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Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.

Genetic engineering of eggplant using fruit-specific EEF48 promoter-driven bacterial PSY gene, crtB, confers β-carotene accumulation in fruit. Eggplant (Solanum melongena L.) is globally cultivated especially in Asia and is an important source of nutrients in the diets of low-income consumers in developing countries. Since fruits of eggplant have low provitamin A carotenoid content, it is expected to develop eggplant with high carotenoid content for combatting vitamin A deficiency. To achieve this, the present study implemented a metabolic engineering strategy to modify the carotenoid biosynthetic pathway in eggplant. Expression analysis of carotenogenic genes in eggplant tissues showed that the expression of the endogenous phytoene synthase (PSY) was low in fruit and callus. Orange-colored calluses were generated from ectopic expression of crtB gene, which encodes bacterial PSY, in eggplant cells. The orange calluses accumulated > 20 μg g−1 FW of β-carotene, which was approximately 150-fold higher than that of the untransformed calluses. These observations suggest that the PSY expression is the rate-limiting step for β-carotene production in callus and fruit. Since the orange calluses did not regenerate plants, we chose eggplant EEF48 gene, which is presumably expressed in fruit. We amplified its promoter region by TAIL-PCR and showed that the EEF48 promoter is indeed active in eggplant fruit. Subsequently, transgenic eggplant lines having EEF48 promoter-driven crtB were produced. Among the transgenic lines produced, one line set fruit containing 1.50 μg g−1 FW of β-carotene, which was 30-fold higher than that of the untransformed fruits (0.05 μg g−1 FW). The self-pollinated progenies showed a 3:1 segregation ratio for the presence and absence of the transgene, which was linked to the β-carotene accumulation in fruit. These results provide a strategy for improvement of carotenoid content in eggplant fruit.

Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.

Objective microRNA (miRNA) is a small non-coding RNA that regulates gene expression by sequence-dependent binding to protein-coding mRNA in eukaryotic cells. In plants, miRNA plays important roles in a plethora of physiological processes, including abiotic and biotic stress responses. The present study was conducted to investigate whether miRNA-mediated regulation is important for the endoplasmic reticulum (ER) stress response in Arabidopsis. Results We found that hyl1 mutant plants are more sensitive to tunicamycin, an inhibitor of N-linked glycosylation that causes ER stress than wild-type plants. Other miRNA-related mutants, se and ago1, exhibited similar sensitivity to the wild-type, indicating that the hypersensitive phenotype is attributable to the loss-of-function of HYL1, rather than deficiency in general miRNA biogenesis and function. However, the transcriptional response of select ER stress-responsive genes in hyl1 mutant plants was indistinguishable from that of wild-type plants, suggesting that the loss-of-function of HYL1 does not affect the ER stress signaling pathways.

DSBs differently affect endoreduplication and organ size in radish cotyledons and hypocotyls in different light conditions, suggesting that DSBs-mediated endoreduplication varies based on different developmental and environmental cues. Endoreduplication induced by DNA double strand breaks (DSBs) in Arabidopsis thaliana roots and cultured cells has been reported in recent years. In this study, we investigated whether DSBs-mediated endoreduplication also occurs in other tissues, such as cotyledons and hypocotyls of radish (Raphanus sativus var. longipinnatus) plants. To induce DSBs, UV irradiation and Zeocin treatment were applied to in vitro-cultured radish seedlings, and ploidy distribution of the treated tissues was analyzed by flow cytometry. Consequently, frequencies of the higher ploidy (8C) cells and cycle values in the cotyledon tissues increased with increasing doses of UV irradiation and concentrations of Zeocin, irrespective of light conditions. UV-stimulated endoreduplication was also observed in four Brassica species. In hypocotyls, UV treatments decreased the frequencies of higher ploidy (32C) cells and cycle values in dark-grown seedlings, whereas Zeocin treatments increased the frequencies of higher ploidy (16C and 32C) cells and cycle values in light- and dark-grown seedlings. Among the treatments, organ sizes did not simply correlate with cycle values. The effects of treatments on endoreduplication and organ size differed based on organ and light conditions, indicating that DSBs-mediated endoreduplication may involve a multifaceted response to different developmental and environmental cues.

