ResearchPad - nutrient-and-storage-proteins https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SNP markers for low molecular glutenin subunits (LMW-GSs) at the <i>Glu-A3</i> and <i>Glu-B3</i> loci in bread wheat]]> https://www.researchpad.co/article/elastic_article_13817 The content and composition of seed storage proteins is largely responsible for wheat end-use quality. They mainly consist of polymeric glutenins and monomeric gliadins. According to their electrophoretic mobility, gliadins and glutenins are subdivided into several fractions. Glutenins are classified as high molecular weight or low molecular weight glutenin subunits (HMW-GSs and LMW-GSs, respectively). LMW-GSs are encoded by multigene families located at the orthologous Glu-3 loci. We designed a set of 16 single-nucleotide polymorphism (SNP) markers that are able to detect SDS-PAGE alleles at the Glu-A3 and Glu-B3 loci. The SNP markers captured the diversity of alleles in 88 international reference lines and 27 Mexican cultivars, when compared to SDS-PAGE and STS markers, however, showed a slightly larger percent of multiple alleles, mainly for Glu-B3. SNP markers were then used to determine the Glu-1 and Glu-3 allele composition in 54 CIMMYT historical lines and demonstrated to be useful tool for breeding programs to improve wheat end product properties.

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<![CDATA[Drought stress affects the protein and dietary fiber content of wholemeal wheat flour in wheat/Aegilops addition lines]]> https://www.researchpad.co/article/5c633936d5eed0c484ae624e

Wild relatives of wheat, such as Aegilops spp. are potential sources of genes conferring tolerance to drought stress. As drought stress affects seed composition, the main goal of the present study was to determine the effects of drought stress on the content and composition of the grain storage protein (gliadin (Gli), glutenin (Glu), unextractable polymeric proteins (UPP%) and dietary fiber (arabinoxylan, β-glucan) components of hexaploid bread wheat (T. aestivum) lines containing added chromosomes from Ae. biuncialis or Ae. geniculata. Both Aegilops parents have higher contents of protein and β-glucan and higher proportions of water-soluble arabinoxylans (determined as pentosans) than wheat when grown under both well-watered and drought stress conditions. In general, drought stress resulted in increased contents of protein and total pentosans in the addition lines, while the β-glucan content decreased in many of the addition lines. The differences found between the wheat/Aegilops addition lines and wheat parents under well-watered conditions were also manifested under drought stress conditions: Namely, elevated β-glucan content was found in addition lines containing chromosomes 5Ug, 7Ug and 7Mb, while chromosomes 1Ub and 1Mg affected the proportion of polymeric proteins (determined as Glu/Gli and UPP%, respectively) under both well-watered and drought stress conditions. Furthermore, the addition of chromosome 6Mg decreased the WE-pentosan content under both conditions. The grain composition of the Aegilops accessions was more stable under drought stress than that of wheat, and wheat lines with the added Aegilops chromosomes 2Mg and 5Mg also had more stable grain protein and pentosan contents. The negative effects of drought stress on both the physical and compositional properties of wheat were also reduced by the addition of these. These results suggest that the stability of the grain composition could be improved under drought stress conditions by the intraspecific hybridization of wheat with its wild relatives.

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<![CDATA[Artificial selection on storage protein 1 possibly contributes to increase of hatchability during silkworm domestication]]> https://www.researchpad.co/article/5c50c457d5eed0c4845e85bf

