Publications by authors named "Ryoichi Sato"

99 Publications

The base and root of domain II loops of Cry toxins contribute to binding to Bombyx mori ABC transporter C2.

FEBS J 2021 Oct 7. Epub 2021 Oct 7.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Japan.

Little information is available regarding the region of Cry toxins involved in binding to their major receptors, the ATP-binding cassette (ABC) transporters. We analyzed which Cry1Aa amino acid residues contribute to binding to Bombyx mori ABC transporter C2 (BmABCC2). Several two oxidized double-cysteine substitution mutant toxins were made. In these, two amino acids at distant positions on toxin loop α8 and loop 2 or loop 2 and loop 3 were substituted with cysteine residues and crosslinked. These mutants exhibited a marked reduction in binding affinity to BmABCC2, suggesting that the binding site comprises complex cavities formed by loops α8, 2, and 3. Loop swapping between Cry1Aa and other BmABCC2-incompatible toxins indicated that loop 2 acts as a binding affinity-generating part of Cry1Aa toxin. Using single amino acid substitution mutants, the results of surface plasmon resonance (SPR) analysis and response assays with BmABCC2-expressing Sf9 cells indicated that Y366, R367, R368, and L447 in the Cry1Aa root and base region of loops 2 and 3 play important roles in binding. Furthermore, SPR analyses of these mutants suggested that a two-state binding model fits best the data obtained. Moreover, complex cavities and the above-mentioned amino acid residues contribute to the generation of multiple binding points and high-affinity binding. Finally, we found that the binding site of B. mori cadherin-like protein consists of complex cavities comprising loops 1, 2, and 3, partially overlapping that of BmABCC2, suggesting that the loop region of Cry1Aa toxin acts as a promiscuous binding site.
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http://dx.doi.org/10.1111/febs.16224DOI Listing
October 2021

Consideration for Affects of an XOR in a Random Number Generator Using Ring Oscillators.

Entropy (Basel) 2021 Sep 5;23(9). Epub 2021 Sep 5.

Department of Electrical Engineering, San José State University, One Washington Square, San José, CA 95192, USA.

A cloud service to offer entropy has been paid much attention to. As one of the entropy sources, a physical random number generator is used as a true random number generator, relying on its irreproducibility. This paper focuses on a physical random number generator using a field-programmable gate array as an entropy source by employing ring oscillator circuits as a representative true random number generator. This paper investigates the effects of an XOR gate in the oscillation circuit by observing the output signal period. It aims to reveal the relationship between inputs and the output through the XOR gate in the target generator. The authors conduct two experiments to consider the relevance. It is confirmed that combining two ring oscillators with an XOR gate increases the complexity of the output cycle. In addition, verification using state transitions showed that the probability of the state transitions was evenly distributed by increasing the number of ring oscillator circuits.
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http://dx.doi.org/10.3390/e23091168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8466240PMC
September 2021

Steel slag quality control for road construction aggregates and its environmental impact: case study of Vietnamese steel industry-leaching of heavy metals from steel-making slag.

Environ Sci Pollut Res Int 2021 Sep 26. Epub 2021 Sep 26.

JFE Steel Corporation, Tokyo, Japan.

Steel slag is an industrial by product of steel manufacturing processes and has been widely utilized within civil and construction materials for road materials and environmental remediation in countries like Japan, USA, and European Union nations. However, the current utilization of steel slag in Vietnam is very low mainly because of lack of quality control of slag treatment and chances for reuse of treated steel slag. This paper presents the up to date steel slag production status in Vietnam through the extensive survey and sampling at seven large steel factories. The paper also highlights the environmental and quality control issues of these steel slags to use as road construction aggregates by assessing the heavy metals concentration in the leachate. The basic oxygen furnace (BOF) and electric arc furnace (EAF) slag samples were collected to evaluate leaching properties of metals leached from the slags. The two standardized batch leaching tests of steel slag roadbed material in Japan (JIS K 0058-1) and toxicity characteristics leaching procedure (TCLP-EPA method 1311) were performed to the evaluated the hazardous metals. The results of the leaching test show that almost all of the concentration of the metals in the leached solution does not exceed the National Standard for Industrial Wastewater Discharge (QCVN 40-2011). The pH and parameters such as total chromium, nickel, copper, lead, arsenic, and manganese differ from the two test methods. The acidic conditions employed in the EPA 1311 were not representative of condition excepted during slag reuse in road constructions because in the operation condition of the road, acidic liquid is absent. The leaching test results confirmed that JIS test which uses deionized water with gentle mixing prevents the slag sample from size degradation is suitable for the environmental assessment of steel slag use for roadbed material. This research suggests that the adjustment of pH value prior to disposal or reuse as base materials and official guideline should be promulgate by the authorities to ensure the leachate meet the surface water quality standard.
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http://dx.doi.org/10.1007/s11356-021-16438-1DOI Listing
September 2021

ATP-binding cassette transporter subfamily C members 2, 3 and cadherin protein are susceptibility-determining factors in Bombyx mori for multiple Bacillus thuringiensis Cry1 toxins.

Insect Biochem Mol Biol 2021 12 22;139:103649. Epub 2021 Sep 22.

