Compensatory Modulation of Seed Storage Protein Synthesis and Alteration of Starch Accumulation by Selective Editing of 13 kDa Prolamin Genes by CRISPR-Cas9 in Rice.

Rice prolamins are categorized into three groups by molecular size (10, 13, or 16 kDa), while the 13 kDa prolamins are assigned to four subgroups (Pro13a-I, Pro13a-II, Pro13b-I, and Pro13b-II) based on cysteine residue content. Since lowering prolamin content in rice is essential to minimize indigestion and allergy risks, we generated four knockout lines using CRISPR-Cas9, which selectively reduced the expression of a specific subgroup of the 13 kDa prolamins. These four mutant rice lines also showed the compensatory expression of glutelins and non-targeted prolamins and were accompanied by low grain weight, altered starch content, and atypically-shaped starch granules and protein bodies. Transcriptome analysis identified 746 differentially expressed genes associated with 13 kDa prolamins during development. Correlation analysis revealed negative associations between genes in Pro13a-I and those in Pro13a-II and Pro13b-I/II subgroups. Furthermore, alterations in the transcription levels of 9 ER stress and 17 transcription factor genes were also observed in mutant rice lines with suppressed expression of 13 kDa prolamin. Our results provide profound insight into the functional role of 13 kDa rice prolamins in the regulatory mechanisms underlying rice seed development, suggesting their promising potential application to improve nutritional and immunological value.

QTL detection for rice grain storage protein content and genetic effect verifications.

Rice grain quality is a multifarious attribute mainly governed by multiple nutritional factors. Grain protein is the central component of rice grain nutrition dominantly affecting eating-cooking qualities. Grain protein content is quantitatively influenced by its protein fractions. Genetic quantification of five protein fractions-albumins, globulins, prolamins, glutelin, and grain protein content-were evaluated by exploiting two BC3F2 mapping populations, derived from Kongyu131/TKM9 (population-I) and Kongyu131/Bg94-1 (population-II), which were grown in a single environment. Correlation studies among protein fractions and grain protein content were thoroughly investigated. A genetic linkage map was developed by using 146 single sequence repeat (SSR) markers in population-I and 167 markers in population-II. In total, 40 QTLs were delineated for five traits in both populations. Approximately 22 QTLs were dissected in population-I, derived from Kongyu131/TKM9, seven QTLs for albumin content, four QTLs for globulin content, three QTLs for prolamin content, four QTLs for glutelin content, and four QTLs for grain protein content. In total, 18 QTLs were detected in population-II, derived from Kongyu131/Bg94-1, five QTLs for albumin content, three QTLs for globulin content, four QTLs for prolamin content, two QTLs for glutelin content, and four QTLs for grain protein content. Three QTLs, qAlb7.1, Alb7.2, and qGPC7.2, derived from population-II (Kongyu131/Bg94-1) for albumin and grain protein content were successfully validated in the near isogenic line (NIL) populations. The localized chromosomal locus of the validated QTLs could be helpful for fine mapping via map-based cloning to discover underlying candidate genes. The functional insights of the underlying candidate gene would furnish novel perceptivity for the foundation of rice grain protein content and trigger the development of nutritionally important rice cultivars by combining marker-assisted selection (MAS) breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01436-7.

Purified Clinoptilolite-Tuff as an Efficient Sorbent for Gluten Derived from Food.

Various gluten-related diseases (celiac disease, wheat allergy, gluten sensitivity) are known and their incidence is growing. Gluten is a specific type of plant storage protein that can impair the health of gluten-prone persons following consumption, depending on the origin. The most severe effects are induced by wheat, barley, and rye. The only treatment is based on the absolute avoidance of those foods, as even traces might have severe effects on human well-being. With the goal of binding gluten impurities after ingestion, an in vitro setting was created. A special processed kind of zeolite, purified clinoptilolite-tuff (PCT), was implemented as an adsorber of gluten derived from different origins. Zeolites are known for their excellent sorption capacities and their applications in humans and animals have been studied for a long time. Tests were also performed in artificial gastric and intestinal fluids, and the adsorption capacity was determined via a certified validated method (ELISA). Depending on the kind of gluten source, 80-130 µg/mg of gluten were bound onto PCT. Hence, purified clinoptilolite-tuff, which was successfully tested for wheat, barley, and rye, proved to be suitable for the adsorption of gluten originating from different kinds of crops. This result might form the basis for an expedient human study in the future.