Like other domesticates, the efficient utilization of nitrogen resources is also important for the only fully domesticated insect, the silkworm. Deciphering the way in which artificial selection acts on the silkworm genome to improve the utilization of nitrogen resources and to advance human-favored domestication traits, will provide clues from a unique insect model for understanding the general rules of Darwin's evolutionary theory on domestication. Storage proteins (SPs), which belong to a hemocyanin superfamily, basically serve as a source of amino acids and nitrogen during metamorphosis and reproduction in insects. In this study, through blast searching on the silkworm genome and further screening of the artificial selection signature on silkworm SPs, we discovered a candidate domestication gene, i.e., the methionine-rich storage protein 1 (SP1), which is clearly divergent from other storage proteins and exhibits increased expression in the ova of domestic silkworms. Knockout of SP1 via the CRISPR/Cas9 technique resulted in a dramatic decrease in egg hatchability, without obvious impact on egg production, which was similar to the effect in the wild silkworm compared with the domestic type. Larval development and metamorphosis were not affected by SP1 knockout. Comprehensive ova comparative transcriptomes indicated significant higher expression of genes encoding vitellogenin, chorions, and structural components in the extracellular matrix (ECM)-interaction pathway, enzymes in folate biosynthesis, and notably hormone synthesis in the domestic silkworm, compared to both the SP1 mutant and the wild silkworm. Moreover, compared with the wild silkworms, the domestic one also showed generally up-regulated expression of genes enriched in the structural constituent of ribosome and amide, as well as peptide biosynthesis. This study exemplified a novel case in which artificial selection could act directly on nitrogen resource proteins, further affecting egg nutrients and eggshell formation possibly through a hormone signaling mediated regulatory network and the activation of ribosomes, resulting in improved biosynthesis and increased hatchability during domestication. These findings shed new light on both the understanding of artificial selection and silkworm breeding from the perspective of nitrogen and amino acid resources.

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<![CDATA[Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition, melanization, coagulants, and hemocyte recruitment]]> https://www.researchpad.co/article/5989db52ab0ee8fa60bdc664

The innate immune system of insects responds to wounding and pathogens by mobilizing multiple pathways that provide both systemic and localized protection. Key localized responses in hemolymph include melanization, coagulation, and hemocyte encapsulation, which synergistically seal wounds and envelop and destroy pathogens. To be effective, these pathways require a targeted deposition of their components to provide protection without compromising the host. Extensive research has identified a large number of the effectors that comprise these responses, but questions remain regarding their post-translational processing, function, and targeting. Here, we used mass spectrometry to demonstrate the integration of pathogen recognition proteins, coagulants, and melanization components into stable, high-mass, multi-functional Immune Complexes (ICs) in Bombyx mori and Aedes aegypti. Essential proteins common to both include phenoloxidases, apolipophorins, serine protease homologs, and a serine protease that promotes hemocyte recruitment through cytokine activation. Pattern recognition proteins included C-type Lectins in B. mori, while A. aegypti contained a protein homologous to Plasmodium-resistant LRIM1 from Anopheles gambiae. We also found that the B. mori IC is stabilized by extensive transglutaminase-catalyzed cross-linking of multiple components. The melanization inhibitor Egf1.0, from the parasitoid wasp Microplitis demolitor, blocked inclusion of specific components into the IC and also inhibited transglutaminase activity. Our results show how coagulants, melanization components, and hemocytes can be recruited to a wound surface or pathogen, provide insight into the mechanism by which a parasitoid evades this immune response, and suggest that insects as diverse as Lepidoptera and Diptera utilize similar defensive mechanisms.

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<![CDATA[Glutamine Supplementation Stimulates Protein-Synthetic and Inhibits Protein-Degradative Signaling Pathways in Skeletal Muscle of Diabetic Rats]]> https://www.researchpad.co/article/5989da08ab0ee8fa60b7664f