Secretory proteins undergo modifications such as glycosylation and disulphide bond formation before proper folding, and move to their final destination via the endomembrane system. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) due to suboptimal environmental conditions triggers a response called the unfolded protein response (UPR), which induces a set of genes that elevate protein folding capacity in the ER. This review aims to establish a connection among ER stress, UPR, and reactive oxygen species (ROS), which remains an unexplored topic in plants. For this, we focused on mechanisms of ROS production originating from ER stress, the interaction between ER stress and overall ROS signalling process in the cell, and the interaction of ER stress with other organellar ROS signalling pathways such as of the mitochondria and chloroplasts. The roles of the UPR during plant hormone signalling and abiotic and biotic stress responses are also discussed in connection with redox and ROS signalling.

This study investigated sequence specificity and perenniality of DNA methylation in the cauliflower mosaic virus (CaMV) 35S promoter of transgenic gentian (Gentiana triflora×G. scabra) plants. Unlike conventional transgene silencing models, 35S promoter hypermethylation in gentian is species-specific and occurs irrespective of the T-DNA copy number and genomic location. Modified 35S promoters were introduced into gentian, and single-copy transgenic lines were selected for methylation analysis. Modified 35S promoter lacking a core (-90) region [35S(Δcore)] in gentian conferred hypermethylation and high levels of de novo methylation of the CpHpH/CpCpG sites in the 35S enhancer regions (-298 to -241 and -148 to -85). Therefore, promoter transcription may not be an absolute requirement for the methylation machinery. In vitro, de novo methylation persisted for more than eight years. In another modified 35S promoter, two "GAAGA" motifs (-268 to -264 and -135 to -131) were replaced by "GTTCA" in the two highly de novo methylated regions. It did not support hypermethylation and showed transgene expression. A 64-bp fragment of the 35S enhancer region (-148 to -85) was introduced into gentian and the resultant transgenic lines analyzed. The 64-bp region exhibited hypermethylation at the CpG/CpWpG sites, but the CpHpH/CpCpG methylation frequency was lower than those of the unmodified 35S- and 35S(Δcore) promoters. Nevertheless, a distinct CpHpH/CpCpG methylation peak was found in the 64-bp region of all single-copy transgenic lines. These results suggest that the 64-bp region may contain an element required for 35S methylation but insufficient for high de novo methylation compared with those in the unmodified 35S and 35S(Δcore) promoters.

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P.

The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response occurs when folding and maturation of secretory and membrane proteins are impaired in the ER. The UPR induces a number of genes that encode ER-localized molecular chaperones and folding enzymes to increase folding capacity in the ER. We have identified Tunicamycin Induced 1 (TIN1), an Arabidopsis gene that is highly induced during the UPR. We have shown that TIN1 protein is localized in the ER but its physiological function remains to be elucidated. In the present study we generated and analyzed transgenic Arabidopsis plants expressing TIN1 under CaMV35S promoter to obtain insights into the physiological role of TIN1. We found that although TIN1-overexpressing plants grew as did wild-type plants under ambient laboratory conditions, their pollen grains exhibited abnormal surface morphology. The result suggests a specific role of TIN1 in secretion of proteins and/or lipids during pollen development.

The unfolded protein response (UPR) mitigates stress caused by accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) is the most conserved sensor of the UPR with ribonuclease activity that mediates cytoplasmic splicing and decay of mRNA encoding secretory and membrane proteins. In the present study, we demonstrate that the Arabidopsis mutant defective in two IRE1 genes exhibit retarded growth of primary roots under moderate salt stress, although such grow retardation is not observed in wild type plants. Microscopic observation showed decrease in the number of meristematic cells in the mutant under salt stress. This finding suggests that IRE1 plays a role in the maintenance of root meristems under salt stress. Possible connections between the function of IRE1 and the salt sensitivity are discussed.

The Arabidopsis unfolded protein response transducer IRE1 contributes to male gametophyte development using an alternative activation mechanism bypassing the unfolded protein-sensing domain., In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay under ER stress, whereas the mutant does not exhibit severe developmental defects under normal conditions. In this study, we focused on the Arabidopsis IRE1C gene, whose product lacks a sensor domain. We found that the ire1a/b/c triple mutant is lethal, and heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate regulated IRE1-dependent decay but not bZIP60 splicing. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.

