Prevalence, complete genome, and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge, Terpios hoshinota.

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that 'Paraprochloron' and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of 'Paraprochloron terpiosi' in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.

Transcriptome Profile of the Variegated Ficus microcarpa c.v. Milky Stripe Fig Leaf.

Photosynthetic properties and transcriptomic profiles of green and white sectors of Ficus microcarpa (c.v. milky stripe fig) leaves were examined in naturally variegated plants. An anatomic analysis indicated that chloroplasts of the white sectors contained a higher abundance of starch granules and lacked stacked thylakoids. Moreover, no photosynthetic rate was detected in the white sectors. Transcriptome profile and differential expressed gene (DEG) analysis showed that genes encoding PSII core proteins were down-regulated in the white sectors. In genes related to chlorophyll metabolism, no DEGs were identified in the biosynthesis pathway of chlorophyll. However, genes encoding the first step of chlorophyll breakdown were up-regulated. The repression of genes involved in N-assimilation suggests that the white sectors were deprived of N. The mutation in the transcription factor mitochondrial transcription termination factor (mTERF) suggests that it induces colorlessness in leaves of the milky stripe fig.

Enhancing the abiotic stress tolerance of plants: from chemical treatment to biotechnological approaches.

Abiotic stresses affect crop plants and cause decreases in plant quality and productivity. Plants can overcome environmental stresses by activating molecular networks, including signal transduction, stress perception, metabolite production and expressions of specific stress-related genes. Recent research suggests that chemical priming is a promising field in crop stress management because plants can be primed by chemical agents to increase their tolerance to various environmental stresses. We present a concept to meet this objective and protect plants through priming of existing defense mechanisms avoiding manipulation of the genome. In addition, recent developments in plant molecular biology include the discovery of genes related to stress tolerance, including functional genes for protecting cells and regulatory genes for regulating stress responses. Therefore, enhancing abiotic stress tolerance using a transgenic approach to transfer these genes into plant genomes has attracted more investigations. Both chemical priming agents and genetic engineering can enhance regulatory and functional genes in plants and increase stress tolerance of plants. This review summarizes the latest findings of chemical priming agents and major achievements in molecular approaches that can potentially enhance the abiotic stress tolerance of plants.

Biochemical and Physiological Characteristics of Photosynthesis in Plants of Two Calathea Species.

Plants of the genus Calathea possess many leaf colors, and they are economically important because they are widely used as ornamentals for interior landscaping. Physiological performances and photosynthetic capacities of C. insignis and C. makoyana were investigated. The photosynthetic efficiencies of C. insignis and C. makoyana were significantly increased when the photosynthetic photon flux density (PPFD) increased from 0 to 600 µmol photons·m-2·s-1 and became saturated with a further increase in the PPFD. The two Calathea species had lower values of both the light saturation point and maximal photosynthetic rate, which indicated that they are shade plants. No significant differences in predawn Fv/Fm values (close to 0.8) were observed between dark-green (DG) and light-green (LG) leaf sectors in all tested leaves. However, the effective quantum yield of photosystem II largely decreased as the PPFD increased. An increase in the apparent photosynthetic electron transport rate was observed in both species to a maximum at 600 µmol·m-2·s-1 PPFD, following by a decrease to 1500 µmol·m-2·s-1 PPFD. Compared to LG leaf extracts, DG leaf extracts contained higher levels of chlorophyll (Chl) a, Chl b, Chls a + b, carotenoids (Cars), anthocyanins (Ants), flavonoids (Flas), and polyphenols (PPs) in all plants, except for the Ant, Fla and PP contents of C. insignis plants. Calathea insignis also contained significantly higher levels of total protein than did C. makoyana. The adjusted normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), red-green, and flavonol index (FlavI) were significantly correlated to leaf Chls a + b, Cars, Ants, and Flas in C. makoyana, respectively, and can be used as indicators to characterize the physiology of these plants.

Structural, biochemical, and physiological characterization of photosynthesis in leaf-derived cup-shaped galls on Litsea acuminata.