In this study, we investigated the effect of glutamine (Gln) supplementation on the signaling pathways regulating protein synthesis and protein degradation in the skeletal muscle of rats with streptozotocin (STZ)-induced diabetes. The expression levels of key regulatory proteins in the synthetic pathways (Akt, mTOR, GSK3 and 4E-BP1) and the degradation pathways (MuRF-1 and MAFbx) were determined using real-time PCR and Western blotting in four groups of male Wistar rats; 1) control, non-supplemented with glutamine; 2) control, supplemented with glutamine; 3) diabetic, non-supplemented with glutamine; and 4) diabetic, supplemented with glutamine. Diabetes was induced by the intravenous injection of 65 mg/kg bw STZ in citrate buffer (pH 4.2); the non-diabetic controls received only citrate buffer. After 48 hours, diabetes was confirmed in the STZ-treated animals by the determination of blood glucose levels above 200 mg/dL. Starting on that day, a solution of 1 g/kg bw Gln in phosphate buffered saline (PBS) was administered daily via gavage for 15 days to groups 2 and 4. Groups 1 and 3 received only PBS for the same duration. The rats were euthanized, and the soleus muscles were removed and homogenized in extraction buffer for the subsequent measurement of protein and mRNA levels. The results demonstrated a significant decrease in the muscle Gln content in the diabetic rats, and this level increased toward the control value in the diabetic rats receiving Gln. In addition, the diabetic rats exhibited a reduced mRNA expression of regulatory proteins in the protein synthesis pathway and increased expression of those associated with protein degradation. A reduction in the skeletal muscle mass in the diabetic rats was observed and was alleviated partially with Gln supplementation. The data suggest that glutamine supplementation is potentially useful for slowing the progression of muscle atrophy in patients with diabetes.

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<![CDATA[Circulating Levels of Adiponectin, Leptin, Fetuin-A and Retinol-Binding Protein in Patients with Tuberculosis: Markers of Metabolism and Inflammation]]> https://www.researchpad.co/article/5989da80ab0ee8fa60b9a6aa

Background

Wasting is known as a prominent feature of tuberculosis (TB). To monitor the disease state, markers of metabolism and inflammation are potentially useful. We thus analyzed two major adipokines, adiponectin and leptin, and two other metabolic markers, fetuin-A and retinol-binding protein 4 (RBP4).

Methods

The plasma levels of these markers were measured using enzyme-linked immunosorbent assays in 84 apparently healthy individuals ( = no-symptom group) and 46 patients with active pulmonary TB around the time of treatment, including at the midpoint evaluation ( = active-disease group) and compared them with body mass index (BMI), C-reactive protein (CRP), chest radiographs and TB-antigen specific response by interferon-γ release assay (IGRA).

Results

In the no-symptom group, adiponectin and leptin showed negative and positive correlation with BMI respectively. In the active-disease group, at the time of diagnosis, leptin, fetuin-A and RBP4 levels were lower than in the no-symptom group [adjusted means 2.01 versus 4.50 ng/ml, P<0.0001; 185.58 versus 252.27 µg/ml, P<0.0001; 23.88 versus 43.79 µg/ml, P<0.0001, respectively]. High adiponectin and low leptin levels were associated with large infiltrates on chest radiographs even after adjustment for BMI and other covariates (P = 0.0033 and P = 0.0020). During treatment, adiponectin levels increased further and then decreased. Leptin levels remained low. Initial low levels of fetuin-A and RBP4 almost returned to the normal reference range in concert with reduced CRP.

Conclusions

Our data and recent literature suggest that low fat store and underlying inflammation may regulate these metabolic markers in TB in a different way. Decreased leptin, increased adiponectin, or this ratio may be a promising marker for severity of the disease independent of BMI. We should further investigate pathological roles of the balance between these adipokines.

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<![CDATA[Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach]]> https://www.researchpad.co/article/5989da26ab0ee8fa60b80dcb

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.

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<![CDATA[Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism]]> https://www.researchpad.co/article/5989daa5ab0ee8fa60ba7354

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

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<![CDATA[LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3]]> https://www.researchpad.co/article/5989d9e7ab0ee8fa60b6ba3d

Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori.