Prolamin-based complexes: Structure design and food-related applications.

Prolamins are a group of safe food additives that are biocompatible, biodegradable, and sustainable. Zein, gliadin, kafirin, and hordein are common prolamins that have been extensively studied, particularly as these form colloidal particles because of their amphiphilic properties. Prolamin-based binary/ternary complexes, which have stable physicochemical properties and superior functionality, are formed by combining prolamins with polysaccharides, polyphenols, water-soluble proteins, and surfactants. Although the combination of prolamins with other components has received attention, the relationship between the structural design of prolamin-based complexes and their functionalities remains uncertain. This review discusses the production methods of prolamin-based complexes, the factors influencing their structural characteristics, and their applications in the food industry. Further studies are needed to elucidate the structure-function relationships between prolamins and other biopolymers, as well as the toxicological effects of these complexes in food.

Over-Expressing TaSPA-B Reduces Prolamin and Starch Accumulation in Wheat (Triticum aestivum L.) Grains.

Starch and prolamin composition and content are important indexes for determining the processing and nutritional quality of wheat (Triticum aestivum L.) grains. Several transcription factors (TFs) regulate gene expression during starch and protein biosynthesis in wheat. Storage protein activator (TaSPA), a member of the basic leucine zipper (bZIP) family, has been reported to activate glutenin genes and is correlated to starch synthesis related genes. In this study, we generated TaSPA-B overexpressing (OE) transgenic wheat lines. Compared with wild-type (WT) plants, the starch content was slightly reduced and starch granules exhibited a more polarized distribution in the TaSPA-B OE lines. Moreover, glutenin and ω- gliadin contents were significantly reduced, with lower expression levels of related genes (e.g., By15, Dx2, and ω-1,2 gliadin gene). RNA-seq analysis identified 2023 differentially expressed genes (DEGs). The low expression of some DEGs (e.g., SUSase, ADPase, Pho1, Waxy, SBE, SSI, and SS II a) might explain the reduction of starch contents. Some TFs involved in glutenin and starch synthesis might be regulated by TaSPA-B, for example, TaPBF was reduced in TaSPA-B OE-3 lines. In addition, dual-luciferase reporter assay indicated that both TaSPA-B and TaPBF could transactivate the promoter of ω-1,2 gliadin gene. These results suggest that TaSPA-B regulates a complex gene network and plays an important role in starch and protein biosynthesis in wheat.

Modeling to Understand Plant Protein Structure-Function Relationships-Implications for Seed Storage Proteins.

Proteins are among the most important molecules on Earth. Their structure and aggregation behavior are key to their functionality in living organisms and in protein-rich products. Innovations, such as increased computer size and power, together with novel simulation tools have improved our understanding of protein structure-function relationships. This review focuses on various proteins present in plants and modeling tools that can be applied to better understand protein structures and their relationship to functionality, with particular emphasis on plant storage proteins. Modeling of plant proteins is increasing, but less than 9% of deposits in the Research Collaboratory for Structural Bioinformatics Protein Data Bank come from plant proteins. Although, similar tools are applied as in other proteins, modeling of plant proteins is lagging behind and innovative methods are rarely used. Molecular dynamics and molecular docking are commonly used to evaluate differences in forms or mutants, and the impact on functionality. Modeling tools have also been used to describe the photosynthetic machinery and its electron transfer reactions. Storage proteins, especially in large and intrinsically disordered prolamins and glutelins, have been significantly less well-described using modeling. These proteins aggregate during processing and form large polymers that correlate with functionality. The resulting structure-function relationships are important for processed storage proteins, so modeling and simulation studies, using up-to-date models, algorithms, and computer tools are essential for obtaining a better understanding of these relationships.

ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) ß-carotene hydroxylase 2 gene.

The maize (Zea mays) enzyme ß-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of ß-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036 bp of the 5'-flanking region containing the 263 bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the ß-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.

Physicochemical measurements of Japonica rice cultivars in Heilongjiang Province.

We here characterized 27 japonica rice cultivars grown in Heilongjiang province and evaluated the relationship among their iodine absorption curve, physical properties, and ratio of 13 kDa prolamin. We developed the novel estimation formulae for ratio of 13 kDa prolamin and overall hardness (H2) with the use of Aλmax and λmax.

Compensatory rebalancing of rice prolamins by production of recombinant prolamin/bioactive peptide fusion proteins within ER-derived protein bodies.

KEY MESSAGE: Bioactive peptide was produced by fusion to rice prolamins in transgenic rice seeds. Their accumulation levels were affected by their deposition sites and by compensatory rebalancing between prolamins within PB-Is. Peptide immunotherapy using analogue peptide ligands (APLs) is one of promising treatments against autoimmune diseases. Use of seed storage protein as a fusion carrier is reasonable strategy for production of such small size bioactive peptides. In this study, to examine the efficacy of various rice prolamins deposited in ER-derived protein bodies (PB-Is), the APL12 from the Glucose-6-phosphate isomerase (GPI325-339) was expressed by fusion to four types of representative prolamins under the control of the individual native promoters. When the 14 and 16 kDa Cys-rich prolamins, which were localized in middle layer of PB-Is, were used for production of the APL12, they highly accumulated in transgenic rice seeds (~ 200 µg/grain). By contrast, fusion to the 10 and 13 kDa prolamins, which were localized in the core and outermost layer of PB-Is, resulted in lower levels of accumulation (~ 40 µg/grain). These results suggest that accumulation levels were highly affected by their deposition sites. Next, when different prolamin/APL12 fusion proteins were co-expressed to increase accumulation levels, they could not be increased so much as their expected additive levels. High accumulation of one type prolamin/APL12 led to reduction of other type(s) prolamin/APL12 to maintain the limited amounts of prolamins that can be deposited in PB-Is. Moreover, suppression of endogenous seed proteins by RNA interference also did not significantly enhance the accumulation levels of prolamin/APL12. These findings suggest that there may be compensatory rebalancing mechanism that controls the accumulation levels of prolamins deposited within PB-Is.

Fermentation and aerobic stability of rehydrated corn grain silage treated with different doses of Lactobacillus buchneri or a combination of Lactobacillus plantarum and Pediococcus acidilactici.

We investigated the effects of different types and doses of inoculants for ensiling rehydrated corn grain. Shelled corn was finely ground and rehydrated to 35% moisture. Treatments were as follows: (1) control (no additives); (2) Lactobacillus plantarum and Pediococcus acidilactici (LPPA) at a theoretical application rate of 1 × 105 cfu/g; (3) LPPA at 5 × 105 cfu/g; (4) LPPA at 1 × 106 cfu/g; (5) Lactobacillus buchneri (LB) at 1 × 105 cfu/g; (6) LB at 5 × 105 cfu/g; and (7) LB at 1 × 106 cfu/g. We detected no effect of inoculant dose. Gas losses were greater in silages treated with LB compared with control and LPPA silages. Treating silages with LB reduced the concentrations of lactic acid and ethanol and increased silage pH and concentrations of acetic acid, propionic acid, and 1,2-propanediol. At silo opening, silages treated with LB had higher counts of lactic acid bacteria but lower yeast counts than the control silage. Aerobic stability was greater for silages treated with LB and lower for silages treated with LPPA compared with the control. The LB reduced dry matter (DM) losses during aerobic exposure, whereas LPPA increased them. Prolamin content was lower in silages treated with LB compared with the control, resulting in greater ruminal in situ DM degradability. Inoculating LB to a dose of 1 × 105 cfu/g increased aerobic stability and ruminal in situ DM degradability of rehydrated corn grain silage. The addition of LPPA did not alter the fermentation process and worsened the aerobic stability of rehydrated corn grain silage. Further studies are warranted to confirm these conclusions in other corn hybrids, inoculants, and their combinations.