The feasibility of magnetic anchor-guided endoscopic submucosal dissection (MAG-ESD) using a neodymium magnet for colorectal tumors has not been evaluated. The aim of this study was to clarify the feasibility of MAG-ESD for colorectal tumors. This prospective trial was conducted at Yamashita Hospital. MAG-ESD was performed for 49 colorectal tumors. The magnetic anchor comprised an internal magnet attached to an endoclip with 3-0 silk. Both external and internal magnets were made using neodymium magnets. The feasibility of traction achieved using MAG-ESD, en bloc resection rate, complete en bloc resection rate, time required for preparation and attachment of the magnetic anchor, procedure time, rate of retrieval of magnetic anchors, and adverse events were evaluated. MAG-ESDs were successfully performed for 48 colorectal tumors except for a rectal case in which the internal magnet stuck to the endoscope. En bloc resections and complete en bloc resections were achieved in all cases. Attaching the magnetic anchor required a median of 8 min (range 3–37 min). Median procedure time was 76 min (range 28–283 min) and the magnetic anchors were retrieved in all cases without adverse events. MAG-ESD is feasible and safe in the colon and may facilitate the treatment of all difficult lesions. (UMIN000024100).

Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role.

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.

Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site. In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. ‘SR-1’) was achieved using binary vectors comprising two (T1–T2) or three (T1–T2–T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRBW568L, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1–T2-derived lines and two T1–T2–T3-derived lines. Of these mutations, four T1–T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5′ homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1–T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (

The unfolded protein response (UPR) occurs when protein folding and maturation are disturbed in the endoplasmic reticulum (ER). During the UPR, a number of genes including those encoding ER-resident molecular chaperones are induced. In Arabidopsis, BiP3 has been used as a UPR marker gene whose expression is strongly induced in response to ER stress. In this study, we mutagenized Arabidopsis plants expressing β-glucuronidase (GUS) gene under the control of BiP3 promoter and isolated a mutant that exhibits strong GUS activity without treatment with ER stress inducers. By whole genome sequencing, we identified a causal gene in the mutant as SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY2 (SSI2), which encodes stearoyl-acyl carrier protein desaturase that converts stearic acids to oleic acids in the chloroplasts. In addition to GUS proteins, the ssi2 mutant accumulates endogenous BiP3 proteins without treatment by any stress reagents. Interestingly, although the degree of endogenous BiP3 protein accumulation in the ssi2 mutant was comparable to that in wild-type plants treated with the ER stress inducer tunicamycin, much less BiP3 transcripts were detected in the ssi2 mutant compared to tunicamycin-treated wild-type plants. Our finding suggests a genetic link between fatty acid metabolism in the chloroplasts and ER functions.

We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

Under the unfolded protein response (UPR), transcripts encoding the endoplasmic reticulum (ER) chaperones are increased and those encoding proteins synthesized in the ER are decreased. To reproducibly detect such changes of an expression profile, homogeneous growth of plants is desired. In addition, uniform treatment with drugs inducing the UPR is also necessary. Here we describe our methods of plant culture and drug treatment, and procedure to detect gene expression by quantitative RT-PCR.

The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response occurs when folding and maturation of secretory and membrane proteins are impaired in the ER. The UPR induces a number of genes that encode ER-localized molecular chaperones and folding enzymes to increase folding capacity in the ER. We have identified Tunicamycin Induced 1 (TIN1), an Arabidopsis gene that is highly induced during the UPR. We have shown that TIN1 protein is localized in the ER but its physiological function remains to be elucidated. In the present study we generated and analyzed transgenic Arabidopsis plants expressing TIN1 under CaMV35S promoter to obtain insights into the physiological role of TIN1. We found that although TIN1-overexpressing plants grew as did wild-type plants under ambient laboratory conditions, their pollen grains exhibited abnormal surface morphology. The result suggests a specific role of TIN1 in secretion of proteins and/or lipids during pollen development.

We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

In mammalian herbivores, faecal particle size indicates chewing efficiency. Proboscis monkeys (Nasalis larvatus) are foregut fermenters in which regurgitation and remastication (i.e. rumination) was observed in the wild, but not with the same consistency as found in ruminants and camelids. To test whether this species has exceptional chewing efficiency among primates, as ruminants have among mammals, we compared faecal particle size in free-ranging specimens with those of 12 other primate species. The discrete mean faecal particle size (dMEAN) increased with body mass (M) as dMEAN (mm) = 0.65 (95% confidence interval 0.49-0.87) M((0.33 (0.23-0.43)) in simple-stomached species. At 0.53 ± 0.09 mm, dMEAN of proboscis monkeys was particularly small for their average M (15 kg) and significantly smaller than values of two other foregut fermenting primate species. While we cannot exclude other reasons for the exceptional chewing efficiency in proboscis monkeys, this represents circumstantial evidence for regular use of rumination in this species. Thus, proboscis monkeys might be a model for convergent evolution towards rumination in a non-ungulate taxon.