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<![CDATA[Proteomic analysis of injured storage roots in cassava (Manihot esculenta Crantz) under postharvest physiological deterioration]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcdb3

Postharvest physiological deterioration (PPD) is a global challenge in the improvement of cassava value chain. However, how to reduce cassava spoilage and reveal the mechanism of injured cassava storage roots in response to PPD were poorly understood. In the present study, we investigated the activities of antioxidant enzymes of cassava injured storage roots in PPD-susceptible (SC9) and PPD-tolerant (QZ1) genotypes at the time-points from 0h to 120h, and further analyzed their proteomic changes using two-dimensional electrophoresis (2-DE) in combination with MALDI-TOF-MS/MS. Ninety-nine differentially expressed proteins were identified from SC9 and QZ1 genotypes in the pairwise comparison of 24h/0h, 48h/0h, 72h/0h and 96h/0h. Of those proteins were associated with 13 biological functions, in which carbohydrate and energy metabolism related proteins were the biggest amount differential proteins in both genotypes, followed by chaperones, DNA and RNA metabolism, and defense system. We speculated that SOD in combination with CAT activities would be the first line of defense against PPD to support PPD-tolerant cassava varieties. The four hub proteins including CPN60B, LOS2, HSC70-1 and CPN20B, produced from the network of protein-protein interaction, will be the candidate key proteins linked with PPD. This study provides a new clue to improve cassava PPD-tolerant varieties and would be helpful to much better understand the molecular mechanism of PPD of cassava injured storage roots.

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<![CDATA[TBC-2 Is Required for Embryonic Yolk Protein Storage and Larval Survival during L1 Diapause in Caenorhabditis elegans]]> https://www.researchpad.co/article/5989da3eab0ee8fa60b892c2

C. elegans first stage (L1) larvae hatched in the absence of food, arrest development and enter an L1 diapause, whereby they can survive starvation for several weeks. The physiological and metabolic requirements for survival during L1 diapause are poorly understood. However, yolk, a cholesterol binding/transport protein, has been suggested to serve as an energy source. Here, we demonstrate that C. elegans TBC-2, a RAB-5 GTPase Activating Protein (GAP) involved in early-to-late endosome transition, is important for yolk protein storage during embryogenesis and for L1 survival during starvation. We found during embryogenesis, that a yolk::green fluorescent protein fusion (YP170::GFP), disappeared much more quickly in tbc-2 mutant embryos as compared with wild-type control embryos. The premature disappearance of YP170::GFP in tbc-2 mutants is likely due to premature degradation in the lysosomes as we found that YP170::GFP showed increased colocalization with Lysotracker Red, a marker for acidic compartments. Furthermore, YP170::GFP disappearance in tbc-2 mutants required RAB-7, a regulator of endosome to lysosome trafficking. Although tbc-2 is not essential in fed animals, we discovered that tbc-2 mutant L1 larvae have strongly reduced survival when hatched in the absence of food. We show that tbc-2 mutant larvae are not defective in maintaining L1 diapause and that mutants defective in yolk uptake, rme-1 and rme-6, also had strongly reduced L1 survival when hatched in the absence of food. Our findings demonstrate that TBC-2 is required for yolk protein storage during embryonic development and provide strong correlative data indicating that yolk constitutes an important energy source for larval survival during L1 diapause.

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<![CDATA[Enhanced Gastrointestinal Expression of Cytosolic Malic Enzyme (ME1) Induces Intestinal and Liver Lipogenic Gene Expression and Intestinal Cell Proliferation in Mice]]> https://www.researchpad.co/article/5989dafcab0ee8fa60bc5290

The small intestine participates in lipid digestion, metabolism and transport. Cytosolic malic enzyme 1 (ME1) is an enzyme that generates NADPH used in fatty acid and cholesterol biosynthesis. Previous work has correlated liver and adipose ME1 expression with susceptibility to obesity and diabetes; however, the contributions of intestine-expressed ME1 to these conditions are unknown. We generated transgenic (Tg) mice expressing rat ME1 in the gastrointestinal epithelium under the control of the murine villin1 promoter/enhancer. Levels of intestinal ME1 protein (endogenous plus transgene) were greater in Tg than wildtype (WT) littermates. Effects of elevated intestinal ME1 on body weight, circulating insulin, select adipocytokines, blood glucose, and metabolism-related genes were examined. Male Tg mice fed a high-fat (HF) diet gained significantly more body weight than WT male littermates and had heavier livers. ME1-Tg mice had deeper intestinal and colon crypts, a greater intestinal 5-bromodeoxyuridine labeling index, and increased expression of intestinal lipogenic (Fasn, Srebf1) and cholesterol biosynthetic (Hmgcsr, Hmgcs1), genes. The livers from HF diet-fed Tg mice also exhibited an induction of cholesterol and lipogenic pathway genes and altered measures (Irs1, Irs2, Prkce) of insulin sensitivity. Results indicate that gastrointestinal ME1 via its influence on intestinal epithelial proliferation, and lipogenic and cholesterologenic genes may concomitantly impact signaling in liver to modify this tissue’s metabolic state. Our work highlights a new mouse model to address the role of intestine-expressed ME1 in whole body metabolism, hepatomegaly, and crypt cell proliferation. Intestinal ME1 may thus constitute a therapeutic target to reduce obesity-associated pathologies.