BACKGROUND: The source and sink relationships between insect-induced galls and host plant leaves are interesting. In this research, we collected cup-like galls induced by Bruggmanniella sp. (Diptera: Cecidomyiidae) on host leaves of Litsea acuminata and assessed them to investigate source-sink relationships between galls and host leaves. We characterized several of their photosynthetic characteristics including chlorophyll fluorescence (Fv/Fm), stomatal conductance, and photosynthetic capacity, biochemical components such as total soluble sugar, starches, free amino acids, and soluble proteins. The structural analyses were performed under confocal, light, and scanning electron microscopies. RESULTS: Compared with host leaves, galls exhibited slightly lower chlorophyll fluorescence; however, stomatal conductance and photosynthetic capacity were not detected at all. Galls accumulated higher total soluble sugars and free amino acids but less soluble proteins than host leaves. No stomata was observed on exterior or interior gall surfaces under light or scanning electron microscopy, but their inner surfaces were covered with fungal hyphae. Confocal imagery showed a gradient of chloroplasts distribution between gall outer and inner surfaces. CONCLUSIONS: Our results strongly suggest that leaf-derived cecidomyiid galls are a type of chlorophyll-deficient non-leaf green tissue and consists on a novel sink in L. acuminate.

Leaf-derived cecidomyiid galls are sinks in Machilus thunbergii (Lauraceae) leaves.

Three relevant hypotheses - nutrition, environment and the enemies hypothesis - often invoked to explore source and sink relationships between galls and their host plants are still under dispute. In this research, chlorophyll fluorescence, gas exchange capacity, stomatal conductance, total carbon and nitrogen, total soluble sugars and starches, and scanning and transmission electron microscopy of two types of galls were used to investigate source-sink relationships. Compared with host leaves, these galls demonstrated slightly lower chlorophyll fluorescence; however, gas exchange capacity and stomatal conductance were not detected at all. Scanning electron micrographs demonstrated that the abaxial epidermis of host leaves contain normal amounts of stomata, whereas no stomata were observed on the exterior and interior surfaces of both types of galls. In addition, gall inner surfaces were covered with many kinds of fungal hyphae. Gall total carbon (C) and nitrogen (N) levels were lower but the C/N ratio was higher in galls than host leaves. Both types of galls accumulated higher total soluble sugars and starches than host leaves. Transmission electron micrographs also revealed that both types of galls contain plastoglobuli and giant starch granules during gall development. Results strongly indicate that leaf-derived cecidomyiid galls are sinks in Machilus thunbergii leaves. However, it is perplexing how larvae cycle and balance CO(2) and O(2) in gall growth chambers without stomata.

Organ-specific distribution of chlorophyll-related compounds from dietary spinach in rabbits.

The distribution of chlorophyll-related compounds (CRCs) derived from dietary spinach was investigated in different organs the rabbits. The rabbits in the experimental group consumed 100 g of freeze-dried spinach powder after a 24 h fasting period and sacrificed 2, 4, 8, 12 and 24 h later and in the control group sacrificed after the 24 h fasting period. The main CRCs in the liver were found to be chlorophyll (Chl a) and b, chlorophyllide (Chlide) a and b, pheophytin (Phe) a and b and pheophorbide (Pho) a and b, which reached their peak values at 8 h post-feeding. The gallbladder contained mainly Chlide a and a', Pho a and a', Pho b and b', which peaked their values at 2 h post-feeding. Pho a and b were consistently observed in the blood and peaked at 12 h post-feeding. The earlier appearance of Chlide a', Pho a' and Pho b' in the gallbladder compared to the liver indicated that these CRCs were compartmentalized differently and might undergo the same type of vectorialized transport as characterized for the bile salts. Pho levels peaked later in the blood compared to the liver, suggesting that Pho might be released into the peripheral blood circulation from the liver. In conclusion, Chlide and Pho were the principal Chl metabolites in the rabbits. Our data may expand our understanding of the metabolism and biodistribution of CRCs in the human body. A number of biological functions, including anti-oxidation, anti-tumor and anti-aging have recently been attributed to CRCs, it will be interesting to explore, if the binding of Chlide and Pho to other nutrients or trace metal ions in the body mediate their biological functions.