Institute of Agrobiological Sciences, NARO, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan. Electronic address:

Field-evolved resistance of insect pests to Bacillus thuringiensis (Bt) toxins (Cry toxins) is a threat to the efficacy of Bt-based bio-insecticides and transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) and cadherin-like receptor play important roles in conferring susceptibility to Cry1 toxins. However, the receptors involved in Bt susceptibility in each insect remain unclear. To determine the receptors that are involved in the susceptibility of Bombyx mori to Cry1 toxins (1Ab, 1Ac and 1Fa), we conducted diet overlay bioassay using B. mori strains disrupted with one or two receptor (s) among BmABCC2, BmABCC3, and cadherin-like receptor (BtR175) generated by transcription activator-like effector nuclease (TALEN)-mediated gene editing. The single-knockout strains for BmABCC2 showed resistance to Cry1Ab and Cry1Ac, whereas only strains with double knockout of BmABCC2 and BmABCC3 exhibited high resistance to Cry1Fa. Progeny populations generated from the crossing of heterozygotes for BtR175 knockout allele included 25% theoretical homozygotes for the BtR175 knockout allele and they showed resistance to Cry1Ab and Cry1Ac. Then, through a cell swelling assay using Sf9 cells ectopically expressing the receptor, we analyzed the mechanisms underlying the different contributions of BmABCC2, BmABCC3, and BtR175 to larval susceptibility. The receptor activity of BmABCC2 for Cry1Ab and Cry1Ac was far higher than that of BmABCC3, and BtR175 synergistically enhanced the receptor activity of BmABCC2. This result well explained the important involvement of BmABCC2 and BtR175 in the larval susceptibility to Cry1A toxins. By contrast, the receptor activities of BmABCC2 and BmABCC3 for Cry1Fa were observed at a similar level and synergistic effect of BtR175 was small. This finding explains the equal importance of BmABCC2 and BmABCC3 and very small contribution of BtR175 on larval susceptibility to Cry1Fa. Thus, we demonstrated the different importance of BmABCC2, BmABCC3, and BtR175 to various Cry1 toxins as susceptibility-determining factors in B. mori larvae and the underlying basis for the observed differences. Furthermore, a weak correlation was indicated between the binding affinity and receptor activities of BmABCC2 and BmABCC3 to Cry1 toxins.
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http://dx.doi.org/10.1016/j.ibmb.2021.103649DOI Listing
December 2021

Ultrasensitive detection by maxillary palp neurons allows non-host recognition without consumption of harmful allelochemicals.

J Insect Physiol 2021 07 27;132:104263. Epub 2021 May 27.

Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan. Electronic address:

Most lepidopteran insect larvae exhibit stepwise feeding behaviors, such as palpation using the maxillary palps (MPs) followed by test biting and persistent biting. However, the purpose of palpation has been unclear. In particular, nothing is known about the neurons in the MP and their mode of recognition of undesired plants, although such neurons have been suggested to exist. In this study, we used larvae of the stenophagous insect Bombyx mori and compared the roles of palpation and test biting in the selection of feeding behavior. When the larvae were given non-host plant leaves, they did not initiate test biting, indicating that non-host plant leaves were recognized via palpation without biting, and that this behavior resulted in a lack of persistent biting, as the leaves were judged non-suitable for consumption. Surface extracts of inedible leaves significantly suppressed test biting of mulberry leaves, a host plant of B. mori, suggesting that secondary metabolites on the leaf surface of inedible leaves function as test biting suppressors, even when another conditions are suitable for test biting. The allelochemical coumarin, which is found in the inedible leaves of cherry, Cerasus speciosa, significantly suppressed test biting of mulberry leaves, suggesting that coumarin is a possible deterrent to the eating of cherry leaves. Using the electrophysiological method of tip recording and a leaf-surface extract as the test material, leaf-surface compound-responsive neurons were identified in the MP. In addition, several neurons that respond to coumarin in the attomolar range were identified, suggesting that the larvae use ultrasensitive neurons in the MP to recognize inedible leaves. In the HEK293T cell heterologous expression system, the B. mori gustatory receptors BmGr53 and BmGr19, which were previously found to be expressed in the MP and to respond to coumarin in the attomolar range, responded to a leaf-surface extract of C. speciosa, suggesting that these receptors may be present on the inedible-leaf-recognizing neurons of the MP. These findings suggest that ultrasensitive plant secondary metabolite-recognizing neurons in the MP allow for the recognition of non-host plants via palpation without risking damage caused by ingesting harmful allelochemicals.
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http://dx.doi.org/10.1016/j.jinsphys.2021.104263DOI Listing
July 2021

A humoral factor, hemolymph proteinase 8, elicits a cellular defense response of nodule formation in Bombyx mori larvae in association with recognition by C-type lectins.

J Insect Physiol 2021 07 20;132:104252. Epub 2021 May 20.

Graduate School of Bio-Application and Systems Engineering, Tokyo University of Agriculture and Technology, Naka-cho 2-24-16, Koganei, Tokyo 184-8588, Japan. Electronic address:

Previously, we found that nodule formation, a cellular defense response in insects, is regulated by humoral factors called C-type lectins in the hemolymph. To elucidate the factors that elicit nodule formation following the recognition of microorganisms by C-type lectins, a reproducible quantitative in vitro assay system was constructed. Then, using this system, the inhibitory activities of antisera raised against hemolymph proteases (HPs), serine protease homologues (SPHs), and pathogen-associated molecular pattern (PAMP)-recognition proteins were assessed. Among the antisera raised against HP and SPH, only that against HP8, a terminal proteinase that activates Spätzle, consistently inhibited in-vitro nodule-like aggregate formation in all three tested microorganisms, Micrococcus luteus, Escherichia coli, and Saccharomyces cerevisiae. Antisera raised against C-type lectins, BmLBP, and BmMBP also inhibited nodule-like aggregate formation, while those against β-glucan recognition proteins and peptidoglycan recognition protein-S1 did not. Microorganisms pretreated with hemolymph, which contains HP8 and C-type lectins, also induced nodule-like aggregate formation, indicating that nodulation factors are present on microbial cells. Furthermore, antisera raised against HP8, BmLBP, and BmMBP showed inhibitory activities in the in vivo nodule formation system using Bombyx mori larvae. Thus, two humoral factors in the hemolymph of B. mori larvae, BmHP8 and C-type lectins, were found to play significant roles in eliciting the cellular defense response of nodule formation.
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http://dx.doi.org/10.1016/j.jinsphys.2021.104252DOI Listing
July 2021

