RNAi-mediated suppression of endogenous storage proteins leads to a change in localization of overexpressed cholera toxin B-subunit and the allergen protein RAG2 in rice seeds

Key message

RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2.

Abstract

A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the α-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.     

Correlation between Muscle Strength and Muscle Mass,and Their Association with Walking Speed,in Community-Dwelling Elderly Japanese Individuals

Objectives

We aimed to assess the correlation between muscle strength and muscle mass based on sex and age, and their association with walking speed, which is a health predictor for independent living, in elderly Japanese individuals.

Methods

The participants included 318 (111 men, 207 women) community-dwelling elderly Japanese individuals aged ≥65 years. Knee extension strength was assessed as an indicator of muscle strength, and bioelectrical impedance analysis was used to measure muscle mass. The maximum walking speed of participants was recorded. All measurements were categorized based on sex and age groups as follows: young-old (age, 65–74 years) and old-old (age, ≥75 years).

Results

Appendicular muscle mass and knee extension strength decreased with age in both men and women. In men, knee extension strength showed significant positive correlations with leg and appendicular muscle mass in both young-old and old-old age groups. However, in women, only the old-old age group showed significant positive correlations between knee extension strength and leg and appendicular muscle mass. Muscle strength was significantly positively correlated with maximum walking speed in all groups, whereas muscle mass was not significantly correlated with maximum walking speed in men and women.

Conclusions

Muscle strength was significantly correlated with muscle mass in both age groups in men. However, in women, the correlation between muscle strength and muscle mass differed according to age. This finding suggests that the relationship between muscle strength and muscle mass differs according to sex and age. Muscle strength showed significant correlation with walking speed in both men and women in both age groups. These findings suggest that it is necessary to recognize that muscle strength is different from muscle mass, and that an individualized approach to prevent decline of muscle strength and muscle mass is necessary for health promotion in elderly.     

Marine Hydroquinone Zonarol Prevents Inflammation and Apoptosis in Dextran Sulfate Sodium-Induced Mice Ulcerative Colitis

Background and Aim

We previously identified an anti-inflammatory compound, zonarol, a hydroquinone isolated from the brown algae Dictyopteris undulata as a marine natural product. To ascertain the in vivo functions of zonarol, we examined the pharmacological effects of zonarol administration on dextran sulfate sodium (DSS)-induced inflammation in a mouse model of ulcerative colitis (UC). Our goal is to establish a safe and effective cure for inflammatory bowel disease (IBD) using zonarol.

Methods and Results

We subjected Slc:ICR mice to the administration of 2% DSS in drinking water for 14 days. At the same time, 5-aminosalicylic acid (5-ASA) at a dose of 50 mg/kg (positive control) and zonarol at doses of 10 and 20 mg/kg, were given orally once a day. DSS-treated animals developed symptoms similar to those of human UC, such as severe bloody diarrhea, which were evaluated by the disease activity index (DAI). Treatment with 20 mg/kg of zonarol, as well as 5-ASA, significantly suppressed the DAI score, and also led to a reduced colonic ulcer length and/or mucosal inflammatory infiltration by various immune cells, especially macrophages. Zonarol treatment significantly reduced the expression of pro-inflammatory signaling molecules, and prevented the apoptosis of intestinal epithelial cells. Finally, zonarol protected against in vitro lipopolysaccharide (LPS)-induced activation in the RAW264.7 mouse macrophage cell line.

Conclusions

This is the first report that a marine bioproduct protects against experimental UC via the inhibition of both inflammation and apoptosis, very similar to the standard-of-care sulfasalazine, a well-known prodrug that releases 5-ASA. We believe that the oral administration of zonarol might offer a better treatment for human IBDs than 5-ASA, or may be useful as an alternative/additive therapeutic strategy against UC, without any evidence of side effects.     

