a significant antitumor effect on colon cancer cells SW480, and it has the potential to be developed as an anticancer agent.

Pollination Drop Proteome and Reproductive Organ Transcriptome Comparison in Gnetum Reveals Entomophilous Adaptation.

Gnetum possesses morphologically bisexual but functionally unisexual reproductive structures that exude sugary pollination drops to attract insects. Previous studies have revealed that the arborescent species (G. gnemon L.) and the lianoid species (G. luofuense C.Y.Cheng) possess different pollination syndromes. This study compared the proteome in the pollination drops of these two species using label-free quantitative techniques. The transcriptomes of fertile reproductive units (FRUs) and sterile reproductive units (SRUs) for each species were furthermore compared using Illumina Hiseq sequencing, and integrated proteomic and transcriptomic analyses were subsequently performed. Our results show that the differentially expressed proteins between FRUs and SRUs were involved in carbohydrate metabolism, the biosynthesis of amino acids and ovule defense. In addition, the differentially expressed genes between the FRUs and SRUs (e.g., MADS-box genes) were engaged in reproductive development and the formation of pollination drops. The integrated protein-transcript analyses revealed that FRUs and their exudates were relatively conservative while the SRUs and their exudates were more diverse, probably functioning as pollinator attractants. The evolution of reproductive organs appears to be synchronized with changes in the pollination drop proteome of Gnetum, suggesting that insect-pollinated adaptations are not restricted to angiosperms but also occur in gymnosperms.

Ratio of sugar concentrations in the phloem sap and the cytosol of mesophyll cells in different tree species as an indicator of the phloem loading mechanism.

MAIN CONCLUSION: Sucrose concentration in phloem sap was several times higher than in the cytosol of mesophyll cells. The results suggest that phloem loading involves active steps in the analyzed tree species. Phloem loading in source leaves is a key step for carbon partitioning and passive symplastic loading has been proposed for several tree species. However, experimental evidence to prove the potential for sucrose diffusion from mesophyll to phloem is rare. Here, we analyzed three tree species (two angiosperms, Fagus sylvatica, Magnolia kobus, and one gymnosperm, Gnetum gnemon) to investigate the proposed phloem loading mechanism. For this purpose, the minor vein structure and the sugar concentrations in phloem sap as well as in the subcellular compartments of mesophyll cells were investigated. The analyzed tree species belong to the open type minor vein subcategory. The sucrose concentration in the cytosol of mesophyll cells ranged between 75 and 165 mM and was almost equal to the vacuolar concentration. Phloem sap could be collected from F. sylvatica and M. kobus and the concentration of sucrose in phloem sap was about five- and 11-fold higher, respectively, than in the cytosol of mesophyll cells. Sugar exudation of cut leaves was decreased by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton transporter. The results suggest that phloem loading of sucrose in the analyzed tree species involves active steps, and apoplastic phloem loading seems more likely.

Melinjo (Gnetum gnemon) Seed Extract Consumption during Lactation Improved Vasodilation and Attenuated the Development of Hypertension in Female Offspring of Fructose-Fed Pregnant Rats.

BACKGROUND: Fructose intake has been correlated with increased prevalence of metabolic disorders including hypertension. In pregnant rats, fructose intake has been reported to have adverse effects on the health of its offspring. This study investigated the effects of gestational maternal fructose consumption and if supplementation with melinjo seed extracts to the maternal diet during lactation could benefit the offspring in later life. METHODS: Pregnant rats were randomly divided into three groups: untreated (CC), fructose-treated (FC), and fructose and melinjo-treated (FM). FC and FM groups received 100 g/L of D(-)-fructose solution by means of the drinking water during gestation while CC received normal drinking water. During lactation, CC and FC groups were given standard commercial laboratory diet, while the FM group was given commercial laboratory diet with 0.1% melinjo seed extracts. After weaning, the offspring were given normal drinking water and standard commercial diet until week 17. The blood pressure of the offspring was monitored until the 16th week. During week 17, the offspring were killed, and the kidneys were collected and analyzed. RESULTS: The level of renal phosphorylated AMP-activated protein kinase (pAMPK) in FM of 17-week female offspring was significantly higher compared with FC and CC groups. Maternal fructose intake down-regulated the renal endothelial isoform of nitric oxide synthetase expression in FC and maternal melinjo seed extract consumption maintained renal endothelial isoform of nitric oxide synthetase expression in FM of 17-week female offspring. In addition, maternal melinjo seed extract intake during lactation lowered the systolic blood pressure in FM of 17-week female offspring. CONCLUSION: Female offspring were more vulnerable to the effects of placental fructose and melinjo seed extracts, suggesting sex-specific sensitivities. In summary, our data show that melinjo seed extract consumption during lactation improved vasodilation and attenuated the development of hypertension in the 17-week female offspring of fructose-fed pregnant rats. Birth Defects Research 110:27-34, 2018. © 2017 Wiley Periodicals, Inc.

