Wheat Germ Extract Research Articles

Mechanism of the anti-angiogenic effect of Avemar on tumor cells.

Avemar, a derivative of fermented wheat germ extract, is a non-toxic and natural compound that is used as a dietary supplement by cancer patients undergoing chemotherapy and radiotherapy. Avemar has numerous biological activities, and several recent studies have reported that it may also have metastatic and anti-angiogenic effects. In the present study, the mechanism of the anti-angiogenic effect of Avemar on human cancer cells was investigated. The human cell lines NCI-N87 (gastric tubular adenocarcinoma), PC3 (prostate carcinoma), HeLa (endocervical adenocarcinoma) and A549 (lung adenocarcinoma) were treated with various doses (400, 800, 1,600 and 3,200 µg/ml) of Avemar, and the changes in mRNA and protein levels of two important markers of angiogenesis, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (Cox-2), were assessed by reverse transcription-quantitative polymerase chain reaction and ELISA. VEGF and Cox-2 protein and mRNA levels were significantly lower in Avemar-treated cells than in untreated cells. The data suggest that Avemar may exert an anti-angiogenic effect on cancer cells. Thus, it is suggested to medical doctors as a potential agent for the anti-angiogenic treatment of cancer.

Isolation and purification of wheat germ agglutinin and analysis of its properties

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Effects of G-quadruplex topology on translational inhibition by tRNA fragments in mammalian and plant systems in vitro.

The folding of tRNA fragments (tRFs) into G-quadruplex structures and the implications of G-quadruplexes in translational inhibition have been studied mainly in mammalian systems. To increase our knowledge of these phenomena, we determined the influence of human and plant tRFs and model G-quadruplexes on translation in rabbit reticulocyte lysate and wheat germ extract. The efficiency of translational inhibition in the mammalian system was strongly associated with the type of G-quadruplex topology. In the plant system, the ability of a small RNA to adopt the G-quadruplex conformation was not sufficient to repress translation, indicating the importance of other structural determinants.

Anti-Inflammatory Activity of Citric Acid-Treated Wheat Germ Extract in Lipopolysaccharide-Stimulated Macrophages.

Until recently, fermentation was the only processing used to improve the functionality of wheat germ. The release of 2,6-dimethoxy-1,4-benzoquinone (DMBQ) from hydroquinone glycosides during the fermentation process is considered a marker of quality control. Here, we treated wheat germ extract with citric acid (CWG) to release DMBQ and examined the anti-inflammatory activity of this extract using a lipopolysaccharide-activated macrophage model. Treatment of wheat germ with citric acid resulted in detectable release of DMBQ but reduced total phenolic and total flavonoid contents compared with untreated wheat germ extract (UWG). CWG inhibited secretion of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-6, and IL-12 and the synthesis of cyclooxygenase-2, while UWG only decreased IL-12 production. CWG and UWG induced high levels of anti-inflammatory IL-10 and heme oxygenase-1. CWG specifically inhibited phosphorylation of NF-κB p65 and p38 kinase at 15 min after LPS stimulation. Our study showed that citric acid treatment enhanced the anti-inflammatory activity of wheat germ extract.

Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System.

We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5' cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.

Position-dependent effects of regioisomeric methylated adenine and guanine ribonucleosides on translation.

Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of adenine and guanine in mRNA affects translation. When situated at the third codon position, the methylated nucleosides did not compromise the speed or accuracy of translation under most circumstances. When located at the first or second codon position, N1-methyladenosine (m1A) and m1G constituted robust blocks to both Escherichia coli and wheat germ extract translation systems, whereas N2-methylguanosine (m2G) moderately impeded translation. While m1A, m2G and N6-methyladenosine (m6A) did not perturb translational fidelity, O6-methylguanosine (m6G) at the first and second codon positions was strongly and moderately miscoding, respectively, and it was decoded as an adenosine in both systems. The effects of methylated ribonucleosides on translation could be attributed to the methylation-elicited alterations in base pairing properties of the nucleobases, and the mechanisms of ribosomal decoding contributed to the position-dependent effects. Together, our study afforded important new knowledge about the modulation of translation by methylation of purine nucleobases in mRNA.

The Triticum Mosaic Virus 5' Leader Binds to Both eIF4G and eIFiso4G for Translation.

We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5’ untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5’ UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5’ UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

Ligand-responsive upregulation of 3' CITE-mediated translation in a wheat germ cell-free expression system.