Insights into intramolecular Trp and His side-chain orientation and stereospecific π interactions surrounding metal centers: an investigation using protein metal-site mimicry in solution.

Metal-binding scaffolds incorporating a Trp/His-paired epitope are instrumental in giving novel insights into the physicochemical basis of functional and mechanistic versatility conferred by the Trp-His interplay at a metal site. Herein, by coupling biometal site mimicry and (1)H and (13)C NMR spectroscopy experiments, modular constructs EDTA-(L-Trp, L-His) (EWH; EDTA=ethylenediamino tetraacetic acid) and DTPA-(L-Trp, L-His) (DWH; DTPA=diethylenetriamino pentaacetic acid) were employed to dissect the static and transient physicochemical properties of hydrophobic/hydrophilic aromatic interactive modes surrounding biometal centers. The binding feature and identities of the stoichiometric metal-bound complexes in solution were investigated by using (1)H and (13)C NMR spectroscopy, which facilitated a cross-validation of the carboxylate, amide oxygen, and tertiary amino groups as the primary ligands and indole as the secondary ligand, with the imidazole (Im) N3 nitrogen being weakly bound to metals such as Ca(2+) owing to a multivalency effect. Surrounding the metal centers, the stereospecific orientation of aromatic rings in the diastereoisomerism is interpreted with the Ca(2+)-EWH complex. With respect to perturbed Trp side-chain rotamer heterogeneity, drastically restricted Trp side-chain flexibility and thus a dynamically constrained rotamer interconversion due to π interactions is evident from the site-selective (13)C NMR spectroscopic signal broadening of the Trp indolyl C3 atom. Furthermore, effects of Trp side-chain fluctuation on indole/Im orientation were the subject of a 2D NMR spectroscopy study by using the Ca(2+)-bound state; a C-H2(indolyl)/C-H5(Im(+)) connectivity observed in the NOESY spectra captured direct evidence that the N-H1 of the Ca(2+)-Im(+) unit interacted with the pyrrole ring of the indole unit in Ca(2+)-bound EWH but not in DWH, which is assignable to a moderately static, anomalous, T-shaped, interplanar π(+)-π stacking alignment. Nevertheless, a comparative (13)C NMR spectroscopy study of the two homologous scaffolds revealed that the overall response of the indole unit arises predominantly from global attractions between the indole ring and the entire positively charged first coordination sphere. The study thus demonstrates the coordination-sphere/geometry dependence of the Trp/His side-chain interplay, and established that π interactions allow (13)C NMR spectroscopy to offer a new window for investigating Trp rotamer heterogeneity near metal-binding centers.

Potential of galled leaves of Goji (Lycium chinense) as functional food.

BACKGROUND: Goji (Lycium) is a popular traditional health food, and its fruit and root extracts have been found to possess antioxidant, anti-inflammatory, and hypocholesterolemia-inducing abilities. Goji leaves also contain high amounts of phenolic compounds, similar to its fruit, and their extracts also exhibit several pharmaceutical effects. The induction of galls on Goji leaves reduces their photosynthetic ability and fruit yield, which raise their farming costs, thereby leading to economic loss. However, the defense mechanisms induced by infection may elevate the secondary metabolite content of the leaves, which might provide more nutritive compounds. METHOD: Content of chlorophyll, carotenoids, polyphenols, and flavonoids in the extracts of normal and infected Goji leaves (L. chinense) were analyzed. The relative content of chlorogenic acid and rutin, two major phenolic compounds in Goji leaves, were determined by LC-MS/MS. Antioxidant activity was presented by demonstrating the DPPH scavenging percentage. The extract of Goji fruit (L. barbarum) was also analyzed to show a comparative result. RESULTS: In this study, we found that in infected Goji leaves, the polyphenol content was significantly increased. The level of chlorogenic acid was increased by 36% in galled leaves. The content of rutin in galled leaves was also elevated. Testing the antioxidant activities also showed that the extracts of galled leaves have higher DPPH scavenging abilities. CONCLUSIONS: Our results demonstrated that galled Goji leaves have higher functional value, and may have potential as being consumed as health food.

Metagenomic, phylogenetic, and functional characterization of predominant endolithic green sulfur bacteria in the coral Isopora palifera.