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<![CDATA[Characterization of PHB1 and Its Role in Mitochondrial Maturation and Yolk Platelet Degradation during Development of Artemia Embryos]]> https://www.researchpad.co/article/5989da0fab0ee8fa60b78f39

Background

To cope with harsh environments, crustaceans such as Artemia produce diapause gastrula embryos (cysts) with suppressed metabolism. Metabolism and development resume during post-diapause development, but the mechanism behind these cellular events remains largely unknown.

Principal Finding

Our study investigated the role of prohibitin 1 (PHB1) in metabolic reinitiation during post-diapause development. We found that PHB1 was developmentally regulated via changes in phosphorylation status and localization. Results from RNA interference experiments demonstrated PHB1 to be critical for mitochondrial maturation and yolk degradation during development. In addition, PHB1 was present in yolk platelets, and it underwent ubiquitin-mediated degradation during the proteolysis of yolk protein.

Conclusions/Significance

PHB1 has an indispensable role in coordinating mitochondrial maturation and yolk platelet degradation during development in Artemia. This novel function of PHB1 provides new clues to comprehend the roles of PHB1 in metabolism and development.

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<![CDATA[Differential Roles of Iron Storage Proteins in Maintaining the Iron Homeostasis in Mycobacterium tuberculosis]]> https://www.researchpad.co/article/5989d9efab0ee8fa60b6ddef

Ferritins and bacterioferritins are iron storage proteins that represent key players in iron homeostasis. Several organisms possess both forms of ferritins, however, their relative physiological roles are less understood. Mycobacterium tuberculosis possesses both ferritin (BfrB) and bacterioferritin (BfrA), playing an essential role in its pathogenesis as reported by us earlier. This study provides insights into the role of these two proteins in iron homeostasis by employing M. tuberculosis bfr mutants. Our data suggests that BfrA is required for efficient utilization of stored iron under low iron conditions while BfrB plays a crucial role as the major defense protein under excessive iron conditions. We show that these two proteins provide protection against oxidative stress and hypoxia. Iron incorporation study showed that BfrB has higher capacity for storing iron than BfrA, which augurs well for efficient iron quenching under iron excess conditions. Moreover, iron release assay demonstrated that BfrA has 3 times superior ability to release stored iron emphasizing its requirement for efficient iron release under low iron conditions, facilitated by the presence of heme. Thus, for the first time, our observations suggest that the importance of BfrA or BfrB separately might vary depending upon the iron situation faced by the cell.

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<![CDATA[Transgenic Biofortification of the Starchy Staple Cassava (Manihot esculenta) Generates a Novel Sink for Protein]]> https://www.researchpad.co/article/5989daa8ab0ee8fa60ba864b

Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz). (Montagnac JA, Davis CR, Tanumihardjo SA. (2009) Compr Rev Food Sci Food Saf 8:181–194). Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

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<![CDATA[Maximal Load of the Vitamin B12 Transport System: A Study on Mice Treated for Four Weeks with High-Dose Vitamin B12 or Cobinamide]]> https://www.researchpad.co/article/5989dad3ab0ee8fa60bb726a