We have rationally constructed a novel regulation-type of artificial riboswitch that ligand-dose dependently upregulates translation initiation mediated by a 3' cap-independent translation element (3' CITE) with no major hybridization switches in a plant expression system (wheat germ extract).

The Effect of Fermented Wheat Germ Extract on Biochemical, Physiological and Performance Parameters of Broiler Chickens

Fermented wheat germ extract (FWGE) is a multisubstance composition contains 2-methoxy benzoquinone and 2, 6-dimethoxy benzoquinone which are likely to exert some of its biological effects as well as it is a concentrated source of vitamins, minerals, and protein. An experimental trial of FWGE supplementation to broiler feed from one day old with a rate of 0.5, 1.5 and 3 g/kg feed was tried. Results revealed that all doses of FWGE increased body weight significantly (p≤0.05), especially with the dose of 3 g/kg feed. Also, FCR values decreased significantly (p≤0.05) in the FWGE treated groups. Regarding biochemical analysis at 35 days old, the most significant results obtained with the doses of 1.5 and 3 g FWGE/kg feed especially in SGOT, SGPT, creatinine, uric acid, total protein, glucose, and triglycerides levels. Physiologically, FWGE only increased hemoglobin concentration significantly (p≤0.05) without alteration of red blood and white blood cells counts. There was also a significant increase (p≤0.05) in the intestinal weight in relation to carcass weight% and a significant decrease (p≤0.05) in the liver and total body fat weights in relation to carcass weight%. At 45 days (10 days after vNDV challenge), the mortality rates were 60% in the non-treated non-vaccinated challenged chicken group 5 and 4% in the non-treated vaccinated challenged chicken group 4 without appearance of any mortality in the 3 FWGE treated groups. Also, addition of FWGE had a positive effect on HI titers for NDV in the collected serum samples at 45 days old.

EIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation.

Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5'-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation.

Production of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione from rice germ and wheat germ extracts by Mycobacterium sp.

To study the biotransformation of phytosterol and phytosterol-containing rice germ and wheat germ ethanolic extracts to produce 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacterium sp. DSM 2966 using phytosterol to hydroxypropyl-β-cyclodextrin (2:1, 1:1 and 1:2mol/mol) and 2% (w/v) Tween 80 as solubilizing agents. A maximum yield of 180±27mgADl(-1) and 31±11.4mgADDl(-1) with a total conversion of 65% (day 12) was obtained using 1g phytosterol l(-1) and hydroxypropyl-β-cyclodextrin (2 : 1mol/mol) with 2% (w/v) Tween 80 in the fermentation medium. The most appropriate conditions for rice germ extract and wheat germ extract which gave the maximum conversion of 22 and 43% (day 14) were obtained by using 2% (w/v) Tween 80. Phytosterol and wheat germ are effective sources for AD and ADD production while rice germ required further development. Hydroxypropyl-β-cyclodextrin (2 :1mol/mol) and/or 2% (w/v) Tween 80 in the biotransformation process could improve AD and ADD yields, depending on substrates and biotransformation conditions.

Biofunction-assisted DNA detection through RNase H-enhanced 3′ processing of a premature tRNA probe in a wheat germ extract

We have developed a novel type of biofunction-assisted, signal-turn-on sensor for simply and homogenously detecting DNA. This sensor system is composed of two types of in vitro-transcribed label-free RNAs (a 3′ premature amber suppressor tRNA probe and an amber-mutated mRNA encoding a reporter protein), RNase H, and a wheat germ extract (WGE). A target DNA induces the 3′ end maturation of the tRNA probe, which is enhanced by RNase H and leads to the expression of a full-length reporter protein through amber suppression in WGE, while there is almost no expression without the target due to the inactivity of the premature probe. Therefore, the target can be readily detected with the activity of the translated reporter. The catalytic reuse of the target with the help of RNase H in addition to various bioprocesses in WGE enables this sensor system to exhibit relatively high selectivity and sensitivity.

Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

BackgroundViral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus.ResultsHere, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures.ConclusionsThese results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner.ReviewersReviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers’ comments section.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0126-5) contains supplementary material, which is available to authorized users.

