Scavenging Glyoxal and Methylglyoxal by Synephrine Alone or in Combination with Neohesperidin at High Temperatures.

α-Dicarbonyl compounds, such as glyoxal (GO) and methylglyoxal (MGO), are a series of chemical hazards that exist in vivo and in vitro, posing a threat to human health. We aimed to explore the scavenging effects on GO/MGO by synephrine (SYN) alone or in combination with neohesperidin (NEO). First, through LC-MS/MS, we confirmed that both SYN and NEO could effectively remove GO and form GO adducts, while NEO could also clear MGO by forming MGO adducts, and its ability to clear MGO was stronger than that of GO. Second, a synergistic inhibitory effect on GO was found when SYN and NEO were used in combination by using the Chou-Talalay method; on the other hand, SYN could promote NEO to clear more MGO, although SYN could not capture MGO. Third, after synthesizing four GO/MGO-adducts (SYN-GO-1, SYN-GO-3, NEO-GO-7, and NEO-MGO-2) and identifying their structure through NMR, strict correlations between the GO/MGO-adducts and the GO/MGO-clearance rate were found when using SYN and NEO alone or in combination. Furthermore, it was inferred that the synergistic effect between SYN and NEO stems from their mutual promotion in capturing more GO by the quantitative analysis of the adducts in the combined model. Finally, a study was conducted on flowers of Citrus aurantium L. var. amara Engl. (FCAVA, an edible tea) rich in SYN and NEO, which could serve as an effective GO and MGO scavenger in the presence of both GO and MGO. Therefore, our study provided well-defined evidence that SYN and NEO, alone or in combination, could efficiently scavenge GO/MGO at high temperatures, whether in the pure form or located in FCAVA.

Boosting Activity and Selectivity of UiO-66 through Acidity/Alkalinity Functionalization in Dimethyl Carbonate Catalysis.

The acid-base properties of supports have an enormous impact on catalytic reactions to regulate the selectivity and activity of supported catalysts. Herein, a train of Pd-X-UiO-66 (X = NO2 , NH2 , and CH3 ) catalysts with different acidity/alkalinity functional groups and encapsulated Pd(II) species is first developed, whose activities in dimethyl carbonate (DMC) catalysis are then investigated in details. Thereinto, the Pd-NO2 -UiO-66 catalyst with acidity functionalization exhibits the best catalytic behavior: the DMC selectivity stemmed from methyl nitrite (MN) is up to 68%, the conversion of CO is 73.4%. The obtained experimental results demonstrate that the NO2 group not only affected the interaction between X-UiO-66 and Pd(II) active sites but also play an indispensable role in the adsorption and activation of MN and CO, which remarkably promote the formation of the COOCH3 * intermediate and DMC product.

Insights into angiosperm evolution, floral development and chemical biosynthesis from the Aristolochia fimbriata genome.

Aristolochia, a genus in the magnoliid order Piperales, has been famous for centuries for its highly specialized flowers and wide medicinal applications. Here, we present a new, high-quality genome sequence of Aristolochia fimbriata, a species that, similar to Amborella trichopoda, lacks further whole-genome duplications since the origin of extant angiosperms. As such, the A. fimbriata genome is an excellent reference for inferences of angiosperm genome evolution, enabling detection of two novel whole-genome duplications in Piperales and dating of previously reported whole-genome duplications in other magnoliids. Genomic comparisons between A. fimbriata and other angiosperms facilitated the identification of ancient genomic rearrangements suggesting the placement of magnoliids as sister to monocots, whereas phylogenetic inferences based on sequence data we compiled yielded ambiguous relationships. By identifying associated homologues and investigating their evolutionary histories and expression patterns, we revealed highly conserved floral developmental genes and their distinct downstream regulatory network that may contribute to the complex flower morphology in A. fimbriata. Finally, we elucidated the genetic basis underlying the biosynthesis of terpenoids and aristolochic acids in A. fimbriata.

Ergosterol peroxide from marine fungus Phoma sp. induces ROS-dependent apoptosis and autophagy in human lung adenocarcinoma cells.