Rapid evolutionary dynamics in a 2.8-Mb chromosomal region containing multiple prolamin and resistance gene families in Aegilops tauschii.

Prolamin and resistance gene families are important in wheat food use and in defense against pathogen attacks, respectively. To better understand the evolution of these multi-gene families, the DNA sequence of a 2.8-Mb genomic region, representing an 8.8 cM genetic interval and harboring multiple prolamin and resistance-like gene families, was analyzed in the diploid grass Aegilops tauschii, the D-genome donor of bread wheat. Comparison with orthologous regions from rice, Brachypodium, and sorghum showed that the Ae. tauschii region has undergone dramatic changes; it has acquired more than 80 non-syntenic genes and only 13 ancestral genes are shared among these grass species. These non-syntenic genes, including prolamin and resistance-like genes, originated from various genomic regions and likely moved to their present locations via sequence evolution processes involving gene duplication and translocation. Local duplication of non-syntenic genes contributed significantly to the expansion of gene families. Our analysis indicates that the insertion of prolamin-related genes occurred prior to the separation of the Brachypodieae and Triticeae lineages. Unlike in Brachypodium, inserted prolamin genes have rapidly evolved and expanded to encode different classes of major seed storage proteins in Triticeae species. Phylogenetic analyses also showed that the multiple insertions of resistance-like genes and subsequent differential expansion of each R gene family. The high frequency of non-syntenic genes and rapid local gene evolution correlate with the high recombination rate in the 2.8-Mb region with nine-fold higher than the genome-wide average. Our results demonstrate complex evolutionary dynamics in this agronomically important region of Triticeae species.

Accumulation of foreign polypeptides to rice seed protein body type I using prolamin portion sequences.

KEY MESSAGE: Rice prolamins are accumulated in endoplasmic reticulum (ER)-derived proteins bodies, although conserved sequences retained in ER are not confirmed. We investigated portion sequences of prolamins that must accumulate in PB-Is. Rice seed prolamins are accumulated in endoplasmic reticulum (ER)-derived protein body type I (PB-I), but ER retention sequences in rice prolamin polypeptides have not been confirmed. Here we investigated the lengths of the prolamin portion sequences required for accumulation in PB-Is. Of the rice prolamins, we compared 13a and 13b prolamins because the amino acid sequences of these prolamins are quite similar except for the presence or absence of Cys-residues. We also generated and analyzed transgenic rice expressing several prolamin portion sequence-GFP fusion proteins. We observed that in 13a prolamin, when the portion sequences were extended more than the 68th amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. In 13b prolamin, when the portion sequences were extended by more than the 82nd amino acid residue from the initiating methionine, the prolamin portion sequence-GFP fusion proteins were accumulated in PB-Is. When those fusion proteins were extracted under non-reduced or reduced conditions, the 13a prolamin portion sequence-GFP fusion proteins in PB-Is were soluble under only the reduced condition. In contrast, 13b prolamin portion sequence-GFP fusion proteins were soluble under both non-reduced and reduced conditions. These results suggest that the accumulation of 13a prolamin in PB-Is is associated with the formation of disulfide bonds and/or hydrophobicity in 13a prolamin polypeptide, whereas the accumulation of 13b prolamin in PB-Is was less involved in the formation of disulfide bonds.

Short communication: Influence of various proteolytic sources during fermentation of reconstituted corn grain silages.

The objective of this study was to determine the contribution of corn kernel enzymes, bacteria, fungi, and fermentation end-products (main acids and ethanol) to protein solubilization during fermentation of reconstituted corn grain silage. Flint corn kernels were ground (5-mm sieve), rehydrated to 32% of moisture, and treated with no additives (control), gamma irradiation (32 kGy), gamma irradiation + fermentation end-products (1% of lactic acid, 0.3% of acetic acid, and 0.7% of ethanol, as fed), and natamycin (1% as fed). Treated grains were ensiled in nylon-polyethylene bags and stored for 90 d. Protein solubilization was calculated for each treatment and the contributions of proteolytic sources were determined. Bacterial activity was the main contributor to proteolysis (60%) followed by corn kernel enzymes (30%), whereas fungi and fermentation end-products had only minor contributions (∼5% each).