Seroprevalence of antibodies to SARS-CoV-2 in healthcare workers in a nonepidemic region, Japan: A hospital-based study on May, 2020.

J Med Virol 2021 07 3;93(7):4608-4611. Epub 2021 Apr 3.

Department of Clinical Laboratory, Iwate Prefectural Central Hospital, Morioka, Iwate, Japan.

The polymerase chain reaction (PCR) testing rate is low in our local area and the true rate of coronavirus disease 2019 (COVID-19) infection may include many asymptomatic individuals. We conducted a serosurveillance using antibody testing in an area where official report of COVID-19 infection is not done yet. Blood samples were obtained from 1404 healthcare workers (41 ± 11 years) in our hospital on May 29-31, 2020. First, the potential infection frequency was confirmed using two quantitative antibody tests. In addition, the usefulness of rapid antibody kit testing for COVID-19 serosurveillance was examined. A COVID-19-indected case was defined as showing positive results in both quantitative tests. None of 1404 samples had positive results from the two quantitative tests. The false positive rates were 0.36% and 0.07%, whereas those in rapid antibody kits were 3.3% and 3.0%. In conclusion, as of May, 2020, potential spread mainly by asymptomatic individuals infected with COVID-19 was not found in our local area where there was no official report of COVID-19, even if the PCR testing rate was low. Rapid antibody kits might not be useful due to the high false positive rate in an area with a low incidence of COVID-19 infected individuals.
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http://dx.doi.org/10.1002/jmv.26962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8250612PMC
July 2021

Orthogonal antibody testing for COVID-19 among healthcare workers in a non-epidemic place and time:Japan's Iwate Prefecture, May 18-31, 2020.

Fukushima J Med Sci 2021 Apr 26;67(1):27-32. Epub 2021 Feb 26.

Department of Gastroenterological Surgery, Iwate Prefectural Central Hospital.

Of the 47 prefectures in Japan, Iwate had the fewest cases of coronavirus disease 2019 (COVID-19), with the first diagnosis officially confirmed on July 28, 2020. A baseline serological survey of COVID-19 antibodies is essential to accurately evaluate an epidemic outbreak. The primary purpose of this study was to determine pre-epidemic prevalence of COVID-19 antibodies among healthcare workers, using two laboratory-based quantitative tests. In addition, a point-of-care (POC) qualitative test, rapid, simple, and convenient for primary care clinics, was compared with the laboratory-based tests. All antibody tests were performed on serum from 1,000 healthcare workers (mean age, 40 ± 11 years) in Iwate Prefectural Central Hospital, May 29-31, 2020. A COVID-19 case was defined as showing positive results in both laboratory-based quantitative tests. None of 1,000 samples had positive results in both of the laboratory immunoassays. The POC test showed positive results in 33 of 1,000 samples (3.3%) (95% confidence interval:2.19-4.41), but no samples were simultaneously positive in both laboratory-based tests. In conclusion, COVID-19 cases were not serologically confirmed by a baseline control study of healthcare workers at our hospital in late May, 2020. Moreover, the POC qualitative test may offer no advantage in areas with very low prevalence of COVID-19, due to higher false-positive reactions compared with laboratory-based quantitative immunoassays.
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http://dx.doi.org/10.5387/fms.2020-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8075555PMC
April 2021

Diet choice: The two-factor host acceptance system of silkworm larvae.

PLoS Biol 2020 09 16;18(9):e3000828. Epub 2020 Sep 16.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.

Many herbivorous insects are mono- or oligophagous, having evolved to select a limited range of host plants. They specifically identify host-plant leaves using their keen sense of taste. Plant secondary metabolites and sugars are thought to be key chemical cues that enable insects to identify host plants and evaluate their quality as food. However, the neuronal and behavioral mechanisms of host-plant recognition are poorly understood. Here, we report a two-factor host acceptance system in larvae of the silkworm Bombyx mori, a specialist on several mulberry species. The first step is controlled by a chemosensory organ, the maxillary palp (MP). During palpation at the leaf edge, the MP detects trace amounts of leaf-surface compounds, which enables host-plant recognition without biting. Chemosensory neurons in the MP are tuned with ultrahigh sensitivity (thresholds of attomolar to femtomolar) to chlorogenic acid (CGA), quercetin glycosides, and β-sitosterol (βsito). Only if these 3 compounds are detected does the larva make a test bite, which is evaluated in the second step. Low-sensitivity neurons in another chemosensory organ, the maxillary galea (MG), mainly detect sucrose in the leaf sap exuded by test biting, allowing larvae to accept the leaf and proceed to persistent biting (feeding). The two-factor host acceptance system reported here may commonly underlie stereotyped feeding behavior in many phytophagous insects and determine their feeding habits.
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http://dx.doi.org/10.1371/journal.pbio.3000828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494105PMC
September 2020

Photoprotective Capabilities of Light-Harvesting Complex II Trimers in the Green Alga .