BACKGROUND: Endolithic microbes in coral skeletons are known to be a nutrient source for the coral host. In addition to aerobic endolithic algae and Cyanobacteria, which are usually described in the various corals and form a green layer beneath coral tissues, the anaerobic photoautotrophic green sulfur bacteria (GSB) Prosthecochloris is dominant in the skeleton of Isopora palifera. However, due to inherent challenges in studying anaerobic microbes in coral skeleton, the reason for its niche preference and function are largely unknown. RESULTS: This study characterized a diverse and dynamic community of endolithic microbes shaped by the availability of light and oxygen. In addition, anaerobic bacteria isolated from the coral skeleton were cultured for the first time to experimentally clarify the role of these GSB. This characterization includes GSB's abundance, genetic and genomic profiles, organelle structure, and specific metabolic functions and activity. Our results explain the advantages endolithic GSB receive from living in coral skeletons, the potential metabolic role of a clade of coral-associated Prosthecochloris (CAP) in the skeleton, and the nitrogen fixation ability of CAP. CONCLUSION: We suggest that the endolithic microbial community in coral skeletons is diverse and dynamic and that light and oxygen are two crucial factors for shaping it. This study is the first to demonstrate the ability of nitrogen uptake by specific coral-associated endolithic bacteria and shed light on the role of endolithic bacteria in coral skeletons.

Transcriptome profile of cup-shaped galls in Litsea acuminata leaves.

BACKGROUND: Insect galls are atypical plant tissues induced by the invasion of insects. Compared to the host leaf, gall tissues lose photosynthetic ability, but have higher soluble sugar content. Although the physiological and biochemical regulation of gall tissues have been demonstrated, the mechanism of genetic regulation has only been analyzed in few studies. RESULTS: In this study, the transcriptome of cup-shaped galls and its host leaf were de novo assembled. Cellular functional enrichment and differentially expressed gene groups in the gall tissues were analyzed. The genes associated with primary metabolism, including photosynthesis, cell wall turnover, and sugar degradation, were expressed differently in galls and leaves. The examination of gene expression demonstrated that the genes involved in brassinosteroid synthesis and responses exhibited a remarkable modulation in cup-shaped galls, suggesting a potential role of steroid hormones in regulating gall development. CONCLUSIONS: This study revealed the genetic responses, including those involved in source-sink reallocation and phytohormone metabolism, of galls induced by a dipteran insect.

Eliminating interference by anthocyanins when determining the porphyrin ratio of red plant leaves.

Anthocyanins (Ants) are water-soluble secondary metabolites that are responsible for red colour of plant leaves. To determine photosynthetic pigments, 80% acetone was used to extract Ants from Ant-containing leaves of test plants. However, using the 80% acetone extraction method can lead to interference between chlorophylls (Chls) and Ants. Porphyrins, such as protoporphyrin IX (PPIX), Mg-protoporphyrin IX (MgPP), and protochlorophyllide (Pchlide), are Chl biosynthetic intermediates and demonstrate photospectrometric characteristics similar to those of Chl. Although the ether/water extraction method was able to remove Ants interference when detecting porphyrins, the porphyrins extraction efficiency was lower than that of the 80% acetone extraction method. Low Ants levels interfered with individual porphyrin ratios, and the extent of the effect was correlated with Ants concentrations. We developed the three equations could eliminate interference by Ants when determining the porphyrin molecular percentage (%) and were comprehensively applied to all tested species of Ants-containing leaves.

Effects of chlorophyll-related compounds on hydrogen peroxide induced DNA damage within human lymphocytes.