As part of our ongoing search for novel therapeutic structures from microorganism, the chemical examination of marine fungus Phoma sp. resulted in the isolation of ergosterol, ergosterol peroxide (EP), and 9,11-dehydroergosterol peroxide (DEP). The bioassay results demonstrated that the three isolates reduced the viability of various cancer cells, with EP being highest in human lung cancer cell line A549 cells. EP induced caspase-dependent apoptosis through mitochondrial damage in A549 cells. Additionally, EP-induced ROS generation and apoptosis were attenuated by ROS-generating enzymes inhibitors and antioxidant N-acetylcysteine, indicated that ROS played an important role in EP-mediated apoptosis in A549 cells. Furthermore, it was observed that EP induced ROS-dependent autophagy, which attenuated apoptosis in A549 cells. On the other hand, EP reduced the LPS/ATP-induced proliferation and migration of A549 cells through attenuated NLRP3 inflammasome activity. Additionally, EP showed synergistic cytotoxic effect with antitumor drug Sorafenib in A549 cell viability inhibition. Furthermore, Micro-Western Array and Western blot analyses demonstrated that the protein levels of EGFR, HSP27, MEK5, AKT1, mTOR, Smad2, Smad3, TAB1, NF-κB, and HIF1-α decreased, while the levels of p-p38α, p-ERK1/2, p-JNK, fibronectin and p27 increased. Collectively, the results of this study demonstrated that EP might be useful to develop a therapeutic candidate for lung cancer complications.

Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis.

SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a ß-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

Cloning, characterization and functional analysis of two type 1 diacylglycerol acyltransferases (DGAT1s) from Tetraena mongolica.

Two cDNAs encoding putative type 1 acyl-CoA: diacylglycerol acyltransferases (DGAT1, EC 2.3.1.20), were cloned from Tetraena mongolica, an extreme xerophyte with high oil content in the stems. The 1 488-bp and 1 485-bp of the open reading frame (ORF) of the two cDNAs, designated as TmDGAT1a and TmDGAT1b, were both predicted to encode proteins of 495 and 494 amino acids, respectively. Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in the T. mongolica genome. In addition to ubiquitous expression with different intensity in different tissues, including stems, leaves and roots, TmDGAT1a and TmDGAT1b, were found to be strongly induced by high salinity, drought and osmotic stress, resulting in a remarkable increase of triacylglycerol (TAG) accumulation in T. mongolica plantlets. TmDGAT1a and TmDGAT1b activities were confirmed in the yeast H1246 quadruple mutant (DGA1, LRO1, ARE1, ARE2) by restoring DGAT activity of the mutant host to produce TAG. Overexpression of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as in T. mongolica calli both resulted in an increase in oil content (ranging from 37% to 108%), accompanied by altered fatty acid profiles.

Monogalactosyldiacylglycerol deficiency in tobacco inhibits the cytochrome b6f-mediated intersystem electron transport process and affects the photostability of the photosystem II apparatus.

Monogalactosyldiacylglycerol (MGDG) is the most abundant lipid component of the thylakoid membrane. Although MGDG is believed to be important in sustaining the structure and function of the photosynthetic membrane, its exact role in photosynthesis in vivo requires further investigation. In this study, the transgenic tobacco plant M18, which has an MGDG deficiency of approximately 53%, and which contains many fewer thylakoid membranes and exhibits retarded growth and a chlorotic phenotype, was used to investigate the role of MGDG. Chlorophyll fluorescence analysis of the M18 line revealed that PSII activity was inhibited when the plants were exposed to light. The inactive linear electron transport found in M18 plants was mainly attributed to a block in the intersystem electron transport process that was revealed by P700 redox kinetics and PSI light response analysis. Immunoblotting and Blue Native SDS-PAGE analysis suggested that a reduction in the accumulation of cytochrome b6f in M18 plants is a direct structural effect of MGDG deficiency, and this is likely to be responsible for the inefficiency observed in intersystem electron transport. Although drastic impairments of PSII subunits were detected in M18 plants grown under normal conditions, further investigations of low-light-grown M18 plants indicated that the impairments are not direct structural effects. Instead, they are likely to result from the cumulative photodamage that occurs due to impaired photostability under long-term exposure to relatively high light levels. The study suggests that MGDG plays important roles in maintaining both the linear electron transport process and the photostability of the PSII apparatus.