Knockout of PARG110 confers resistance to cGMP-induced toxicity in mammalian photoreceptors

Hereditary retinal degeneration (RD) relates to a heterogeneous group of blinding human diseases in which the light sensitive neurons of the retina, the photoreceptors, die. RD is currently untreatable and the underlying cellular mechanisms remain poorly understood. However, the activity of the enzyme poly-ADP-ribose polymerase-1 (PARP1) and excessive generation of poly-ADP-ribose (PAR) polymers in photoreceptor nuclei have been shown to be causally involved in RD. The activity of PARP1 is to a large extent governed by its functional antagonist, poly-ADP-glycohydrolase (PARG), which thus also may have a role in RD. To investigate this, we analyzed PARG expression in the retina of wild-type (wt) mice and in the rd1 mouse model for human RD, and detected increased PARG protein in a subset of degenerating rd1 photoreceptors. Knockout (KO) animals lacking the 110 kDa nuclear PARG isoform were furthermore analyzed, and their retinal morphology and function were indistinguishable from wild-type animals. Organotypic wt retinal explants can be experimentally treated to induce rd1-like photoreceptor death, but PARG110 KO retinal explants were unexpectedly highly resistant to such treatment. The resistance was associated with decreased PAR accumulation and low PARP activity, indicating that PARG110 may positively regulate PARP1, an event that therefore is absent in PARG110 KO tissue. Our study demonstrates a causal involvement of PARG110 in the process of photoreceptor degeneration. Contrasting its anticipated role as a functional antagonist, absence of PARG110 correlated with low PARP activity, suggesting that PARG110 and PARP1 act in a positive feedback loop, which is especially active under pathologic conditions. This in turn highlights both PARG110 and PARP1 as potential targets for neuroprotective treatments for RD.     

The Arabidopsis ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development

In animals and yeasts, the DEAH-box RNA-dependent ATPase Prp16 facilitates pre-mRNA splicing. However, in Chlamydomonas reinhardtii and Caenorhabditis elegans, Prp16 orthologs are not important for general pre-mRNA splicing, but are required for gene silencing and sex determination, respectively. The CLUMSY VEIN (CUV) gene, which encodes a unique Prp16 ortholog in Arabidopsis thaliana, influences auxin-mediated development. A loss-of-function cuv-1 mutation tells us that CUV does not facilitate splicing of pre-mRNA substrates indiscriminately, but differentially effects splicing and expression of genes. Here we show that CUV influences root-meristem maintenance and planar polarity of root-hair positioning, both of which are processes regulated by auxin. We propose that Arabidopsis PRP16/CUV differentially facilitates the expression of genes, including genes involved in auxin biosynthesis, transport, perception and signaling, and that in this way it influences auxin-mediated development.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Comparison of the biological properties of purified natural and recombinant human interleukin-2

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.     

Involvement of Cyclic AMP in Adventitious Bud Initiation of Torenia Stem Segments

Adventitous bud initiation in epidermis of Torenia stem segmentscultured in vitro, which is usually induced by cytokinin, couldbe induced by application of dibutyryl cyclic AMP in the absenceof cytokinin. Similar stimulatory effects on bud initiationwere observed when various promoting reagents to accumulateendogenous cyclic AMP were added, such as activators for adenylatecyclase, forskolin and prostaglandin E₁, and inhibitors of cyclicnucleotide phospho-diesterase, theophylline and isobutyl methylxanthine. Endogenous content of cyclic AMP in Torenia stem segmentswere increased by the application of the above chemicals, calciumionophore or cytokinin. These results suggested that endogenousconcentrations of cyclic AMP were involved in adventitious budinitiation of Torenia stem segments. Furthermore, some inhibitorsof protein kinases inhibited bud initiation induced by cyclicAMP-accumulating reagents. (Received August 15, 1989; Accepted November 4, 1989)     

Plant regeneration from Bulgarian rose callus

Plant regeneration capacity of Bulgarian rose callus tissue was examined. Adventitious bud formation could be successfully attained, depending on the kinds of mineral salts used in the medium, auxin and cytokinin used. When callus tissues were cultured on the medium without ammonium nitrate and contained indoleacetic acid and benzyladenine, buds were formed in the callus. The number of buds were significantly increased by the simultaneous addition of calcium ionophore. When the cultures were transferred to the medium without cytokinin, roots were formed in the basal part of the buds.Abbreviations BA benzyladenine - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid