Changes in Molecular Characteristics of Cereal Carbohydrates after Processing and Digestion

Different extraction, purification and digestion methods were used to investigate the molecular properties of carbohydrates in arabinoxylan and β-glucan concentrates, dietary fiber (DF) rich breads and ileum content of bread fed pigs. The breads studied were: a low DF wheat bread (WF), whole meal rye bread (GR), rye bread with kernels (RK), wheat bread supplemented with wheat arabinoxylan concentrate (AX) and wheat bread supplemented with oat β-glucan concentrate (BG). The weight average molecular weight (M_w) of extractable carbohydrates in β-glucan concentrate decreased eight-fold after inclusion in the BG bread when exposed to in vitro digestion, while the M_w of purified extractable carbohydrates in AX bread was reduced two-fold, and remained almost unaffected until reaching the terminal ileum of pigs. Similarly, the M_w of purified extractable carbohydrates in GR and RK bread was not significantly changed in the ileum. The AX bread resulted in the highest concentration of dissolved arabinoxylan in the ileum among all the breads that caused a substantial increased in ileal AX viscosity. Nevertheless, for none of the breads, the M_w of extractable carbohydrates was related neither to the bread extract nor ileal viscosity.     

MAP3Kε1/2 Interact with MOB1A/1B and Play Important Roles in Control of Pollen Germination through Crosstalk with JA Signaling in Arabidopsis

Restriction of pollen germination before the pollen grain is pollinated to stigma is essential for successful fertilization in angiosperms. However, the mechanisms underlying the process remain poorly understood. Here, we report functional characterization of the MAPKKK kinases, MAP3Kε1 and MAP3Kε2, involve in control of pollen germination in Arabidopsis. The two genes were expressed in different tissues with higher expression levels in the tricellular pollen grains. The map3kε1 map3kε2 double mutation caused abnormal callose accumulation, increasing level of JA and precocious pollen germination, resulting in significantly reduced seed set. Furthermore, the map3kε1 map3kε2 double mutations obviously upregulated the expression levels of genes in JA biosynthesis and signaling. The MAP3Kε1/2 interacted with MOB1A/1B which shared homology with the core components of Hippo singling pathway in yeast. The Arabidopsis mob1a mob1b mutant also exhibited a similar phenotype of precocious pollen germination to that in map3kε1 map3kε2 mutants. Taken together, these results suggested that the MAP3Kεs interacted with MOB1s and played important role in restriction of the precocious pollen germination, possibly through crosstalk with JA signaling and influencing callose accumulation in Arabidopsis.     

Development and Molecular Cytogenetic Identification of Two Wheat-Aegilops geniculata Roth 7Mg Chromosome Substitution Lines with Resistance to Fusarium Head Blight,Powdery Mildew and Stripe Rust

Fusarium head blight (Fhb), powdery mildew, and stripe rust are major wheat diseases globally. Aegilops geniculata Roth (U^gU^gM^gM^g, 2n = 4x = 28), a wild relative of common wheat, is valuable germplasm of disease resistance for wheat improvement and breeding. Here, we report the development and characterization of two substitution accessions with high resistance to powdery mildew, stripe rust and Fhb (W623 and W637) derived from hybrid progenies between Ae. geniculata and hexaploid wheat Chinese Spring (CS). Fluorescence in situ hybridization (FISH), Genomic in situ hybridizations (GISH), and sequential FISH-GISH studies indicated that the two substitution lines possess 40 wheat chromosomes and 2 Ae. geniculata chromosomes. Furthermore, compared that the wheat addition line parent W166, the 2 alien chromosomes from W623 and W637 belong to the 7M^g chromosomes of Ae. geniculata via sequential FISH-GISH and molecular marker analysis. Nullisomic-tetrasomic analysis for homoeologous group-7 of wheat and FISH revealed that the common wheat chromosomes 7A and 7B were replaced in W623 and W637, respectively. Consequently, lines W623, in which wheat chromosomes 7A were replaced by a pair of Ae. geniculata 7M^g chromosomes, and W637, which chromosomes 7B were substituted by chromosomes 7M^g, with resistance to Fhb, powdery mildew, and stripe rust. This study has determined that the chromosome 7M^g from Ae. geniculata exists genes resistant to Fhb and powdery mildew.     

Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.     