Purpose Effectiveness of differential diagnosis of gallbladder polypoid lesions using contrast enhanced ultrasound (CEUS) examination by perflubutane (Sonazoid®:Daiichi-Sankyo, Tokyo). Material & Methods CEUS was performed on 183 patients with gallbladder polypoid lesions.It consists of 10 adenocarcinomas, 2 adenomas, 129 benign polyps and 42 adenomyomatosis (fundal type). For benigndiseases, we examined cases where the size was 6 mm or more and could be observed for more than one year. Ultrasound was performed using TUS-A500 (Aplio 500) (Canon MedicalSystems. Tokyo) and prosound alpha10 (Hitachi Aloka Medical,Ltd.Tokyo). First, the largest cross section of a lesion was visualized using B-mode and infusion of Sonazoid. The characteristics and behavior of blood flow in the lesion in the early vascular phase were examined over approximately 30 sec. Thereafter, observation was continued accordingly for about 3 min. Sonazoid infusion using contrast harmonic imaging and real-time perfusion Imaging. Results Gallbladder carcinoma showed hypervascular staining and “eruption sign” was apparent in 60%. The “eruption sign” showed an image of a vigorously boiling water that was splashing up from the entire lesion. Blood flow imaging revealed irregular size and buckling, including tortuous vessels and erratic blood flow in 70%. Gallbladder adenoma showed “eruption sign” was apparent in 50%. The irregular size, tortuous vessels and eruption signs of blood flow imaging were 83.3% as an index of neoplastic. Gallbladder benign polyp, staining was scattered within them, and “flicker sign” was observed in 93.8%. The “flicker sign” showed an image of bubbling pre-boiled hot water. Five cases (3.9%) showed a diffused and hypervascular staining pattern. The staining time was shorter than gallbladder carcinoma. Blood flow image showed normal vessels without buckling. Linear blood flow of the stalk of gallbladder polyp was also characteristic. Gallbladder adenomyomatosis (fundal type), staining was scattered and mild flicker signs were observed in 85.7%. Blood flow at the surface of the prominence and internal Rokitansky-Aschoff sinus was observed clearity. Conclusions CEUS can clarify the hemodynamics and blood flow images and are useful for the differential diagnosis of gallbladder polypoid lesions.

Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.

The male dispersal patterns of western lowland gorillas (WLGs, Gorilla gorilla gorilla) are not well understood. To determine whether most silverbacks stay close to their relatives, we analyzed autosomal and Y-chromosomal microsatellites (STRs) in wild WLGs at Moukalaba, Gabon. We obtained STR genotypes for 38 individuals, including eight silverbacks and 12 adult females in an approximately 40 km2 area. Among them, 20 individuals were members of one identified group (Group Gentil; GG), including one silverback and six adult females. The silverback sired all 13 of the offspring in GG and no Y-STR polymorphism within GG was found, as expected in a one-male group structure. Over all silverbacks sampled, Y-STR diversity was high considering the limited sampling area, and silverbacks with similar Y-STR haplotypes were not always located in nearby areas. Although the misclassification rate of kinship estimates in this study was not negligible, there were no kin dyads among all silverbacks sampled. These results suggest that silverbacks born in the same group do not stay close to each other after maturation. The Y-STR diversity in this study was similar to that of a previous study conducted in an area that was approximately 150 times larger than our study area. Similarity of WLG Y-STR diversity between studies at different sampling scales suggests that male gene flow may not be geographically limited. These results suggest that WLG males normally disperse from their natal areas after maturation, at least, in Moukalaba. Am J Phys Anthropol 151:583–588, 2013. © 2013 Wiley Periodicals, Inc.

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response.