Chlorophylls (Chl's) are the most abundant natural plant pigments. Four chlorophyll-related compounds (CRCs), including chlorophyllide a and b (Chlide a and b) and pheophorbide a and b (Pho a and b), were investigated for their antioxidative capacities to protect human lymphocyte DNA from hydrogen peroxide (H(2)O(2)) induced strand breaks and oxidative damage ex vivo. Lymphocytes exposed to H(2)O(2) at concentrations of 10 and 50 microM revealed an increased frequency of DNA single-strand breaks (ssb's; as measured by the comet assay) and also an increased level of oxidized nucleoside (as measured by 8-hydroxydeoxyguanosine, 8-OHdG). All Chl's reduced the level of DNA ssb's and 8-OHdG within human lymphocytes following exposure to 10 microM H(2)O(2). Only Pho a and b were able to decrease DNA ssb's and 8-OHdG following treatment of lymphocytes with 50 microM H(2)O(2), in a concentration-dependent fashion. It was demonstrated herein that Pho a and b were more antioxidative than others. We applied DPPH free-radical scavenge assays in vitro, and got similar results. Pho a and b had higher ability in scavenging capacities than others. We conclude that water-extract Chl's are able to enhance the ability of human lymphocytes to resist H(2)O(2)-induced oxidative damage, especially for Pho a and b.

A rationally designed novel receptor for probing cooperative interaction between metal ions and bivalent tryptophan side chain in solution.

Cooperative interactions between metal ions and bivalent tryptophan side chain are identified in water by fluorescence quenching of a designed novel receptor, EDTA-bis(L-tryptophan methyl ester), supplemented by a circular dichroism study; results revealed that the receptor is capable of distinguishing a variety of metal ions on the basis of their abilities in quenching tryptophan fluorescence and the relative magnitude of these interactions is Cu2+ approximately Fe2+ >> Co2+ > Ni2+ >> Mn2+ > Zn2+ > La3+ > Al3+.

Antioxidant activity in extracts of 27 indigenous Taiwanese vegetables.

The objectives of this study were to identify the antioxidants and antioxidant axtivity in 27 of Taiwan's indigenous vegetables. Lycium chinense (Lc), Lactuca indica (Li), and Perilla ocymoides (Po) contained abundant quercetin (Que), while Artemisia lactiflora (Al) and Gynura bicolor (Gb) were rich in morin and kaempferol, respectively. Additionally, Nymphoides cristata (Nc) and Sechium edule (Se)-yellow had significantly higher levels of myricetin (Myr) than other tested samples. Cyanidin (Cyan) and malvidin (Mal) were abundant in Gb, Abelmoschus esculentus Moench (Abe), Po, Anisogonium esculentum (Retz.) Presl (Ane), Ipomoea batatas (Ib)-purple, and Hemerocallis fulva (Hf)-bright orange. Relatively high levels of Trolox equivalent antioxidant capacity (TEAC), oxygen radical absorption capacity (ORAC), and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenger were generated from extracts of Toona sinensis (Ts) and Po. Significant and positive correlations between antioxidant activity and polyphenols, anthocyanidins, Que, Myr, and morin were observed, indicating that these phytochemicals were some of the main components responsible for the antioxidant activity of tested plants. The much higher antioxidant activity of Po, Ts, and Ib (purple leaf) may be related to their higher Cyan, Que, and polyphenol content.

Eliminating interference by anthocyanin in chlorophyll estimation of sweet potato (Ipomoea batatas L.) leaves.

BACKGROUND: Spectral reflectance was evaluated for its usefulness as a nondestructive estimation of chlorophyll (Chl) content from three cultivars of sweet potato (Ipomoea batatas L.) with green, yellow, and purple leaves grown in a greenhouse for 22 days. While the green and yellow leaves contain variant amount of photosynthetic pigments without or with little level of anthocyanins, the purple leaves, except large amount of photosynthetic pigments, have high quantity of anthocyanins. RESULTS: For green and yellow leaves, the reciprocal reflectance (R-1) and derived indices incorporating near infrared (NIR) reflectance, [(Rλ)-1 - (RNIR)-1] and [(RNIR/Rλ) - 1], in the green and red edge spectral ranges were shown to be strongly correlated (r2 = 0.8 ~ 0.9) with the chlorophyll content. The root mean square error (RMSE) of the chlorophyll content estimation using these indices was < 50 mg m-2. However, when purple leaves containing high levels of anthocyanins were included in the sample, R-1 in the green spectral range and the above-mentioned indices displayed much weaker correlations with the chlorophyll content. The RMSE of chlorophyll estimation using these indices in the green spectral range sharply increased to > 110 mg m-2 when the sample included purple leaves. The new index, [1 - (Rλ/RNIR)], was therefore inferred and developed to eliminate the distorting effect of anthocyanins on chlorophyll content estimation using reflectance in the green spectral range. For leaves with high levels of anthocyanins, the correlation between [1 - (Rλ/RNIR)] and the chlorophyll content remained strong (r2 = 0.8 ~ 0.9) in the green spectral range, and the RMSE was minimal. CONCLUSION: The reflectance index, [1 - (Rλ/RNIR)], therefore represents a new and useful parameter for estimating leaf chlorophyll content in leaves with any level of anthocyanins such as purple rice leaf.