Split Enzyme-Based Biosensors for Structural Characterization of Soluble and Insoluble β-Glucans

β-Glucan is widely distributed in various plants and microorganisms and is composed of β-1,3-linked d-glucose units. It may have a branched short or long side chain of glucose units with β-1,6- or β-1,4-linkage. Numerous studies have investigated different β-glucans and revealed their bioactivities. To understand the structure-function relationship of β-glucan, we constructed a split-luciferase complementation assay for the structural analysis of long-chain β-1,6-branched β-1,3-glucan. The N- and C-terminal fragments of luciferase from deep-sea shrimp were fused to insect-derived β-1,3-glucan recognition protein and fungal endo-β-1,6-glucanase (Neg1)-derived β-1,6-glucan recognition protein, respectively. In this approach, two β-glucan recognition proteins bound to β-glucan molecules come into close proximity, resulting in the assembly of the full-length reporter enzyme and induction of transient luciferase activity, indicative of the structure of β-glucan. To test the applicability of this assay, β-glucan and two β-glucan recognition proteins were mixed, resulting in an increase in the luminescence intensity in a β-1,3-glucan with a long polymer of β-1,6-glucan in a dose-dependent manner. This simple test also allows the monitoring of real-time changes in the side chain structure and serves as a convenient method to distinguish between β-1,3-glucan and long-chain β-1,6-branched β-1,3-glucan in various soluble and insoluble β-glucans.     

Identification of Novel Chromosomal Aberrations Induced by 60Co-γ-Irradiation in Wheat-Dasypyrum villosum Lines

Mutations induced by radiation are widely used for developing new varieties of plants. To better understand the frequency and pattern of irradiation-induced chromosomal rearrangements, we irradiated the dry seeds of Chinese Spring (CS)-Dasypyrum villosum nullisomic-tetrasomic (6A/6D) addition (6V) line (2n = 44), WD14, with ⁶⁰Co-γ-rays at dosages of 100, 200, and 300 Gy. The M₀ and M₁ generations were analyzed using Feulgen staining and non-denaturing fluorescence in situ hybridization (ND-FISH) by using oligonucleotide probes. Abnormal mitotic behavior and chromosomes with structural changes were observed in the M₀ plants. In all, 39 M₁ plants had structurally changed chromosomes, with the B genome showing the highest frequency of aberrations and tendency to recombine with chromosomes of the D genome. In addition, 19 M₁ plants showed a variation in chromosome number. The frequency of chromosome loss was considerably higher for 6D than for the alien chromosome 6V, indicating that 6D is less stable after irradiation. Our findings suggested that the newly obtained γ-induced genetic materials might be beneficial for future wheat breeding programs and functional gene analyses.     

Biotin Transport-Targeting Polysaccharide-Modified PAMAM G3 Dendrimer as System Delivering α-Mangostin into Cancer Cells and C. elegans Worms

The natural xanthone α-mangostin (αM) exhibits a wide range of pharmacological activities, including antineoplastic and anti-nematode properties, but low water solubility and poor selectivity of the drug prevent its potential clinical use. Therefore, the targeted third-generation poly(amidoamine) dendrimer (PAMAM G3) delivery system was proposed, based on hyperbranched polymer showing good solubility, high biocompatibility and low immunogenicity. A multifunctional nanocarrier was prepared by attaching αM to the surface amine groups of dendrimer via amide bond in the ratio 5 (G3^2B12gh5M) or 17 (G3^2B10gh17M) residues per one dendrimer molecule. Twelve or ten remaining amine groups were modified by conjugation with D-glucoheptono-1,4-lactone (gh) to block the amine groups, and two biotin (B) residues as targeting moieties. The biological activity of the obtained conjugates was studied in vitro on glioma U-118 MG and squamous cell carcinoma SCC-15 cancer cells compared to normal fibroblasts (BJ), and in vivo on a model organism Caenorhabditis elegans. Dendrimer vehicle G3^2B12gh at concentrations up to 20 µM showed no anti-proliferative effect against tested cell lines, with a feeble cytotoxicity of the highest concentration seen only with SCC-15 cells. The attachment of αM to the vehicle significantly increased cytotoxic effect of the drug, even by 4- and 25-fold for G3^2B12gh5M and G3^2B10gh17M, respectively. A stronger inhibition of cells viability and influence on other metabolic parameters (proliferation, adhesion, ATP level and Caspase-3/7 activity) was observed for G3^2B10gh17M than for G3^2B12gh5M. Both bioconjugates were internalized efficiently into the cells. Similarly, the attachment of αM to the dendrimer vehicle increased its toxicity for C. elegans. Thus, the proposed α-mangostin delivery system allowed the drug to be more effective in the dendrimer-bound as compared to free state against both cultured the cancer cells and model organism, suggesting that this treatment is promising for anticancer as well as anti-nematode chemotherapy.     