High temperature and UV-C treatments affect stilbenoid accumulation and related gene expression levels in Gnetum parvifolium

Background: Gnetum parvifolium stems and roots have been used for a long time in traditional Chinese medicines. Stilbenes are bioactive compounds present in G. parvifolium plants, and they possess antioxidative and anticancer properties. However, little is known about the responses of G. parvifolium stilbene biosynthetic pathways to stress conditions. Therefore, we investigated stilbene biosynthesis, including the expression of relevant genes, in G. parvifolium exposed to high-temperature and ultraviolet-C treatments. Results: High temperatures did not influence the accumulation of total stilbenes in stems but decreased stilbene concentrations in roots at 3 h, with a subsequent restoration to control levels. In contrast, ultraviolet irradiation induced the accumulation of total stilbenes in stems but not in roots. We also observed that high temperatures inhibited the production of resveratrol and piceatannol in G. parvifolium stems and roots, whereas ultraviolet treatments initially inhibited their accumulation (up to 6 h) but induced their production at later time points. Analyses of specific genes (i.e., PAL, C4H, 4CL, STS, and CYP) revealed that their expression levels generally increased in stress-treated stems and roots, although there was some variability in the expression profiles during treatments. Conclusions: Our results indicated that high temperatures and ultraviolet irradiation differentially affect the biosynthesis of specific stilbenes in G. parvifolium stems and roots. Therefore, cultivating G. parvifolium seedlings under optimal stress conditions may increase the biosynthesis of specific stilbene compounds.

Resveratrol Derivative-Rich Melinjo Seed Extract Attenuates Skin Atrophy in Sod1-Deficient Mice.

The oxidative damages induced by a redox imbalance cause age-related changes in cells and tissues. Superoxide dismutase (SOD) enzymes play a pivotal role in the antioxidant system and they also catalyze superoxide radicals. Since the loss of cytoplasmic SOD (SOD1) resulted in aging-like phenotypes in several types of murine tissue, SOD1 is essential for the maintenance of tissue homeostasis. Melinjo (Gnetum gnemon Linn) seed extract (MSE) contains trans-resveratrol (RSV) and resveratrol derivatives, including gnetin C, gnemonoside A, and gnemonoside D. MSE intake also exerts no adverse events in human study. In the present studies, we investigated protective effects of MSE on age-related skin pathologies in mice. Orally MSE and RSV treatment reversed the skin thinning associated with increased oxidative damage in the Sod1 (-/-) mice. Furthermore, MSE and RSV normalized gene expression of Col1a1 and p53 and upregulated gene expression of Sirt1 in skin tissues. In vitro experiments revealed that RSV significantly promoted the viability of Sod1 (-/-) fibroblasts. These finding demonstrated that RSV in MSE stably suppressed an intrinsic superoxide generation in vivo and in vitro leading to protecting skin damages. RSV derivative-rich MSE may be a powerful food of treatment for age-related skin diseases caused by oxidative damages.

Molecular interactions of orthologues of floral homeotic proteins from the gymnosperm Gnetum gnemon provide a clue to the evolutionary origin of 'floral quartets'.

Several lines of evidence suggest that the identity of floral organs in angiosperms is specified by multimeric transcription factor complexes composed of MADS-domain proteins. These bind to specific cis-regulatory elements ('CArG-boxes') of their target genes involving DNA-loop formation, thus constituting 'floral quartets'. Gymnosperms, angiosperms' closest relatives, contain orthologues of floral homeotic genes, but when and how the interactions constituting floral quartets were established during evolution has remained unknown. We have comprehensively studied the dimerization and DNA-binding of several classes of MADS-domain proteins from the gymnosperm Gnetum gnemon. Determination of protein-protein and protein-DNA interactions by yeast two-hybrid, in vitro pull-down and electrophoretic mobility shift assays revealed complex patterns of homo- and heterodimerization among orthologues of floral homeotic class B, class C and class E proteins and B(sister) proteins. Using DNase I footprint assays we demonstrate that both orthologues of class B with C proteins, and orthologues of class C proteins alone, but not orthologues of class B proteins alone can loop DNA in floral quartet-like complexes. This is in contrast to class B and class C proteins from angiosperms, which require other factors such as class E floral homeotic proteins to 'glue' them together in multimeric complexes. Our findings suggest that the evolutionary origin of floral quartet formation is based on the interaction of different DNA-bound homodimers, does not depend on class E proteins, and predates the origin of angiosperms.

Hydraulic design of leaves: insights from rehydration kinetics.

We examined the leaf hydraulic design in 10 species based on their rehydration kinetics. In all cases, a biphasic response described the temporal pattern of water uptake, with time constants of approximately 30 to 800 s and approximately 800 to 8000 s. The time constants of the fast phase were significantly shorter in the six angiosperms (30 to 110 s) compared with the two single-veined conifer species (>400 s) examined, while the two multi-veined gymnosperm species, Gnetum gnemon and Ginkgo biloba, had time constants for the fast phase of approximately 150 s. Among angiosperm species, the fast phase constituted 50-90% of the total water absorbed, whereas in gymnosperms 70-90% of the water uptake could be assigned to the slow phase. In the four gymnosperms, the relative water uptake corresponding to the fast phase matched to a good degree the relative volume of the venation and bundle sheath extension; whereas in the angiosperm species, the relatively larger water influx during the fast phase was similar in relative volume to the combined venation, bundle sheath extension, epidermis and (in four species) the spongy mesophyll. This suggests a general trend from a design in which the epidermis is weakly connected to the veins (all four gymnosperms), to a design with good hydraulic connection between epidermis and veins that largely bypasses the mesophyll (four of six angiosperms), to a design in which almost the entire leaf appears to function as a single pool.

Distinct MADS-box gene expression patterns in the reproductive cones of the gymnosperm Gnetum gnemon.

Expression patterns from in situ hybridization of four MADS-box genes (GGM7, GGM9, GGM11, and GGM15) from the gymnosperm species Gnetum gnemon are presented. Together with previously published data about putative orthologs of floral homeotic genes from G. gnemon (GGM2, GGM3, GGM13), we describe seven temporally and spatially distinct expression patterns in male, female or both types of reproductive units which very likely reflect the diversity of MADS-box gene function in gymnosperm cones. There is evidence that some aspects of the observed differential expression have been conserved since the last common ancestor of extant angiosperms and gymnosperms about 300 million years ago.