J Phys Chem Lett 2020 Sep 2;11(18):7755-7761. Epub 2020 Sep 2.

Division of Environmental Photobiology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

Major light-harvesting complex (LHCII) trimers in plants induce the thermal dissipation of absorbed excitation energy against photooxidative damage under excess light conditions. LHCII trimers in green algae have been thought to be incapable of energy dissipation without additional quencher proteins, although LHCIIs in plants and green algae are homologous. In this study, we investigated the energy-dissipative capabilities of four distinct types of LHCII trimers isolated from the model green alga using spectroscopic analysis. Our results revealed that the LHCII trimers possessing LHCII type II (LHCBM5) and LHCII type IV (LHCBM1) had efficient energy-dissipative capabilities, whereas LHCII type I (LHCBM3/4/6/8/9) and type III (LHCBM2/7) did not. On the basis of the amino acid sequences of LHCBM5 and LHCBM1 compared with the other LHCBMs, we propose that positively charged extra N-terminal amino acid residues mediate the interactions between LHCII trimers to form energy-dissipative states.
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http://dx.doi.org/10.1021/acs.jpclett.0c02098DOI Listing
September 2020

Regulation of light-induced H extrusion and uptake by cyanobacterial homologs of the plastidial FLAP1, DLDG1, and Ycf10 in Synechocystis sp. PCC6803.

Biochim Biophys Acta Bioenerg 2020 10 30;1861(10):148258. Epub 2020 Jun 30.

Department of Life Science and Technology, Tokyo Institute of Technology, Yokohama 226-8501, Japan. Electronic address:

Upon a dark-to-light transition, multiple species of cyanobacteria release a certain amount of H from the inside to the outside of their cells. Previous studies revealed the plasma membrane-localizing Proton exchange A (PxcA) is involved in the light-induced H extrusion in the cyanobacterium Synechocystis sp. PCC6803. Among oxygenic phototrophs, two PxcA homologs are conserved; they are the nuclear-encoded Day-length-dependent delayed-greening1 (DLDG1) and the plastid-encoded Ycf10 in Arabidopsis thaliana. We previously identified the putative DLDG1/Ycf10-interacting protein, Fluctuating-light acclimation protein1 (FLAP1), required for pH regulation in Arabidopsis chloroplasts. Synechocystis has PxcA and FLAP1 homologs designated here as PxcA like (PxcL) and FLAP1 homolog A (FlpA). Synechocystis mutants lacking pxcA, pxcL, and flpA were constructed and characterized to gain more insight into regulatory mechanisms of light-induced H extrusion in cyanobacteria. pH change kinetics of the extracellular solvent after shifting Synechocystis cells from dark to light indicated that PxcA is essential for the light-induced H extrusion, and both PxcA and PxcL were involved in H uptake. Mutational loss of flpA resulted in altered PxcA- and PxcL-dependent H efflux/influx activities, and the flpA-null mutant showed inhibited growth under dark-light cycles, indicating the importance of FlpA function for photosynthetic growth under fluctuating light. Collectively, these data suggest that PxcA is involved in H efflux immediately after light irradiation for the rapid formation of the H concentration gradient across the thylakoid membranes, PxcL is involved in H influx for activation of the Calvin-Benson-Bassham cycle, and FlpA controls the H transport under fluctuating light.
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http://dx.doi.org/10.1016/j.bbabio.2020.148258DOI Listing
October 2020

ATP-Binding Cassette Subfamily A Member 2 is a Functional Receptor for Cry2A Toxins in , but not for Cry1A, Cry1C, Cry1D, Cry1F, or Cry9A Toxins.

Toxins (Basel) 2020 02 6;12(2). Epub 2020 Feb 6.

Institute of Agrobiological Sciences, NARO, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.

Cry toxins are insecticidal proteins produced by (Bt). They are used commercially to control insect pests since they are very active in specific insects and are harmless to the environment and human health. The gene encoding ATP-binding cassette subfamily A member 2 (ABCA2) was identified in an analysis of Cry2A toxin resistance genes. However, we do not have direct evidence for the role of ABCA2 for Cry2A toxins or why Cry2A toxin resistance does not cross to other Cry toxins. Therefore, we performed two experiments. First, we edited the sequence in using transcription activator-like effector-nucleases (TALENs) and confirmed the susceptibility-determining ability in a diet overlay bioassay. Strains with C-terminal half-deleted BmABCA2 showed strong and specific resistance to Cry2A toxins; even strains carrying a deletion of 1 to 3 amino acids showed resistance. However, the C-terminal half-deleted strains did not show cross-resistance to other toxins. Second, we conducted a cell swelling assay and confirmed the specific ability of BmABCA2 to Cry2A toxins in HEK239 cells. Those demonstrated that BmABCA2 is a functional receptor for Cry2A toxins and that BmABCA2 deficiency-dependent Cry2A resistance does not confer cross-resistance to Cry1A, Cry1F, Cry1Ca, Cry1Da, or Cry9Aa toxins.
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http://dx.doi.org/10.3390/toxins12020104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076765PMC
February 2020

Changes in surface properties of dental alloys with atmospheric plasma irradiation.

Dent Mater J 2020 Jun 19;39(3):375-380. Epub 2019 Dec 19.

Department of Dental Materials, Nihon University School of Dentistry.