Several studies suggest that the vitamin B12 (B12) transport system can be used for the cellular delivery of B12-conjugated drugs, also in long-term treatment Whether this strategy will affect the endogenous metabolism of B12 is not known. To study the effect of treatment with excess B12 or an inert derivative, we established a mouse model using implanted osmotic minipumps to deliver saline, cobinamide (Cbi) (4.25 nmol/h), or B12 (1.75 nmol/h) for 27 days (n = 7 in each group). B12 content and markers of B12 metabolism were analysed in plasma, urine, kidney, liver, and salivary glands. Both Cbi and B12 treatment saturated the transcobalamin protein in mouse plasma. Cbi decreased the content of B12 in tissues to 33–50% of the level in control animals but did not influence any of the markers examined. B12 treatment increased the tissue B12 level up to 350%. In addition, the transcript levels for methylenetetrahydrofolate reductase in kidneys and for transcobalamin and transcobalamin receptor in the salivary glands were reduced. Our study confirms the feasibility of delivering drugs through the B12 transport system but emphasises that B12 status should be monitored because there is a risk of decreasing the transport of endogenous B12. This risk may lead to B12 deficiency during prolonged treatment.

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<![CDATA[The Ileal Lipid Binding Protein Is Required for Efficient Absorption and Transport of Bile Acids in the Distal Portion of the Murine Small Intestine]]> https://www.researchpad.co/article/5989db03ab0ee8fa60bc782a

The ileal lipid binding protein (ilbp) is a cytoplasmic protein that binds bile acids with high affinity. However evidence demonstrating the role of this protein in bile acid transport and homeostasis is missing. We created a mouse strain lacking ilbp (Fabp6−/− mice) and assessed the impact of ilbp deficiency on bile acid homeostasis and transport in vivo. Elimination of ilbp increased fecal bile acid excretion (54.2%, P<0.05) in female but not male Fabp6−/− mice. The activity of cholesterol 7α-hydroxylase (cyp7a1), the rate-controlling enzyme of the classical bile acid biosynthetic pathway, was significantly increased in female (63.5%, P<0.05) but not in male Fabp6−/− mice. The amount of [3H]taurocholic acid (TCA) excreted by 24 h after oral administration was 102% (P<0.025) higher for female Fabp6−/− mice whereas it was 57.3% (P<0.01) lower for male Fabp6−/− mice, compared to wild-type mice. The retained fraction of the [3H]TCA localized in the small and large intestines was increased by 22% (P<0.02) and decreased by 62.7% (P<0.01), respectively, in male Fabp6−/− mice relative wild-type mice, whereas no changes were seen in female Fabp6−/− mice. Mucosal to serosal bile acid transport using everted distal gut sacs was decreased by 74% (P<0.03) in both sexes of Fabp6−/− mice as compared to wild-type mice. The results demonstrate that ilbp is involved in the apical to basolateral transport of bile acids in ileal enterocytes, and is vital for the maintenance of bile acid homeostasis in the enterohepatic circulation (EHC) in mice.

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<![CDATA[Polymorphisms in Thioredoxin Reductase and Selenoprotein K Genes and Selenium Status Modulate Risk of Prostate Cancer]]> https://www.researchpad.co/article/5989d9f2ab0ee8fa60b6ee2d

Increased dietary intake of Selenium (Se) has been suggested to lower prostate cancer mortality, but supplementation trials have produced conflicting results. Se is incorporated into 25 selenoproteins. The aim of this work was to assess whether risk of prostate cancer is affected by genetic variants in genes coding for selenoproteins, either alone or in combination with Se status. 248 cases and 492 controls from an EPIC-Heidelberg nested case-control study were subjected to two-stage genotyping with an initial screening phase in which 384 tagging-SNPs covering 72 Se-related genes were determined in 94 cases and 94 controls using the Illumina Goldengate methodology. This analysis was followed by a second phase in which genotyping for candidate SNPs identified in the first phase was carried out in the full study using Sequenom. Risk of high-grade or advanced stage prostate cancer was modified by interactions between serum markers of Se status and genotypes for rs9880056 in SELK, rs9605030 and rs9605031 in TXNRD2, and rs7310505 in TXNRD1. No significant effects of SNPs on prostate cancer risk were observed when grade or Se status was not taken into account. In conclusion, the risk of high-grade or advanced-stage prostate cancer is significantly altered by a combination of genotype for SNPs in selenoprotein genes and Se status. The findings contribute to explaining the biological effects of selenium intake and genetic factors in prostate cancer development and highlight potential roles of thioredoxin reductases and selenoprotein K in tumour progression.

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<![CDATA[Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily]]> https://www.researchpad.co/article/5989da10ab0ee8fa60b7970e

Cupin superfamily is one of the most diverse super families. This study reports the purification and characterization of a novel cupin domain containing protease from rice bran for the first time. Hypothetical protein OsI_13867 was identified and named as cupincin. Cupincin was purified to 4.4 folds with a recovery of 4.9%. Cupincin had an optimum pH and temperature of pH 4.0 and 60°C respectively. Cupincin was found to be a homotrimer, consisting of three distinct subunits with apparent molecular masses of 33.45 kDa, 22.35 kDa and 16.67 kDa as determined by MALDI-TOF, whereas it eluted as a single unit with an apparent molecular mass of 135.33 ± 3.52 kDa in analytical gel filtration and migrated as a single band in native page, suggesting its homogeneity. Sequence identity of cupincin was deduced by determining the amino-terminal sequence of the polypeptide chains and by and de novo sequencing. For understanding the hydrolysing mechanism of cupincin, its three-dimensional model was developed. Structural analysis indicated that cupincin contains His313, His326 and Glu318 with zinc ion as the putative active site residues, inhibition of enzyme activity by 1,10-phenanthroline and atomic absorption spectroscopy confirmed the presence of zinc ion. The cleavage specificity of cupincin towards oxidized B-chain of insulin was highly specific; cleaving at the Leu15-Tyr16 position, the specificity was also determined using neurotensin as a substrate, where it cleaved only at the Glu1-Tyr2 position. Limited proteolysis of the protease suggests a specific function for cupincin. These results demonstrated cupincin as a completely new protease.

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<![CDATA[Dissecting nutrient-related co-expression networks in phosphate starved poplars]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbbbe

Phosphorus (P) is an essential plant nutrient, but its availability is often limited in soil. Here, we studied changes in the transcriptome and in nutrient element concentrations in leaves and roots of poplars (Populus × canescens) in response to P deficiency. P starvation resulted in decreased concentrations of S and major cations (K, Mg, Ca), in increased concentrations of N, Zn and Al, while C, Fe and Mn were only little affected. In roots and leaves >4,000 and >9,000 genes were differently expressed upon P starvation. These genes clustered in eleven co-expression modules of which seven were correlated with distinct elements in the plant tissues. One module (4.7% of all differentially expressed genes) was strongly correlated with changes in the P concentration in the plant. In this module the GO term “response to P starvation” was enriched with phosphoenolpyruvate carboxylase kinases, phosphatases and pyrophosphatases as well as regulatory domains such as SPX, but no phosphate transporters. The P-related module was also enriched in genes of the functional category “galactolipid synthesis”. Galactolipids substitute phospholipids in membranes under P limitation. Two modules, one correlated with C and N and the other with biomass, S and Mg, were connected with the P-related module by co-expression. In these modules GO terms indicating “DNA modification” and “cell division” as well as “defense” and “RNA modification” and “signaling” were enriched; they contained phosphate transporters. Bark storage proteins were among the most strongly upregulated genes in the growth-related module suggesting that N, which could not be used for growth, accumulated in typical storage compounds. In conclusion, weighted gene coexpression network analysis revealed a hierarchical structure of gene clusters, which separated phosphate starvation responses correlated with P tissue concentrations from other gene modules, which most likely represented transcriptional adjustments related to down-stream nutritional changes and stress.

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