Activity of transcription factors must be tightly controlled to regulate gene expression at a proper developmental timing and in response to environmental stimuli. Approximately 10% of plant transcription factors reside as precursor forms in the membranes, such as the endoplasmic reticulum and plasma membranes, and are activated by their liberation from the membranes to translocate into the nucleus where gene expression occurs. Studies in the past decade have facilitated our understanding of the roles of membrane-bound transcription factors in diverse biological phenomena, including abiotic and biotic stress responses, cell division, and hormone signaling. In this chapter we review the molecular and biological functions and the activation mechanism of membrane-bound bZIP and NAC transcription factors in Arabidopsis.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional activation of ER-resident molecular chaperones and folding enzymes to maintain cellular homeostasis. This process is known as the ER stress response or the unfolded protein response. We have identified tunicamycin induced 1 (TIN1), a plant-specific ER stress-inducible Arabidopsis thaliana gene. The TIN1 protein is localized in the ER; however, its molecular function has yet to be clarified. In this study, we performed functional analysis of TIN1 in planta. RT-PCR analysis showed that TIN1 is highly expressed in pollen. Analysis using the β-glucuronidase reporter gene demonstrated that the TIN1 promoter is active throughout pollen development, peaking at the time of flowering and in an ovule of an open flower. Although a T-DNA insertion mutant of TIN1 grows normally under ambient laboratory conditions, abnormal pollen surface morphology was observed under a scanning electron microscope. Based on the current and previous observations, a possible physiological function of TIN1 during pollen development is discussed.

Secretory and membrane proteins of eukaryotic cells must be properly folded and assembled in the endoplasmic reticulum (ER) before translocation to their final destination where they function. Perturbation of this process results in accumulation of unfolded proteins in the ER, so-called ER stress. The cells initiate a protective response to maintain cellular homeostasis, which is termed the ER stress response or the unfolded protein response (UPR). In the present study, we performed time-series transcriptome analysis of the ER stress response in Arabidopsis (Arabidopsis thaliana) with the N-linked glycosylation inhibitor tunicamycin, which causes misfolding of proteins in the ER, and therefore, triggers ER stress. A total of 259 genes were identified as tunicamycin-responsive genes, 175 of which were upregulated and 84 were downregulated. Hierarchical clustering and bioinformatic analysis demonstrated that 259 tunicamycin-responsive genes can be assigned to one of the six distinct expression classes and identified a potential novel cis-element, as well as known cis-elements, i.e., ER stress response element and UPR element. We also observed that a considerable number of tunicamycin-inducible genes, including those encoding the ER chaperone BiP and the membrane-bound transcription factor AtbZIP60, are coordinately upregulated at a late pollen development stage in Arabidopsis. This observation suggests that the ER stress response plays an important role in the development and function of pollens.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional induction of ER-resident molecular chaperones to maintain cellular homeostasis, termed the ER stress response. Previously we isolated AtbZIP60, a membrane-bound transcription factor involved in the Arabidopsis ER stress response whose activity is controlled by proteolytic cleavage. In this study we characterized the active form of AtbZIP60 localized in the nucleus during the ER stress response. Transient assay using Arabidopsis protoplasts revealed that activation of BiP promoters by AtbZIP60 is dependent on the cis-elements plant-unfolded protein response element (P-UPRE) and ER stress response element (ERSE). Transcriptional activation activity of AtbZIP60 was mainly located in the region for amino acids 41-80 of AtbZIP60. Size exclusion chromatography analysis showed that the nuclear form of AtbZIP60 exists as a protein complex of approximately 260 kDa. On the basis of the present study combined with observations described in the literature, possible mechanisms of AtbZIP60's action in the nucleus are discussed.

In the endoplasmic reticulum (ER) stress response, protein folding in the ER is disturbed, resulting in the induction of genes encoding proteins that facilitate correct folding of proteins. Unique mechanism of signaling pathway in the ER stress response has been studied in yeast and animals. In animals, several bZIP type transcription factors with a transmembrane domain controlled by regulated intramembrane proteolysis (RIP) have been reported to be involved in the signaling pathway. Very recently, AtbZIP60, which is a bZIP type transctiption factor with a transmembrane domain, was identified to be involved in the ER stress response in Arabidopsis. Several other transctiption factors that seem to be regulated by RIP have been also reported in Arabidopsis. Analysis of the regulation mechanism of AtbZIP60 will contribute to understanding of the ER stress response and RIP in plants.