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein,Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.     

miR-29b Regulates TGF-β1-Induced Epithelial–Mesenchymal Transition by Inhibiting Heat Shock Protein 47 Expression in Airway Epithelial Cells

Tissue remodeling contributes to ongoing inflammation and refractoriness of chronic rhinosinusitis (CRS). During this process, epithelial-mesenchymal transition (EMT) plays an important role in dysregulated remodeling and both microRNA (miR)-29b and heat shock protein 47 (HSP47) may be engaged in the pathophysiology of CRS. This study aimed to determine the role of miR-29b and HSP47 in modulating transforming growth factor (TGF)-β1-induced EMT and migration in airway epithelial cells. Expression levels of miR-29b, HSP47, E-cadherin, α-smooth muscle actin (α-SMA), vimentin and fibronectin were assessed through real-time PCR, Western blotting, and immunofluorescence staining. Small interfering RNA (siRNA) targeted against miR-29b and HSP47 were transfected to regulate the expression of EMT-related markers. Cell migration was evaluated with wound scratch and transwell migration assay. miR-29b mimic significantly inhibited the expression of HSP47 and TGF-β1-induced EMT-related markers in A549 cells. However, the miR-29b inhibitor more greatly induced the expression of them. HSP47 knockout suppressed TGF-β1-induced EMT marker levels. Functional studies indicated that TGF-β1-induced EMT was regulated by miR-29b and HSP47 in A549 cells. These findings were further verified in primary nasal epithelial cells. miR-29b modulated TGF-β1-induced EMT-related markers and migration via HSP47 expression modulation in A549 and primary nasal epithelial cells. These results suggested the importance of miR-29b and HSP47 in pathologic tissue remodeling progression in CRS.     

3D Structural Insights into β-Glucans and Their Binding Proteins

β(1,3)-glucans are a component of fungal and plant cell walls. The β-glucan of pathogens is recognized as a non-self-component in the host defense system. Long β-glucan chains are capable of forming a triple helix structure, and the tertiary structure may profoundly affect the interaction with β-glucan-binding proteins. Although the atomic details of β-glucan binding and signaling of cognate receptors remain mostly unclear, X-ray crystallography and NMR analyses have revealed some aspects of β-glucan structure and interaction. Here, we will review three-dimensional (3D) structural characteristics of β-glucans and the modes of interaction with β-glucan-binding proteins.     

Heteroalleles in Common Wheat: Multiple Differences between Allelic Variants of the Gli-B1 Locus

The Gli-B1-encoded γ-gliadins and non-coding γ-gliadin DNA sequences for 15 different alleles of common wheat have been compared using seven tests: electrophoretic mobility (EM) and molecular weight (MW) of the encoded major γ-gliadin, restriction fragment length polymorphism patterns (RFLPs) (three different markers), Gli-B1-γ-gliadin-pseudogene known SNP markers (Single nucleotide polymorphisms) and sequencing the pseudogene GAG56B. It was discovered that encoded γ-gliadins, with contrasting EM, had similar MWs. However, seven allelic variants (designated from I to VII) differed among them in the other six tests: I (alleles Gli-B1i, k, m, o), II (Gli-B1n, q, s), III (Gli-B1b), IV (Gli-B1e, f, g), V (Gli-B1h), VI (Gli-B1d) and VII (Gli-B1a). Allele Gli-B1c (variant VIII) was identical to the alleles from group IV in four of the tests. Some tests might show a fine difference between alleles belonging to the same variant. Our results attest in favor of the independent origin of at least seven variants at the Gli-B1 locus that might originate from deeply diverged genotypes of the donor(s) of the B genome in hexaploid wheat and therefore might be called “heteroallelic”. The donor’s particularities at the Gli-B1 locus might be conserved since that time and decisively contribute to the current high genetic diversity of common wheat.     

Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca²⁺ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca²⁺ current (I_CaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of I_CaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of I_CaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.     

Molecular Identification of the G-Protein Genes and Their Expression Profiles in Response to Nitrogen Deprivation in Brassica napus

Heterotrimeric guanine nucleotide binding protein (G-protein) consisting of Gα, Gβ, and Gγ subunits is one of the key signal transducers in plants. Recent studies indicated that G-protein has been proposed as an important mediator of nitrogen responses in rice, wheat, and Arabidopsis. However, little is known about these G-proteins in Brassica napus (B. napus), except for three identified G-proteins, BnGA1, BnGB1, and BnGG2. Therefore, the aim of the present study is to characterize the members of the G-protein gene family in allotetraploid B. napus and to analyze their expression profiles in response to nitrogen deprivation. In total, 21 G-protein family members were identified in B. napus, encoding two Gα, six Gβ, and 13 Gγ. Sequence and phylogenetic analyses showed that although genome-wide triploid events increased the number of genes encoding Gα, Gβ, and Gγ subunits, the gene structure and protein properties of the genes encoding each G-protein subunit were extremely conserved. Collinearity analysis showed that most G-protein genes in B. napus had syntenic relationships with G-protein members of Arabidopsis, Brassica rape (B. rapa), and Brassica oleracea (B. oleracea). Expression profile analysis indicated that Gα and C-type Gγ genes (except BnGG10 and BnGG12 were highly expressed in flower and ovule) were barely expressed in most organs, whereas most Gβ and A-type Gγ genes tended to be highly expressed in most organs. G-protein genes also showed various expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, Gα and five C-type Gγ genes were upregulated initially in roots, while in leaves, Gα was downregulated initially and five C-type Gγ genes were highly expressed in different times. These results provide a complex genetic dissection of G-protein genes in B. napus, and insight into the biological functions of G-protein genes in response to nitrogen deficiency.     