Chemical transitions after atmospheric pressure plasma irradiation were investigated by evaluating intermolecular attractions and atomic and molecular reactions. Gold, titanium and stainless-steel alloy samples were ground with #800 grit SiC waterproof paper and nitrogen gas atmospheric plasma irradiation was conducted. The surface free energies of the treated alloys were calculated and compared statistically. X-ray photoelectron spectroscopy analysis was performed.The surface free energies of all metal surfaces treated by plasma irradiation were 1.5-times higher than those of the untreated metals. The energy of the hydrogen bonding component increased, and all alloy surfaces were coated with metal oxide after only a short period of plasma irradiation. The surfaces oxidized by plasma exhibited a high active energy, mainly due to an increase in the hydrogen bonding component. Reactions with oxygen in the air were promoted on the clean surfaces with exposed reactive elements.
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http://dx.doi.org/10.4012/dmj.2019-023DOI Listing
June 2020

DAY-LENGTH-DEPENDENT DELAYED-GREENING1, the Arabidopsis Homolog of the Cyanobacterial H+-Extrusion Protein, Is Essential for Chloroplast pH Regulation and Optimization of Non-Photochemical Quenching.

Plant Cell Physiol 2019 Dec;60(12):2660-2671

School of Life Science & Technology, Tokyo Institute of Technology, Yokohama, 226-8501 Japan.

Plants convert solar energy into chemical energy through photosynthesis, which supports almost all life activities on earth. Because the intensity and quality of sunlight can change dramatically throughout the day, various regulatory mechanisms help plants adjust their photosynthetic output accordingly, including the regulation of light energy accumulation to prevent the generation of damaging reactive oxygen species. Non-photochemical quenching (NPQ) is a regulatory mechanism that dissipates excess light energy, but how it is regulated is not fully elucidated. In this study, we report a new NPQ-regulatory protein named Day-Length-dependent Delayed-Greening1 (DLDG1). The Arabidopsis DLDG1 associates with the chloroplast envelope membrane, and the dldg1 mutant had a large NPQ value compared with wild type. The mutant also had a pale-green phenotype in developing leaves but only under continuous light; this phenotype was not observed when dldg1 was cultured in the dark for ≥8 h/d. DLDG1 is a homolog of the plasma membrane-localizing cyanobacterial proton-extrusion-protein A that is required for light-induced H+ extrusion and also shows similarity in its amino-acid sequence to that of Ycf10 encoded in the plastid genome. Arabidopsis DLDG1 enhances the growth-retardation phenotype of the Escherichia coli K+/H+ antiporter mutant, and the everted membrane vesicles of the E. coli expressing DLDG1 show the K+/H+ antiport activity. Our findings suggest that DLDG1 functionally interacts with Ycf10 to control H+ homeostasis in chloroplasts, which is important for the light-acclimation response, by optimizing the extent of NPQ.
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http://dx.doi.org/10.1093/pcp/pcz203DOI Listing
December 2019

Superoxide dismutase down-regulation and the oxidative stress is required to initiate pupation in Bombyx mori.

Sci Rep 2019 10 11;9(1):14693. Epub 2019 Oct 11.

Department of United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509, Japan.

Perhaps, oxidative stress progresses pupation in some Lepidopteran insects; however, the reasons for this remain obscure. In our previous study, we clarified Bombyx mori SOD1 (BmSOD1) and B. mori SOD2 (BmSOD2) proteins respond in common to ultraviolet irradiation (UV) oxidative stress and metamorphosis. This result strongly suggested pupation initiates by oxidative stress and might mediate by down-regulation of expression of BmSOD1 and BmSOD2 proteins. Thus, we examined about these relationships in B. mori in this study. In the microarray data reanalysis, we found the Notch signaling pathways as the common pathways in pupation and UV oxidative stress in B. mori. Also, we showed a molting hormone, 20-hydroxyecdysone, leads not only generation of superoxide but also downregulation of the expression of BmSOD proteins during pupation in B. mori. Our findings can contribute to a deeper understanding of how biological defense systems work against environmental oxidative stress.
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http://dx.doi.org/10.1038/s41598-019-51163-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6788986PMC
October 2019

pH-Responsive Binding Properties of Light-Harvesting Complexes in a Photosystem II Supercomplex Investigated by Thermodynamic Dissociation Kinetics Analysis.

J Phys Chem Lett 2019 Jul 17;10(13):3615-3620. Epub 2019 Jun 17.

Division of Environmental Photobiology , National Institute for Basic Biology , Okazaki 444-8585 , Japan.

Reorganization of photosynthetic proteins on the thylakoid membrane is an important regulatory process for photoacclimation in photosynthetic organisms. However, the underlying mechanism has been poorly understood due to the lack of methods to analyze the interactions between membrane proteins. To investigate the mechanism, we demonstrated the binding properties of light-harvesting complex proteins (LHCs) in a photosystem II (PSII) supercomplex regulated by pH conditions, which primarily responded to environmental light conditions, using a thermodynamic dissociation kinetics analysis. The results showed that the strongly bound LHCs (∼60%) were responsive to pH conditions, whereas the moderately and loosely bound LHCs (∼40%) were nonresponsive. This result implies that the pH condition alters the binding properties of LHCs in the PSII supercomplex, inducing the reorganization of protein complexes.
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http://dx.doi.org/10.1021/acs.jpclett.9b01208DOI Listing
July 2019

Function and Role of ATP-Binding Cassette Transporters as Receptors for 3D-Cry Toxins.

Toxins (Basel) 2019 02 19;11(2). Epub 2019 Feb 19.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka 2-24-16, Koganei, Tokyo 184-8588, Japan.