Inhibitory Effects of AVEMAR on Proliferation and Metastasis of Oral Cancer Cells

ABSTRACTOral cancer is keeping its 4th rank on the death causing cancers among Taiwan males, and its metastatic and recurrent rates remain high and a life-threatening issue to the citizens. Fermented wheat germ extract (AVEMAR) is used in clinical cancer nutritional therapy in gastrointestinal cancers but not in oral cancer yet. In this study, the potential of AVEMAR to inhibit tumor proliferation and metastasis of oral cancer was first investigated. Antiproliferative activity of AVEMAR was determined in human oral squamous carcinoma SCC-4 cells by MTT methodology. Wound-healing migration, transwell invasion, and Western blotting assays were carried out to examine the in vitro antimetastatic effects and involved signaling molecules for AVEMAR in oral cancer cells. AVEMAR at 0.2–1.6 mg/ml significantly inhibited the cell viability with IC50 values of 1.19 and 0.98 mg/ml for 24-h and 48-h treatment. Furthermore, AVEMAR could induce cell apoptosis and inhibit the migration and invasion of metastatic SCC-4 cells at a similar dose range. Notably, AVEMAR suppressed the expression of matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator (u-PA), but not MMP-1 or MMP-9, in SCC-4 cells. These results strongly support the antiproliferation and in vitro antimetastatic capacity of AVEMAR which may extend its contributions from cancer nutrition supplements to preventive agent for oral cancer.

The isoquinoline alkaloid sanguinarine which inhibits chaperone activity enhances the production of heat shock proteins in Arabidopsis.

Sanguinarine is an isoquinoline alkaloid produced by Papaveraceae plants. Because sanguinarine has antimicrobial activity, it is believed to be related to the plants' chemical defense systems. However, its action against plants has not been well understood. A previous study reported that among 12 alkaloids, sanguinarine was the only compound which enhanced heat tolerance in Arabidopsis. Here we performed a promoter assay using a heat shock protein gene (HSP17.6C-CI) of Arabidopsis to assess the induction of heat shock responses by alkaloids. Although sanguinarine induced the heat shock response, the other 11 alkaloids did not. Sanguinarine promoted the production of HSP17.6C-CI protein, but berberine and papaverine, which are isoquinoline alkaloids as well as sanguinarine, did not promote it. It is known that geldanamycin, a small molecule chaperone inhibitor, activates the heat shock response in Arabidopsis. Although sanguinarine inhibited the chaperone activities of wheat germ extract much like geldanamycin, berberine and papaverine influenced the activities very little. These results suggest that sanguinarine may promote the heat shock response by regulating the chaperone activities in the way that geldanamycin does in plants.

Rational optimization of amber suppressor tRNAs toward efficient incorporation of a non-natural amino acid into protein in a eukaryotic wheat germ extract.

Amber suppression is a useful method of genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA). A wheat germ extract (WGE) is suitable for this method by virtue of its high productivity and versatility in addition to its advantages as a cell-free translation system. However, in spite of this high potential, a genetic NAA incorporation system in WGE has not been sufficiently optimized in terms of sup-tRNAs, in contrast to that in E. coli and its cell extracts. We herein rationally optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein in WGE, via flexizyme-based aminoacylation. The optimized sup-tRNA (named tLys-opt) that was pre-charged with AcPhe exclusively yielded up to 220 μg mL(-1) of AcPhe-incorporated protein (yellow fluorescent protein, YPet) under the optimal conditions. This high productivity is comparable to the best reported yield of a similar NAA-incorporated protein synthesized with an engineered aminoacyl-tRNA synthetase/sup-tRNA pair in WGE, despite the fact that tLys-opt that has released AcPhe was not reused at all in this study. The results clearly show both the necessity of optimizing sup-tRNAs for efficient NAA incorporation and the validity of our strategy for their optimization. Because the optimization strategy described here is expected to be applicable not only to amber sup-tRNAs for other NAAs but also to ones used in other acylation methods, it would facilitate the synthesis of large amounts of various types of NAA-incorporated proteins in WGE.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

ObjectiveA subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 (LFWGE). MethodsThe HT-29 cells were transplanted via subcutaneous injection of 1×107 cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE (high-dose 2 g/kg/d; low-dose 1 g/kg/d) and for 7 d with 5-fluorouracil (5-FU, 25 mg/kg/d) by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. ResultsTumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group (2 g/kg/d, 60.1%±4.4%; 1 g/kg/d, 58.6%±6.9%) was significantly higher than that of the control group (11.5%±1.6%) and 5-FU group (32.1%±3.5%) as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. ConclusionThe results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.

