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.     

Engineered protein A ligands,derived from a histidine-scanning library,facilitate the affinity purification of IgG under mild acidic conditions

Background

In antibody purification processes, the acidic buffer commonly used to elute the bound antibodies during conventional affinity chromatograph, can damage the antibody. Herein we describe the development of several types of affinity ligands which enable the purification of antibodies under much milder conditions.

Results

Staphylococcal protein A variants were engineered by using both structure-based design and combinatorial screening methods. The frequency of amino acid residue substitutions was statistically analyzed using the sequences isolated from a histidine-scanning library screening. The positions where the frequency of occurrence of a histidine residue was more than 70% were thought to be effective histidine-mutation sites. Consequently, we identified PAB variants with a D36H mutation whose binding of IgG was highly sensitive to pH change.

Conclusion

The affinity column elution chromatograms demonstrated that antibodies could be eluted at a higher pH (?pH**≧2.0) than ever reported (?pH?=?1.4) when the Staphylococcal protein A variants developed in this study were used as affinity ligands. The interactions between Staphylococcal protein A and IgG-Fab were shown to be important for the behavior of IgG bound on a SpA affinity column, and alterations in the affinity of the ligands for IgG-Fab clearly affected the conditions for eluting the bound IgG. Thus, a histidine-scanning library combined with a structure-based design was shown to be effective in engineering novel pH-sensitive proteins.

     

Micronutrient Deficiency and Treatment Adherence in a Randomized Controlled Trial of Micronutrient Supplementation in ART-Na?ve Persons with HIV

Introduction

The MAINTAIN study is an on-going RCT comparing high-dose micronutrient and anti-oxidant supplementation versus recommended daily allowance (RDA) vitamins in slowing HIV immune deficiency progression in ART-naïve people with HIV infection.

Objective

We planned analysis of the first 127 participants to determine the baseline prevalence of serum micronutrient deficiencies and correlates, as well as tolerance and adherence to study interventions.

Methods

Participants receive eight capsules twice daily of 1) high-dose or 2) RDA supplements for two years and are followed-up quarterly for measures of immune deficiency progression, safety and tolerability. Regression analysis was used to identify correlates of micronutrient levels at baseline. Adherence was measured by residual pill count, self-report using the General Treatment Scale (GTS) and short-term recall HIV Adherence Treatment Scale (HATS).

Results

Prior micronutrient supplementation (within 30 days) was 27% at screening and 10% of study population, and was not correlated with baseline micronutrient levels. Low levels were frequent for carotene (24%<1 nmol/L), vitamin D (24%<40 nmol/L) and serum folate (20%<15 nmol/L). The proportion with B₁₂ deficiency (<133 pmol/L) was 2.4%. Lower baseline levels of B₁₂ correlated lower baseline CD4 count (r = 0.21, p = 0.02) with a 21 pmol/L reduction in B₁₂ per 100 cells/µL CD4. Vitamin D levels were higher in men (p<0.001). After a median follow-up of 1.63 years, there were 19 (15%) early withdrawals from the study treatment. Mean treatment adherence using pill count was 88%. Subjective adherence by the GTS was 81% and was moderately but significantly correlated with pill count (r = 0.29, p<0.001). Adherence based on short-term recall (HATS) was >80% in 75% of participants.

Conclusion

Micronutrient levels in asymptomatic HIV+ persons are in keeping with population norms, but micronutrient deficiencies are frequent. Adherence levels are high, and will permit a valid evaluation of treatment effects.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00798772","term_id":"NCT00798772"}}NCT00798772     

Does the Upstream Region Possessing MULE-Like Sequence in Rice Upregulate PsbS1 Gene Expression?

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.     

ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P₁–S1P₅). S1P₁ is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P₁. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P₁ and S1P₅. ASP4058 preferentially activates S1P₁ and S1P₅ compared with S1P_{2, 3, 4} in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.     

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.     