When ABC transporter family C2 (ABCC2) and ABC transporter family B1 (ABCB1) were heterologously expressed in non-susceptible cultured cells, the cells swelled in response to Cry1A and Cry3 toxins, respectively. Consistent with the notion that 3D-Cry toxins form cation-permeable pores, ABCC2 (BmABCC2) facilitated cation-permeable pore formation by Cry1A when expressed in oocytes. Furthermore, BmABCC2 had a high binding affinity () to Cry1Aa of 3.1 × 10 M. These findings suggest that ABC transporters, including ABCC2 and ABCB1, are functional receptors for 3D-Cry toxins. In addition, the Cry2 toxins most distant from Cry1A toxins on the phylogenetic tree used ABC transporter A2 as a receptor. These data suggest that 3D-Cry toxins use ABC transporters as receptors. In terms of inducing cell swelling, ABCC2 has greater activity than cadherin-like receptor. The pore opening of ABC transporters was hypothesized to be linked to their receptor function, but this was repudiated by experiments using mutants deficient in export activity. The synergistic relationship between ABCC2 and cadherin-like receptor explains their ability to cause resistance in one species of insect.
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http://dx.doi.org/10.3390/toxins11020124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409751PMC
February 2019

Genetic characterization of a flap1 null mutation in Arabidopsis npq4 and pgr5 plants suggests that the regulatory role of FLAP1 involves the control of proton homeostasis in chloroplasts.

Photosynth Res 2019 Mar 2;139(1-3):413-424. Epub 2018 Nov 2.

Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.

Precise control of the proton concentration gradient across thylakoid membranes (ΔpH) is essential for photosynthesis and its regulation because the gradient contributes to the generation of the proton motive force used for ATP synthesis and also for the fast and reversible induction of non-photochemical quenching (NPQ) to avoid photoinhibition and photodamage. However, the regulatory mechanism(s) controlling ΔpH in response to fluctuating light has not been fully elucidated. We previously described a new NPQ-regulatory chloroplastic protein, Fluctuating-Light-Acclimation Protein1 (FLAP1), which is important for plant growth and modulation of ΔpH under fluctuating light conditions. For this report, we further characterized FLAP1 activity by individually crossing an Arabidopsis flap1 mutant with npq4 and pgr5 plants; npq4 is defective in PsbS-dependent NPQ, and pgr5 is defective in induction of steady-state proton motive force (pmf) and energy-dependent quenching (qE). Both npq4 and npq4 flap1 exhibited similar NPQ kinetics and other photosynthetic parameters under constant or fluctuating actinic light. Conversely, pgr5 flap1 had recovered NPQ, photosystem II quantum yield and growth under fluctuating light, each of which was impaired in pgr5. Together with other data, we propose that FLAP1 activity controls proton homeostasis under steady-state photosynthesis to manipulate luminal acidification levels appropriately to balance photoprotection and photochemical processes.
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http://dx.doi.org/10.1007/s11120-018-0575-zDOI Listing
March 2019

Role of Bacillus thuringiensis Cry1A toxins domains in the binding to the ABCC2 receptor from Spodoptera exigua.

Insect Biochem Mol Biol 2018 10 2;101:47-56. Epub 2018 Aug 2.

ERI de Biotecnología y Biomedicina (BIOTECMED), Department of Genetics, Universitat de València, 46100 Burjassot, Spain. Electronic address:

Cry proteins from Bacillus thuringiensis (Bt) have been used to control insect pests either as formulated sprays or as in Bt-crops. However, field-evolved resistance to Bt proteins is threatening the long-term use of Bt products. The SeABCC2 locus has been genetically linked to resistance to a Bt bioinsecticide (Xentari™) in Spodoptera exigua (a mutation producing a truncated form of the transporter lacking an ATP binding domain was found in the resistant insects). Here, we investigated the role of SeABCC2 in the mode of action of Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and two Cry1A-1Ca hybrids by expressing the receptor in Sf21 and HEK293T cell lines. Cell toxicity assays showed that Sf21 cells expressing SeABCC2 become susceptible to Cry1A proteins. HEK293T cells expressing the transporter were found susceptible to Cry1A proteins but not to Cry1Ca. The results with the Cry1A-1Ca hybrids suggest that domain II from Cry1Ab/c is crucial for the toxicity to Sf21 cells, whereas domain III from Cry1Aa/b is crucial for the toxicity to HEK293T cells. Binding assays showed that the Cry1Ac binding is of high affinity and specific to cells expressing the SeABCC2 transporter. Heterologous competition experiments support a model in which domain II of Cry1Ab/c has a common binding site in the SeABCC2 protein, whereas domain III of Cry1Aa/b binds to a different binding site in the SeABCC2 protein.
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http://dx.doi.org/10.1016/j.ibmb.2018.07.006DOI Listing
October 2018

Glucose, some amino acids and a plant secondary metabolite, chlorogenic acid induce the secretion of a regulatory hormone, tachykinin-related peptide, from the silkworm midgut.

Peptides 2018 08 19;106:21-27. Epub 2018 Jun 19.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address:

Enteroendocrine cells in the insect midgut are thought to secrete peptide hormones in response to the nutritional state. However, the role of dietary compounds in inducing peptide hormone secretion from enteroendocrine cells in insects remains unknown. In the present study, we demonstrated that several dietary compounds from mulberry leaves, including glucose, amino acids, and the secondary metabolite chlorogenic acid, induced significant secretion of tachykinin-related peptides from isolated silkworm midguts at the luminal concentrations measured in fed larvae. This study provides evidence that the insect midgut senses a non-nutritious secondary metabolite in addition to nutrient metabolites to monitor luminal food status and secretes a feeding regulatory hormone, suggesting that a unique dietary sensory system modulates insect feeding via enteroendocrine control.
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http://dx.doi.org/10.1016/j.peptides.2018.06.004DOI Listing
August 2018

Significance of PGR5-dependent cyclic electron flow for optimizing the rate of ATP synthesis and consumption in Arabidopsis chloroplasts.