Cloning and expression analysis of GmGAL1, SOC1 homolog gene in soybean.

A MADS box gene AGL20/SOC1 is a main integrator in Arabidopsis flowering pathway whose structure and function are highly conserved in many plant species. A soybean MADS box gene GmGAL1 (Glycine max AGAMOUS Like 1) as a homolog of AGL20/SOC1, was cloned from soybean cultivar Kennong18 and its function was investigated in transgenic Arabidopsis lines. Sequence comparisons showed that the closest homolog gene to GmGAL1 is AGL20/SOC1 in Arabidopsis and VuSOC1 in Vigna unguiculata. We investigated the expression level of GmGAL1 using quantitative real-time PCR, and the result showed that GmGAL1 was regulated by a circadian mechanism and its expression oscillated at a cycle of 24 h. The expression level of GmGAL1 was fluctuated in diverse tissues/organs and developmental stages. Considering its expression can be detected in different tissues throughout the life cycle and its protein localized in cytoplasm in Arabidopsis protoplasm, we proposed that GmGAL1 may be a multifunctional gene in the context of the soybean development. Ectopic expression of GmGAL1 in Arabidopsis enhanced flowering under long-day condition and partially rescued soc1 late flowering type.

Theoretical study on the ring-opening isomerization reaction mechanism of the ring isomers of N8H8.

Ring-opening isomerization from ring-shaped isomers to chain-shaped isomers of N(8)H(8) has been studied by a density function B3LYP method at 6-311+ +G** level. 20 ring-shaped isomers have been found to be able to transform into chain-shaped isomers, with 20 possible transition states got by ring-opening structure optimization. Furthermore, the ring-openings have been found in the longer N-N single bond by analyzing the length change of N-N bond of ring-shaped isomers in ring-opening processes. In addition, with the activation energies in ring-opening processes, the differences of the activation energies in isomerization between the isomers have been found according to the classification of rings. The activation energies in ring-opening isomerization of six-membered ring-shaped isomers are higher than that of the four-membered ring-shaped isomers. It indicates that six-membered ring-shaped isomers difficult in ring-opening in the isomerization are the steadiest ring-shaped isomers of N(8)H(8) while four-membered ring-shaped isomers easy in ring-opening are the most unstable.

A phytocyanin-related early nodulin-like gene, BcBCP1, cloned from Boea crassifolia enhances osmotic tolerance in transgenic tobacco.

Using the mRNA differential display combined with 5' rapid amplification of cDNA ends (RACE) technique, an early nodulin-like protein gene (BcBCP1) (accession no. AY243047.1) was isolated from drought-treated Boea crassifolia leaves. The full-length cDNA of BcBCP1 consists of 844 bp nucleotides and has an open reading frame of 606 bp, encoding a putative polypeptide of 201 amino acids with a predicted molecular mass of 22 kDa and a pI of 5.13. The putative protein precursor contains four sequence domains, including a 27 amino acid hydrophobic N-terminal transit peptide, a 100 amino acid phytocyanin-homologous globular domain, a 51 amino acid hydroxyproline-rich cell wall structural protein domain, and a 22 amino acid hydrophobic extension domain. Sequence alignment defined the encoded protein as an early nodulin-like protein, and the absence of key ligands implies that it is unlikely to bind copper. BcBCP1 expression was strongly induced by dehydration, salinity and abscisic acid (ABA), slightly induced by moderate heat shock, and weakly inhibited by low temperature, methyl jasmonic acid (MeJA), and a low concentration of salicylic acid (SA). Overexpression of BcBCP1 in tobacco under the control of CaMV 35S promoter enhanced tolerance to osmotic stress, as indicated by the less impaired growth, less damaged membrane integrity and lower lipid peroxidation levels after osmotic stress. Transgenic tobacco lines overexpressing BcBCP1 showed higher photosynthetic rates, higher antioxidant enzyme activities and higher cytosyl ascorbic peroxidase transcription levels than non-transgenic tobacco plants, both under normal conditions and under osmotic stress.

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.).

A 1.8 kb 5'-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from -986 to -959 and from -472 to -424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative beta-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were approximately 10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves.