Cellular Localization of Wheat High Molecular Weight Glutenin Subunits in Transgenic Rice Grain.

Rice (Oryza sativa L.) is a primary global food cereal. However, when compared to wheat, rice has poor food processing qualities. Dough that is made from rice flour has low viscoelasticity because rice seed lacks storage proteins that are comparable to gluten protein from wheat. Thus, current research efforts aim to improve rice flour processing qualities through the transgenic expression of viscoelastic proteins in rice seeds. In this study, we characterized the transgenic expression of wheat glutenin subunits in rice seeds. The two genes 1Dx5_KK and 1Dy10_JK, which both encode wheat high-molecular-weight glutenin subunits that confer high dough elasticity, were cloned from Korean wheat cultivars KeumKang and JoKyung, respectively. These genes were inserted into binary vectors under the control of the rice endosperm-specific Glu-B1 promoter and were expressed in the high-amylose Korean rice cultivar Koami (Oryza sativa L.). Individual expression of both glutenin subunits was confirmed by SDS-PAGE and immunoblot analyses performed using T3 generation of transgenic rice seeds. The subcellular localization of 1Dx5_KK and 1Dy10_JK in the rice seed endosperm was confirmed by immunofluorescence analysis, indicating that the wheat glutenin subunits accumulate in protein body-II and novel protein body types in the rice seed. These results contribute to our understanding of engineered seed storage proteins in rice.

Comprehensive proteomic analysis of developing protein bodies in maize (Zea mays) endosperm provides novel insights into its biogenesis.

Prolamins, the major cereal seed storage proteins, are sequestered and accumulated in the lumen of the endoplasmic reticulum (ER), and are directly assembled into protein bodies (PBs). The content and composition of prolamins are the key determinants for protein quality and texture-related traits of the grain. Concomitantly, the PB-inducing fusion system provides an efficient target to produce therapeutic and industrial products in plants. However, the proteome of the native PB and the detailed mechanisms underlying its formation still need to be determined. We developed a method to isolate highly purified and intact PBs from developing maize endosperm and conducted proteomic analysis of intact PBs of zein, a class of prolamine protein found in maize. We thus identified 1756 proteins, which fall into five major categories: metabolic pathways, response to stimulus, transport, development, and growth, as well as regulation. By comparing the proteomes of crude and enriched extractions of PBs, we found substantial evidence for the following conclusions: (i) ribosomes, ER membranes, and the cytoskeleton are tightly associated with zein PBs, which form the peripheral border; (ii) zein RNAs are probably transported and localized to the PB-ER subdomain; and (iii) ER chaperones are essential for zein folding, quality control, and assembly into PBs. We futher confirmed that OPAQUE1 (O1) cannot directly interact with FLOURY1 (FL1) in yeast, suggesting that the interaction between myosins XI and DUF593-containing proteins is isoform-specific. This study provides a proteomic roadmap for dissecting zein PB biogenesis and reveals an unexpected diversity and complexity of proteins in PBs.

Deposition mode of transforming growth factor-ß expressed in transgenic rice seed.