African rainforest harbors herbivores at high density. However, because plants and soils typically lack in some essential minerals, rainforest is not always a suitable habitat for herbivores. How they fulfill the mineral requirements is therefore an important question to animal ecology and conservation. Although large marshes, called ‘bais’, are often mentioned as efficient mineral-resource, little information on other sodium resources has still been available. Our laboratory works and field surveys found that a peculiar item, decaying wood stumps of Anthostema aubryanum, played as a major sodium resource for herbivores in Moukalaba-Doudou National Park, Gabon. When A. aubryanum is alive, the sodium content of its bark is low and its latex is toxic. Sodium is accumulated in decaying stumps (mean=1,343 mg/kg dry matter). Eight herbivores visited stumps to ingest the dead wood. Fecal sample analysis revealed that western lowland gorillas, a species most-frequently using the stumps, consumed large amount of the dead wood as regular food. Our findings suggest that decaying A. aubryanum is critical sodium-resources and is a key species for herbivores in our study area. Importance of the A. aubryanum may be particularly large there, because it is a limited sodium-rich material that is available year round. Our study site is known as the site where the densities of several herbivores are among the highest at Central Africa. The relatively high herbivores density in our study site may partly depend on decaying A. aubryanum as sodium resources.

In this paper we describe bed (nest) and bed-site reuse by western lowland gorillas (Gorilla g. gorilla) in Moukalaba-Doudou National Park, south-eastern Gabon. During an eight-month study 44 bed sites and 506 beds were found. Among these, 38.6% of bed sites and 4.1% of beds were reused. We analyzed the monthly frequency of bed-site reuse in relation to rainfall, fruit abundance, and fruit consumption by the gorillas. The different frequency of bed-site reuse in the rainy and dry seasons was not significant. More bed-site reuse was observed during the fruiting season than during the non-fruiting season. Results from fecal analysis suggested that gorillas ate more fruit in the fruiting season than in the non-fruiting season. The frugivorous diet of western gorillas may possibly cause gorillas to stay in some areas and, consequently, reuse their bed sites. Reuse of bed sites by gorillas suggests their frequent return to an area where preferred fruit is readily available. A higher percentage of arboreal beds may also affect bed-site reuse, because of the shortage of bed material.

Analysis of transcripts of 75 genes encoding putative basic leucine zipper (bZIP) transcription factors in the Arabidopsis genome identified AtbZIP60 , which was induced by tunicamycin. AtbZIP60 encodes a predicted protein of 295 aa with a putative transmembrane domain near its C terminus after a bZIP domain. A truncated form of AtbZIP60 without a transmembrane domain (AtbZIP60ΔC) fused with GFP localized to the nucleus, suggesting translocation of native protein to the nucleus by release from the membrane. AtbZIP60 was also induced by DTT and azetidine-2-carboxylate, which induce the endoplasmic reticulum (ER) stress response (also called the unfolded protein response). Expression of AtbZIP60ΔC clearly activated any of three BiP and two calnexin promoters in a dual luciferase assay using protoplasts of cultured cells. The induction was considered to be through cis-elements plant-specific unfolded protein response element and ER stress-response element. Interestingly, AtbZIP60ΔC also appeared to induce the expression of AtbZIP60 through an ER stress-response element-like sequence in the promoter of AtbZIP60 . These characteristics of AtbZIP60 imply a signal transduction pathway of the ER stress response unique to plants.

When correct folding of protein in the endoplasmic reticulum (ER) is prevented, cells respond to overcome the accumulation of unfolded proteins. This cellular response, which includes the induction of ER chaperones, is called an unfolded protein response (UPR). Although a link between the UPR and apoptosis has been reported in mammalian cells, little is known about this mechanism in plant cells. Asparagine (N)-linked glycosylation of proteins is critical for protein folding in the ER; and tunicamycin, a potent inhibitor of N-linked glycosylation, induces UPR. Growth arrest was observed in cultured tobacco cells treated with tunicamycin. Cell death and induction of Hsr203J, a marker for programmed cell death, were observed in the 24-h period after addition of tunicamycin, following UPR that started within 2 h. These results indicate a strong link between UPR and programmed cell death in plant cells.