[Primary targeting of functional regions involved in transcriptional regulation on watermelon fruit-specific promoter WSP].

Fruit ripening is associated with a number of physiological and biochemical changes. They include degradation of chlorophyll, synthesis of flavor compounds, carotenoid biosynthesis, conversion of starch to sugars, cell wall solublisation and fruit softening. These changes are brought about by the expression of specific genes. People are interested in the molecular mechanism involved in the regulation of gene transcription during fruit ripening. Many fruit-specific promoters such as PG, E4, E8, and 2A11 have been characterized and shown to direct ripening-specific expression of reporter genes. AGPase plays the key role in catalyzing the biosynthesis of starch in plants. It is a heterotetrameric enzyme with two small subunits and two large subunits, which are encoded by different genes. In higher plants, small subunits are highly conserved among plant species and expressed in all tissues. And the large subunits are present at multiple isoforms and expressed in a tissue-specific pattern. In fruits, the expression pattern of the large subunits varies with plant species. That made it important to study the transcriptional regulation of the large subunits of AGPase in different plant species. Northern-blot analysis indicates in watermelon, an isoform of the large subunits Wml1 expressed specifically in fruits, not in leaves. The 5' flanking region of Wml1, which covers 1573bp, has been isolated through the method of uneven PCR. And transient expression assay has shown that the 1573bp (named WSP) can direct fruit-specific expression of GUS gene. Our goal in this study was to scan the promoter region for main regulatory regions involved in fruit-specific expression. A chimaeric gene was constructed containing the WSP promoter, the beta-glucuronidase (GUS) structural sequence as a reporter gene and the nopaline synthase polyadenylation site (NOS-ter). The plasmid pSPA was digested with Hind III + Hinc II and promoter fragment of 1573bp (from 180bp to 1752bp) was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-16. The same insert was then cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by HindIII + Bgl II to produce pISPA-16. Three 5'-end deletions of the promoter were obtained and fused to GUS gene in plant transient expression vector pBI426: the 1201bp fragment (from 551bp to 1752bp) was generated by digestion of pBSPA-16 with BamH I + SnaB I, the 898bp fragment (from 854bp to 1752bp) by BamH I + EcoRV. Both fragments were ligated with pBluescript SK(-) digested by BamH I + Sma I, to produce pBSPA-12 and pBS-PA-9. The inserts were cut out with HindmIII + BamH I and ligated with pBI426 digested by Hind III + Bgl II, to produce pISPA-12 and pISPA-9. The 795bp fragment (from 957bp to 1752bp) was generated by digestion of pSPA with Hinc II + EcoR I, promoter fragment was cut out and cloned into Sma I sites of pBluescript SK(-), to produce pBSPA-8. The same insert were cut out with Hind III + BamH I, and ligated with transient expression vector pBI426 digested by Hind III + Bgl II. The 1573bp fragment and three 5'-end deletions were delivered into watermelon leaf, stem, flower and fruit of different development stages (5, 10, 20 days after pollination) via particle bombardment using a biolistic PDS-1000/He particle gun. Bombardment parameters were as follows: a helium pressure of 1200 psi, vacuum of 91432.23Pa, 7 cm between the stopping screen and the plate. Histochemical assay were done on all the tissues bombarded after incubation for 2 days. The 1573bp fragment had the strongest promoter activity, and can induce GUS expression in fruits of 5 and 20 days after anthesis and flowers, but not in fruits of 10 days after anthesis, leaves and stems. Fragments of 1201bp and 898bp can induce GUS expression only in fruits of 20 days after anthesis, and with lower expression levels than 1573bp. Fragment of 795bp was not able to direct GUS expression in any of the tissues bombarded (data not shown). It can be concluded that of the 1573bp, 1201 bp, 898bp Wml1 5'flanking regions include the necessary information directing fruit-specific expression. Deletion from 180bp to 551bp doesn't affect the fruit-specificity of the promoter, but lowered the expression level. There may be some cis-acting elements located in this region, which can enhance external gene expression in later stages of fruit development. Deletion from 854bp and 958bp led to loss of GUS expression. This region includes the necessary information needed for gene expression as well as the regulatory elements for fruit-specific transcription.