Antioxidant activity of herbaceous plant extracts protect against hydrogen peroxide-induced DNA damage in human lymphocytes.

BACKGROUND: Herbaceous plants containing antioxidants can protect against DNA damage. The purpose of this study was to evaluate the antioxidant substances, antioxidant activity, and protection of DNA from oxidative damage in human lymphocytes induced by hydrogen peroxide (H2O2). Our methods used acidic methanol and water extractions from six herbaceous plants, including Bidens alba (BA), Lycium chinense (LC), Mentha arvensis (MA), Plantago asiatica (PA), Houttuynia cordata (HC), and Centella asiatica (CA). METHODS: Antioxidant compounds such as flavonol and polyphenol were analyzed. Antioxidant activity was determined by the inhibition percentage of conjugated diene formation in a linoleic acid emulsion system and by trolox-equivalent antioxidant capacity (TEAC) assay. Their antioxidative capacities for protecting human lymphocyte DNA from H2O2-induced strand breaks was evaluated by comet assay. RESULTS: The studied plants were found to be rich in flavonols, especially myricetin in BA, morin in MA, quercetin in HC, and kaemperol in CA. In addition, polyphenol abounded in BA and CA. The best conjugated diene formation inhibition percentage was found in the acidic methanolic extract of PA. Regarding TEAC, the best antioxidant activity was generated from the acidic methanolic extract of HC. Water and acidic methanolic extracts of MA and HC both had better inhibition percentages of tail DNA% and tail moment as compared to the rest of the tested extracts, and significantly suppressed oxidative damage to lymphocyte DNA. CONCLUSION: Quercetin and morin are important for preventing peroxidation and oxidative damage to DNA, and the leaves of MA and HC extracts may have excellent potential as functional ingredients representing potential sources of natural antioxidants.

Chlorophyll-related compounds inhibit cell adhesion and inflammation in human aortic cells.

The objectives of this study were to investigate the effects of chlorophyll-related compounds (CRCs) and chlorophyll (Chl) a+b on inflammation in human aortic endothelial cells. Adhesion molecule expression and interleukin (IL)-8, nuclear factor (NF)-κB p65 protein, and NF-κB and activator protein (AP)-1 DNA binding were assessed. The effects of CRCs on inflammatory signaling pathways of signal transducers and activators of transcription 3 (STAT3) and mothers against decapentaplegic homolog 4, respectively induced by IL-6 and transforming growth factor (TGF)-ß, in human aortic smooth muscle cells cultured in vitro were also investigated. HAECs were pretreated with 10 µM of CRCs, Chl a+b, and aspirin (Asp) for 18 h followed by tumor necrosis factor (TNF)-α (2 ng/mL) for 6 h, and U937 cell adhesion was determined. TNF-α-induced monocyte-endothelial cell adhesion was significantly inhibited by CRCs. Moreover, CRCs and Chl a+b significantly attenuated vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and IL-8 expressions. Treatments also significantly decreased in NF-κB expression, DNA binding, and AP-1 DNA binding by CRCs and Asp. Thus, CRCs exert anti-inflammatory effects through modulation of NF-κB and AP-1 signaling. Ten micromoles of CRCs and Asp upregulated the expression of mothers against decapentaplegic homolog 4 (Drosophila) (SMAD4) in the TGF-ß receptor signaling pathway, and SMAD3/4 transcription activity was also increased. Ten micromoles of CRCs were able to potently inhibit STAT3-binding activity by repressing IL-6-induced STAT3 expression. Our results provide a potential mechanism that explains the anti-inflammatory activities of these CRCs.

Biometal binding-site mimicry with modular, hetero-bifunctionally modified architecture encompassing a Trp/His motif: insights into spatiotemporal noncovalent interactions from a comparative spectroscopic study.

Metal-site Trp/His interactions are crucial to diverse metalloprotein functions. This paper presents a study using metal-motif mimicry to capture and dissect the static and transient components of physicochemical properties underlying the Trp/His aromatic side-chain noncovalent interactions across the first- and second-coordination spheres of biometal ions. Modular biomimetic constructs, EDTA-(L-Trp, L-His) or EWH and DTPA-(L-Trp, L-His) or DWH, featuring a function-significant Trp/His pair, enabled extracting the putative hydrophobic/hydrophilic aromatic interactions surrounding metal centers. Fluorescence, circular dichroism (CD) spectroscopic titrations and ESI mass spectrometry demonstrated that both the constructs stoichiometrically bind to Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+), Zn(2+), Cd(2+), and Fe(2+), and such binding was strongly coupled to stereospecific side-chain structure reorientations of the Trp indole and His imidazole rings. A mechanistic dichotomy corresponding to the participation of the indole unit in the binding event was revealed by a scaffold-platform correlation of steady-state fluorescence-response landscape, illuminating that secondary-coordination-sphere ligand cation-π interactions were immediately followed by subsequent transient physicochemical processes including through-space energy transfer, charge transfer and/or electron transfer, depending on the type of metals. The fluorescence quenching of Trp side chain by 3d metal ions can be ascribed to through-space d-π interactions. While the fluorescence titration was capable of illuminating a two-component energetic model, clean isosbestic/isodichroic points in the CD titration spectra indicated that the metallo-constructs, such as Cu(2+)-EWH complex, fold thermodynamically by means of a two-state equilibrium. Further, the metal-ion dependence of Trp conformational variation in the modular architecture of metal-bound scaffolds was evidenced unambiguously by the CD spectra and supported by MMFF calculations; both were capable of distinguishing between the coordination geometry and the preference for metal binding mode. The study thus helps understand how aromatic rings around metal-sites have unique capabilities through the control of the spatiotemporal distribution of noncovalent interaction elements to achieve diverse chemical functionality.

Dissecting the general physicochemical properties of noncovalent interactions involving tyrosine side chain as a second-shell ligand in biomolecular metal-binding site mimetics: an experimental study combining fluorescence, 13C NMR spectroscopy and ESI mass spectrometry.

Detailed physicochemical features inherent in the dynamic cation-pi interactions of aromatic amino acid side chains in the secondary coordination spheres around metal ions were extracted and mapped by intrinsic tyrosine fluorescence titration experiments with two homologous, artificially engineered metal-binding scaffolds which mimic metal-binding sites in metalloproteins. A newly formulated method for the treatment of fluorescence titration data allows straightforward assessment of both the magnitudes and properties of metal-chelation-assisted cation-aromatic interactions (K2) underlying a proposed two-step metallosupramolecular association process. The unprecedented linear platform-motif correlations between the two contrasting scaffolds in their changes in tyrosine fluorescence on binding of 3d metal cations help to elucidate the properties of general cation-arene recognition corresponding to the metal-responsive characteristics of the second-shell Tyr residue surrounding the metal-binding sites in the supramolecular context, and thereby define a new noncovalent design principle for metal-ion recognition in aqueous solution. As supported by NMR spectroscopic and ESI-MS analyses and molecular mechanics force field calculations, the systematic study exemplifies the concept of using steady-state tyrosine fluorescence as a powerful tool for comprehensive descriptions of cation-pi interactions in the extended environment of a metal-binding site. We established that the physicochemical properties pertaining to indirect metal-arene interactions are highly dependent on the electronic properties of the metal ions. This work suggests that second-shell cation-pi interactions may play more diverse roles, including modulation of structure, reactivity, and function of metal-binding sites, than the previously well-established direct cation-pi interactions involving hard cations (e.g., alkali metal ions). Moreover, such a study will continue to complement theoretical predications and/or the early experimental investigations in organic solvents.