Photosynth Res 2019 Mar 18;139(1-3):359-365. Epub 2018 Jun 18.

Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama, 226-8501, Japan.

The proton motive force (PMF) across the chloroplast thylakoid membrane that is generated by electron transport during photosynthesis is the driving force for ATP synthesis in plants. The PMF mainly arises from the oxidation of water in photosystem II and from electron transfer within the cytochrome bf complex. There are two electron transfer pathways related to PMF formation: linear electron flow and cyclic electron flow. Proton gradient regulation 5 (PGR5) is a major component of the cyclic electron flow pathway, and the Arabidopsis pgr5 mutant shows a substantial reduction in the PMF. How the PGR5-dependent cyclic electron flow contributes to ATP synthesis has not, however, been fully delineated. In this study, we monitored in vivo ATP levels in Arabidopsis chloroplasts in real time using a genetically encoded bioluminescence-based ATP indicator, Nano-lantern(ATP1). The increase in ATP in the chloroplast stroma of pgr5 leaves upon illumination with actinic light was significantly slower than in wild type, and the decrease in ATP levels when this illumination stopped was significantly faster in pgr5 leaves than in wild type. These results indicated that PGR5-dependent cyclic electron flow around photosystem I helps to sustain the rate of ATP synthesis, which is important for growth under fluctuating light conditions.
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http://dx.doi.org/10.1007/s11120-018-0533-9DOI Listing
March 2019

Extracellular loop structures in silkworm ABCC transporters determine their specificities for Cry toxins.

J Biol Chem 2018 06 17;293(22):8569-8577. Epub 2018 Apr 17.

From the Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan and

Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm () ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.
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http://dx.doi.org/10.1074/jbc.RA118.001761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986200PMC
June 2018

Leptospiral flagellar sheath protein FcpA interacts with FlaA2 and FlaB1 in Leptospira biflexa.

PLoS One 2018 10;13(4):e0194923. Epub 2018 Apr 10.

Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan.

Leptospira spp. are spirochete bacteria that possess periplasmic flagella (PFs) underneath the outer membrane; each flagellum is attached to each end of the protoplasmic cylinder. PFs of Leptospira have a coiled shape that bends the end of the cell body. However, the molecular mechanism by which multiple flagellar proteins organize to form the distinctively curled PF of Leptospira remains unclear. Here we obtained a slow-motility mutant of L. biflexa MD4-3 by random insertion mutagenesis using a Himar1 transposon. In MD4-3, the gene encoding the flagellar sheath protein, flagellar-coiling protein A (FcpA), which was recently identified in L. interrogans, was inactivated. As with L. interrogans ΔfcpA strains, the L. biflexa ΔfcpA strain lacked a distinct curvature at both ends of the cell body, and its motility was significantly reduced as compared with that of the wild-type strain. PFs isolated from the ΔfcpA strain were straight and were thinner than those isolated from the wild-type strain. Western blot analysis revealed that flagellar proteins FlaA1, FlaA2, FlaB1, and FlaB2 were expressed in the ΔfcpA strain but the flagellar proteins, except for FlaB2 were not incorporated in its PFs. Immunoprecipitation assay using anti-FcpA antiserum demonstrated that FcpA associates with FlaA2 and FlaB1. The association between FcpA and FlaA2 was also verified using pull-down assay. The regions of FlaA2 and FlaB1 interacting with FcpA were determined using a bacterial two-hybrid assay. These results suggest that FcpA together with FlaA2, produces coiling of PF of the Leptospira, and the interaction between the sheath and core filament may be mediated by FcpA and FlaB1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194923PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892894PMC
July 2018

Insect taste receptors relevant to host identification by recognition of secondary metabolite patterns of non-host plants.

Biochem Biophys Res Commun 2018 05 7;499(4):901-906. Epub 2018 Apr 7.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo, 184-8588, Japan. Electronic address:

The taste sensing system is crucial for food recognition in insects and other animals. It is commonly believed that insect gustatory receptors (Grs) expressed in gustatory organs are indispensable for host plant selection. Many behavioral studies have shown that mono- or oligo-phagous lepidopteran insects use the balance between feeding attractants and feeding deterrents in host plants and that these are sensed by taste organs for host plant recognition. However, the molecular mechanism underlying taste recognition, especially of feeding deterrents, remains to be elucidated. To better understand this mechanism, we studied orphan Grs, including Bombyx mori Gr (BmGr) 16, BmGr18, and BmGr53, from the mono-phagous insect, Bombyx mori. Using Calcium imaging in mammalian cells, we first confirmed in lepidoptera insects that three of the putative bitter Grs widely responded to structurally different feeding deterrents. Although the phylogenetic distance of these Grs was considerable, they responded to partially overlapping deterrents of plant secondary metabolites. These findings suggest that not only these three Grs but also most of the Grs that have been assigned to putative bitter Grs are feeding-deterrent receptors that play a role in host plant recognition.
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http://dx.doi.org/10.1016/j.bbrc.2018.04.014DOI Listing
May 2018

Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae.