ST6GALNAC5 Expression Decreases the Interactions between Breast Cancer Cells and the Human Blood-Brain Barrier

The ST6GALNAC5 gene that encodes an α2,6-sialyltransferase involved in the biosynthesis of α-series gangliosides, was previously identified as one of the genes that mediate breast cancer metastasis to the brain. We have shown that the expression of ST6GALNAC5 in MDA-MB-231 breast cancer cells resulted in the expression of G_D1α ganglioside at the cell surface. By using a human blood-brain barrier in vitro model recently developed, consisting in CD34⁺ derived endothelial cells co-cultivated with pericytes, we show that ST6GALNAC5 expression decreased the interactions between the breast cancer cells and the human blood-brain barrier.     

Effects of Low Molecular Weight Yeast β-Glucan on Antioxidant and Immunological Activities in Mice

To evaluate the antioxidant and immune effects of low molecular yeast β-glucan on mice, three sulfated glucans from Saccharomyces cerevisiae (sGSCs) with different molecular weight (M_W) and degrees of sulfation (DS) were prepared. The structures of the sGSCs were analyzed through high performance liquid chromatography-gel permeation chromatography (HPLC-GPC) and Fourier transform infrared spectroscopy (FTIR). sGSC1, sGSC2, and sGSC3 had M_W of 12.9, 16.5 and 19.2 kDa, respectively, and DS of 0.16, 0.24 and 0.27, respectively. In vitro and in vivo experiments were conducted to evaluate the antioxidant and immunological activities of the sGSCs. In vitro experiment, the reactive oxygen species (ROS) scavenging activities were determined. In vivo experiment, 50 male BALB/c mice were divided into five groups. The sGSC1, sGSC2 and sGSC3 treatment groups received the corresponding sGSCs at 50 mg/kg/day each. The GSC (glucans from Saccharomyces cerevisiae) treatment group received 50 mg/kg/day GSC. The normal control group received equal volume of physiological saline solution. All treatments were administered intragastrically for 14 day. Results showed that sGSC1, sGSC2 and sGSC3 can scavenge 1,1-diphenyl-2-picryl-hydrazyl (DPPH), superoxide, and hydroxyl radicals in vitro. The strength of the radical scavenging effects of the sGSCs was in the order of sGSC1 > sGSC2 > sGSC3. Oral administration of sGSC1 significantly improved serum catalase (CAT) and glutathione peroxidase (GSH-Px) activities and decreased malondialdehyde (MDA) level in mice. sGSC1 significantly improved the spleen and thymus indexes and the lymphocyte proliferation, effectively enhanced the percentage of CD4⁺ T cells, decreased the percentage of CD8⁺ T cells, and elevated the CD4⁺/CD8⁺ ratio. sGSC1 significantly promoted the secretion of IL-2 and IFN-γ. These results indicate that sGSC1 with low M_W and DS has better antioxidant and immunological activities than the other sGSCs, and sGSC1 could be used as a new antioxidant and immune-enhancing agent.     

Effect of β-Glucan Supplementation on Acute Postprandial Changes in Fatty Acid Profile of Lymph and Serum in Pigs

Triglycerides are absorbed by the lymphatic system and have various functions in the body. It has been shown that some types of β-glucans have a positive effect on the systemic concentrations of cholesterol and lipid, presumably through interference with the absorption of lipid and/or reabsorption of bile acids. In the current study we investigated the acute effects of ingesting 2 g of β-glucan concentrates derived from barley β-(1→3)(1→4)-d-glucan or yeast β-(1→3)(1→6)-d-glucan on fatty acid content and composition in lymph and serum of 10 female pigs (initial weight 34.7 ± 1.1 kg) fitted with a permanent catheter in the jejunal lymphatic trunk in a cross-over design. Lymph was collected continuously for 8 h followed by a spot sample taken 24 h after. A significant effect of time after feeding was observed for all fatty acids in serum and for 18:0, 18:2ω6 and 18:3ω3 in lymph, but a significant effect of β-glucan was only observed for 14:0 (p = 0.049) and 22:6ω3 (p = 0.048) in lymph and 18:0 (p = 0.019) in serum. While the concentration of dietary fatty acids increased postprandially in lymph, the concentration of arachidonic and docahexanoic acid tended to decrease. Furthermore, there was a drop in concentration of all fatty acid in serum 1 h after the meal.