The unfolded protein response (UPR) is a highly conserved cellular response that prevents abnormal maturation of proteins in the endoplasmic reticulum (ER). The expression of genes encoding ER chaperones is induced during the UPR. In the Arabidopsis UPR, two membrane-bound transcription factors, bZIP60 and bZIP28, activate the expression of those genes. bZIP60 is regulated by unconventional cytoplasmic splicing catalyzed by inositol requiring enzyme 1 (IRE1), which is located in the ER membrane. bZIP28 is regulated by intramembrane proteolysis. Pathogen infection and salicylic acid (SA) have been reported to induce the expression of some UPR genes. Here, we show that UPR genes including BiP3, a marker gene of the Arabidopsis UPR, are induced by exogenous SA treatment and activation of bZIP60 in an IRE1-dependent manner. The induction of gene expression and activation of bZIP60 were independent of NPR1 and HsfB1 under these experimental conditions. We generated antibodies to detect the proteolytic products of bZIP28 after SA treatment. An assay using these antibodies showed that SA activated bZIP28, as well as activating bZIP60 through IRE1. Together, these results show that exogenous SA treatment activates two signaling arms of the Arabidopsis UPR. We propose a possible mechanism of activation of the UPR machinery by SA.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers a protective response, termed the ER stress response or the unfolded protein response, to maintain cellular homeostasis. Recently we characterized the Arabidopsis (Arabidopsis thaliana) membrane-bound basic leucine zipper (bZIP) transcription factor AtbZIP60 involved in the ER stress response. We reported that AtbZIP60 is activated by regulated intramembrane proteolysis (RIP), a mechanism by which a membrane-bound transcription factor is released by proteolytic cleavage. We presented evidence that the AtbZIP60 protein resides in the ER membrane under unstressed conditions and is activated by proteolytic cleavage in response to ER stress to translocate into the nucleus where it acts as a transcription factor. Further analysis, however, showed that this cleavage is independent of the function of Arabidopsis homologs of S1P and S2P proteases, which mediate the proteolytic cleavage of the mammalian transcription factor ATF6. Thus AtbZIP60 is an ER stress transducer activated by a novel RIP mechanism that may be unique to plants.

A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.

Fast automated demand response (FastADR) will be one of possible technologies to realize smart grid ancillary services in future. For FastADR control, a statistical prediction model on the nonlinear response of facility loads is necessary. Although neural networks (NNs) can be utilized for nonlinear prediction, they can have harmful exceptional prediction due to bias of the NN on the training data. In this letter, we propose a combination of an autoregressive (AR) model and an NN to avoid the harmful behavior, and discuss the theoretical basis of the method. © 2015 Institute of Electrical Engineers of Japan. Published by John Wiley & Sons, Inc.

Ayu Plecoglossus altivelis is a typical amphidromous fish with ontogenic requirements for habitats among lower reaches of a river, the marine coastal area, and middle reaches of a river at the immature adult stage. Mature fish that had a small body size like ordinary young were found around the spawning ground. In comparison with standard adults, the migratory histories of the dwarf adults were examined using otolith microchemistry analysis. Sr : Ca ratio estimated that the dwarf fish were exposed to saline or brackish water after once moving into the river, whereas the standard-size fish migrated upstream.

In this paper, I describe the food-dropping behavior of western lowland gorillas observed in Moukalaba- Doudou National Park, Gabon. I collected observational data of gorillas eating in trees, and recorded whether any individuals were positioned under the same tree. In 22 of the 24 cases of individuals being present under a tree, I observed the gorilla in a tree dropping food to the individual below. In most cases, the recipient was a silverback or an elder half-sibling of the dropper. The dropper’s elder full-sibling was never a recipient. The food-dropping behavior of Moukalaba western lowland gorillas is likely due to a combination of factors: their frugivory, arboreality, large body size, and the scarcity of terrestrial herbs. It is difficult for multiple gorillas to simultaneously feed in the same tree. Under such limitations, younger gorillas face difficulties in defending their feeding patches from older individuals. Nearly 90 % of the recipients were older than the food droppers. Furthermore, food droppers were significantly younger than non-food droppers who simultaneously fed on the same tree, and most-food recipients were significantly older than least-food recipients on the ground. Food dropping may, therefore, be a tactic employed by younger gorillas to defend a feeding site from older individuals. This study suggests that food dropping may reduce feeding contest competition in a gorilla group in Moukalaba. This is a preliminary study that indicates that food dropping may be intentionally used as a way to reduce feeding competition; nevertheless, further studies are needed.