Selective Increase in S-100β Protein by Aging in Rat Cerebral Cortex

Changes in the concentrations of nervous tissue-related proteins and their isoproteins, such as S-100 proteins (S-100 alpha and S-100 beta), enolase isozymes (alpha-enolase and gamma-enolase), and GTP-binding proteins (Go alpha, Gi2 alpha, and beta-subunits), were determined in the CNS of male rats of various ages (from 2 to 30 months old) by means of enzyme immunoassay. The weights of brains and the concentrations of soluble proteins in the cerebral cortex, cerebellum, and brainstem were constant during the observation period. The concentration of S-100 beta protein, which is predominantly localized in glial cells, increased gradually in the cerebral cortex with age; levels in the 25-month-old rats increased to approximately 150% of the levels in the young (2-month-old) rats. However, the S-100 beta concentrations in the cerebellum and brainstem were relatively constant, showing similar values in rats 2-30 months old. Levels of other proteins, including both neuronal (gamma-enolase and Go alpha) and glial (alpha-enolase and S-100 alpha) marker proteins, did not change significantly with age in the cerebral cortex, cerebellum, and brainstem. These results suggest that there is a close relation between the age-dependent changes of the CNS function and S-100 beta protein levels in the cerebral cortex.     

Photosynthetic responses to photosynthetically active radiation and temperature including chilling‐light stress on the heteromorphic life history stages of a brown alga,Cladosiphon okamuranus (Chordariaceae) from Ryukyu Islands,Japan

Photosynthetic responses to temperature and photosynthetically active radiation (PAR) were investigated on the heteromorphic life history stages (macroscopic and microscopic stages) of an edible Japanese brown alga, Cladosiphon okamuranus from the Ryukyu Islands. Measurements were carried out by using optical dissolved oxygen sensors and a pulse‐amplitude modulated fluorometer. Maximum net photosynthetic rates and other parameters of the Photosynthesis – PAR curves at 28°C were somewhat similar in both life history stages, without characteristic photoinhibition at 1000 μmol photons m^?2 s^?1. Results of oxygenic gross photosynthesis and dark respiration experiments over a temperature range of 8–40°C revealed similar temperature optima for both stages (29.7°C, macroscopic stage; 30.3°C, microscopic stage), which support their observed occurrences in the habitat during summer. Maximum quantum yields of photosystem II (PSII ) (F _v /F _m ) were relatively stable at low temperatures with the highest at 15.1°C for the macroscopic stage and at 16.5°C for the microscopic stage; but dropped at higher temperatures especially above 28°C. Continuous exposures (6 h) to 200 and 1000 μmol photons m^?2 s^?1 at 8, 16, and 28°C revealed greater depressions in effective quantum yields of PSII (Φ _PSII ) of the microscopic stage at 8°C, as well as its F _v /F _m that barely increased after 6 h of dark acclimation. Whereas post‐dark acclimation F _v /F _m of both stages exposed to low PAR fairly recovered at 28°C, suggesting their photosynthetic tolerance to such high temperature. Under natural conditions, both heteromorphic stages of C. okamuranus may persist throughout the year in this region. Beyond its northern limit of distribution, the microscopic stage of this species may suffer from photodamage, as enhanced by low winter temperatures; hence, its restricted occurrence.     

An omnivorous arthropod, <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis>, induces gender-specific plant volatiles to which conspecific males and females respond differently

Nesidiocoris tenuis Reuter (Heteroptera: Miridae) is an omnivorous mirid bug that preys on diverse generalist herbivorous arthropods. N. tenuis adults are attracted to volatiles from plants induced by their prey, such as tobacco cutworms (CCW) (Spodoptera litura larvae) and two-spotted spider mites (Tetranychus urticae). N. tenuis adults also induce volatiles when they infest plants. In this study, we focused on olfactory responses of N. tenuis males and females to volatiles from eggplants and sesame plants induced by conspecifics of the same or different gender by using a Y-tube olfactometer. Males were attracted to volatiles from plants of both species induced by either males or females. The male preference was biased to volatiles from plants of both species induced by females, probably because the biased response would facilitate their mate-finding. Females were attracted only to volatiles from plants of both species induced by females. Mating occurs multiple times in this species. Thus, the responses would indirectly affect mating of males and females. Slight but significant qualitative and quantitative differences were detected between the volatiles of plants of both species induced by N. tenuis females and those of the plants induced by conspecific males. N. tenuis might use such differences in their gender-specific responses.