KEY MESSAGE: Mouse TGF-ß highly accumulated by expressing as a secretory homodimeric protein in transgenic rice endosperm. It was tightly deposited in ER-derived PBs by interaction with cysteine-rich prolamins. TGF-ß is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the induction of regulatory T cells. In order to utilize rice-based TGF-ß as a tool to promote oral immune tolerance induction, high production of TGF-ß is essentially required. When the codon-optimized mTGF-ß was expressed as a secretory protein by ligating an N-terminal signal peptide and C-terminal KDEL ER retention signal under the control of the endosperm-specific rice storage protein glutelin GluB-1 promoter, accumulation level was low in stable transgenic rice seeds. Then, to increase the accumulation level of mTGF-ß, it was expressed as fusion proteins by inserting into the C terminus of acidic subunit of glutelin GluA and the variable region of 26 kDa globulin. When fused with the glutelin, it could accumulate well as visible bands by CBB staining gel, but not for the 26 kDa globulin. Unexpectedly, expression of homodimeric mTGF-ß linked by a 6×Gly1×Ser linker as secretory protein resulted in higher level of accumulation. This expression level was further enhanced by reduction of some endogenous prolamins by RNA interference. The monomeric and dimeric mTGF-ßs were deposited in ER-derived PBs containing prolamins. When highly produced in rice seed, it is notable that most of ER-derived PBs were distorted and granulated. Step-wise extraction of storage proteins from rice seeds suggested that the mTGF-ß strongly interacted with cysteine-rich prolamins via disulfide bonds. This result was also supported by the finding that reducing agent was absolutely required for mTGF-ß extraction.

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed.

KEY MESSAGE: Prolamin-GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin-GFP fusion proteins were gradually digested from outside by pepsin digestion. In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin-GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin-GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin-GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin-GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin-GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.

Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

Formulation of Eco-friendly Medicated Chewing Gum to Prevent Motion Sickness.

An attempt was made to formulate medicated chewing gum to prevent motion sickness using natural gum base for faster onset of action and easy administration, anywhere and anytime, without access to water. To avoid the discard issue of gum cud, natural gum base of Triticum aestivum (wheat grain) was explored because of its biodegradable and biocompatible nature and easy availability. Prolamin, extracted from wheat, showed good chewing capacity, elasticity, high water retention capacity, antifungal activity, and compatibility with the drug. Formulations were prepared based on a two-factor and three-level factorial design. Amount of calcium carbonate (texturizer) and gum base were selected as independent variables. Elasticity and drug release were considered as the dependent variables. All batches were evaluated for the content uniformity, elasticity study, texture study, in vitro drug release study, and chewiness study. Results revealed that medicated chewing gum containing 80 mg of calcium carbonate and 500 mg of gum base showed good elasticity and more than 90% drug release within 16 min. Thus, this study suggested that both good elasticity and chew ability and abundant availability of wheat grain can act as a potential gum base for medicated chewing gum.

Rice prolamin extract ameliorates acute murine colitis by inhibiting nuclear factor-kappa B and modulating intestinal apoptosis and cell proliferation.

We investigated the impact of rice prolamin extract (RPE) on lipopolysaccharide (LPS)-induced nuclear factor (NF)-κB signalling in intestinal epithelial cells and macrophages, and determined the therapeutic efficacy of RPE in acute murine colitis. The effect of RPE on LPS-induced NF-κB signalling and proinflammatory gene expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, immunofluorescence and electrophoretic mobility shift assay (EMSA). The in-vivo efficacy of RPE was assessed in mice with 3% dextran sulphate sodium (DSS)-induced colitis. Apoptotic and cellular proliferative activities were evaluated by immunostaining with cleaved caspase-3 and proliferating cell nuclear antigen (PCNA) antibodies. RPE inhibited LPS-induced expression of monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha and LPS-induced NF-κB signalling in intestinal epithelial cells and macrophages. RPE-fed, DSS-exposed mice showed less weight loss, longer colon length and lower histological score compared to control diet-fed, DSS-exposed mice. Immunostaining analysis revealed a significant decrease of cleaved caspase-3 positive cells in RPE-fed, DSS-exposed mice compared to DSS-exposed mice. Also, the number of PCNA-positive cells within intact colonic crypts decreased significantly in RPE-fed, DSS-exposed mice compared to control diet-fed, DSS-exposed mice. DSS-induced NF-κB signalling was inhibited by RPE. RPE ameliorates intestinal inflammation by inhibiting NF-κB activation and modulating intestinal apoptosis and cell proliferation in an acute murine colitis.