Drug Discov Ther 2018;12(1):7-15

Department of Science of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology.

Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.
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http://dx.doi.org/10.5582/ddt.2018.01004DOI Listing
August 2019

The intracellular region of silkworm cadherin-like protein is not necessary to mediate the toxicity of Bacillus thuringiensis Cry1Aa and Cry1Ab toxins.

Insect Biochem Mol Biol 2018 03 6;94:36-41. Epub 2018 Feb 6.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan. Electronic address:

The cadherin-like protein in lepidopteran insects, known as a receptor for Bacillus thuringiensis Cry1A toxins, is a single-pass membrane protein that can be divided into extracellular and intracellular regions. The extracellular region is important for toxin binding and oligomerization, whereas the role of the intracellular region during Cry1A intoxication is unclear. In the present study, we generated a deletion mutant of Bombyx mori cadherin-like protein (BtR175) that lacked the intracellular region to investigate its role in mediating Cry1A toxicity. Like wild-type BtR175, the mutant protein conferred susceptibility to Cry1Aa and Cry1Ab toxins in Sf9 cells, suggesting that the intracellular region is not required to mediate intoxication. The deletion mutant maintained another role of cadherin-like proteins; that it, synergistic activity with B. mori ABC transporter C2 (ABCC2) when mediating Cry1Aa and Cry1Ab toxicity. In addition, we evaluated the effects of reagents that have been reported to inhibit Cry1A toxicity (e.g., protein kinase A inhibitors, EDTA, and sucrose) on Cry1A toxicity in BtR175-expressing cells. Our results suggest that Cry1Aa-induced cell death in BtR175-expressing cells was not caused by signal transduction but by osmotic lysis. Overall, our data indicate that BtR175 mediates the toxicity of Cry1Aa and Cry1Ab toxins entirely via its extracellular region. They also indicate that the synergism between cadherin-like protein and ABCC2 occurs outside of cells or in the cell membrane.
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http://dx.doi.org/10.1016/j.ibmb.2018.01.005DOI Listing
March 2018

Differential expression of a fructose receptor gene in honey bee workers according to age and behavioral role.

Arch Insect Biochem Physiol 2018 Feb 1;97(2). Epub 2017 Dec 1.

Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Japan.

Honey bee (Apis mellifera) workers contribute to the maintenance of colonies in various ways. The primary functions of workers are divided into two types depending on age: young workers (nurses) primarily engage in such behaviors as cleaning and food handling within the hive, whereas older workers (foragers) acquire floral nutrients beyond the colony. Concomitant with this age-dependent change in activity, physiological changes occur in the tissues and organs of workers. Nurses supply younger larvae with honey containing high levels of glucose and supply older larvae with honey containing high levels of fructose. Given that nurses must determine both the concentration and type of sugar used in honey, gustatory receptors (Gr) expressed in the chemosensory organs likely play a role in distinguishing between sugars. Glucose is recognized by Gr1 in honey bees (AmGr1); however, it remains unclear which Gr are responsible for fructose recognition. This study aimed to identify fructose receptors in honey bees and reported that AmGr3, when transiently expressed in Xenopus oocytes, responded only to fructose, and to no other sugars. We analyzed expression levels of AmGr3 to identify which tissues and organs of workers are involved in fructose recognition and determined that expression of AmGr3 was particularly high in the antennae and legs of nurses. Our results suggest that nurses use their antennae and legs to recognize fructose, and that AmGr3 functions as an accurate nutrient sensor used to maintain food quality in honey bee hives.
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http://dx.doi.org/10.1002/arch.21437DOI Listing
February 2018

Arabidopsis Phosphatidic Acid Phosphohydrolases Are Essential for Growth under Nitrogen-Depleted Conditions.

Front Plant Sci 2017 31;8:1847. Epub 2017 Oct 31.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

The Arabidopsis homologs of mammalian lipin, PAH1 and PAH2, are cytosolic phosphatidic acid phosphohydrolases that are involved in phospholipid biosynthesis and are essential for growth under phosphate starvation. Here, double-knockout mutants were found to be hypersensitive to nitrogen (N) starvation, whereas transgenic plants overexpressing PAH1 or PAH2 in the mutant background showed a similar growth phenotype as compared with wild type (WT) under N starvation. The chlorophyll content of was significantly lower than that of WT, whereas the chlorophyll content and photosynthetic activity of the transgenic plants were significantly higher than those of WT under N-depleted conditions. Membrane glycerolipid composition of the mutants showed a significant decrease in the mole percent of chloroplast lipids to other phospholipids, whereas membrane lipid composition did not differ between transgenic plants and WT plants. Pulse-chase labeling experiments using plants grown under N-depleted conditions showed that, in plants, the labeling percent of chloroplast lipids such as phosphatidylglycerol and monogalactosyldiacylglycerol in the total glycerolipids was significantly lower than in WT. Moreover, N starvation-induced degradation of chloroplast structure was enhanced in mutants, and the membrane structure was recovered by complementation with PAH1. Thus, PAH is involved in maintaining chloroplast membrane structure and is required for growth under N-depleted conditions.
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http://dx.doi.org/10.3389/fpls.2017.01847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671605PMC
October 2017

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

Insect Biochem Mol Biol 2017 12 8;91:44-54. Epub 2017 Nov 8.

Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Naka 2-24-16, Koganei, Tokyo 184-8588, Japan. Electronic address:

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that DYWL of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around DYWL of ECL 4 in the ABCC2.
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http://dx.doi.org/10.1016/j.ibmb.2017.11.002DOI Listing
December 2017
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