Synthesis and Functional Reconstitution of Light-Harvesting Complex II into Polymeric Membrane Architectures.

One of most important processes in nature is the harvesting and dissipation of solar energy with the help of light-harvesting complex II (LHCII). This protein, along with its associated pigments, is the main solar-energy collector in higher plants. We aimed to generate stable, highly controllable, and sustainable polymer-based membrane systems containing LHCII-pigment complexes ready for light harvesting. LHCII was produced by cell-free protein synthesis based on wheat-germ extract, and the successful integration of LHCII and its pigments into different membrane architectures was monitored. The unidirectionality of LHCII insertion was investigated by protease digestion assays. Fluorescence measurements indicated chlorophyll integration in the presence of LHCII in spherical as well as planar bilayer architectures. Surface plasmon enhanced fluorescence spectroscopy (SPFS) was used to reveal energy transfer from chlorophyll b to chlorophyll a, which indicates native folding of the LHCII proteins.

A Unique 5' Translation Element Discovered in Triticum Mosaic Virus.

Several plant viruses encode elements at the 5' end of their RNAs, which, unlike most cellular mRNAs, can initiate translation in the absence of a 5' m7GpppG cap. Here, we describe an exceptionally long (739-nucleotide [nt]) leader sequence in triticum mosaic virus (TriMV), a recently emerged wheat pathogen that belongs to the Potyviridae family of positive-strand RNA viruses. We demonstrate that the TriMV 5' leader drives strong cap-independent translation in both wheat germ extract and oat protoplasts through a novel, noncanonical translation mechanism. Translation preferentially initiates at the 13th start codon within the leader sequence independently of eIF4E but involves eIF4G. We truncated the 5' leader to a 300-nucleotide sequence that drives cap-independent translation from the 5' end. We show that within this sequence, translation activity relies on a stem-loop structure identified at nucleotide positions 469 to 490. The disruption of the stem significantly impairs the function of the 5' untranslated region (UTR) in driving translation and competing against a capped RNA. Additionally, the TriMV 5' UTR can direct translation from an internal position of a bicistronic mRNA, and unlike cap-driven translation, it is unimpaired when the 5' end is blocked by a strong hairpin in a monocistronic reporter. However, the disruption of the identified stem structure eliminates such a translational advantage. Our results reveal a potent and uniquely controlled translation enhancer that may provide new insights into mechanisms of plant virus translational regulation. Many members of the Potyviridae family rely on their 5' end for translation. Here, we show that the 739-nucleotide-long triticum mosaic virus 5' leader bears a powerful translation element with features distinct from those described for other plant viruses. Despite the presence of 12 AUG start codons within the TriMV 5' UTR, translation initiates primarily at the 13th AUG codon. The TriMV 5' UTR is capable of driving cap-independent translation in vitro and in vivo, is independent of eIF4E, and can drive internal translation initiation. A hairpin structure at nucleotide positions 469 to 490 is required for the cap-independent translation and internal translation initiation abilities of the element and plays a role in the ability of the TriMV UTR to compete against a capped RNA in vitro. Our results reveal a novel translation enhancer that may provide new insights into the large diversity of plant virus translation mechanisms.

The Medical Use of Wheatgrass: Review of the Gap Between Basic and Clinical Applications

A wide range of health benefits have been attributed to wheatgrass, the young grass of the common wheat plant Triticum aestivum. Its components include chlorophyll, flavonoids, and vitamins C and E. Forms of wheatgrass include fresh juice, frozen juice, tablets, and powders, with compositions varying according to their production processes, as well as to the growing conditions of the wheatgrass. Laboratory in vitro studies, mostly using the fermented wheat germ extract, have demonstrated anti-cancer potential and have identified apoptosis as a possible mechanism. In animal experiments, wheatgrass demonstrated benefits in cancer prevention and as an adjunct to cancer treatment, as well as benefits to immunological activity and oxidative stress. Clinical trials show that wheatgrass may induce synergistic benefits to chemotherapy and may attenuate chemotherapy-related side effects, as well as benefit rheumatoid arthritis, ulcerative colitis, hematological diseases, diabetes, obesity, and oxidative stress. However, all the trials were small and a number of methodological problems arose. No adverse events of wheatgrass have been reported, although some forms pose problems of tolerability. The popularity of wheatgrass continues to grow. Nevertheless, the advantages seen in the clinical trials need to be proved in larger studies before clinical recommendations for the public can be given.