Search

Objectives: This study compared the prognostic role of high on-treatment platelet reactivity (HTPR) in predicting thrombotic events in a Korean population undergoing percutaneous coronary intervention (PCI) in the acute myocardial infarction (AMI) and non-AMI setting. Background: The prognostic significance and optimal cutoff of HTPR might differ according to a given clinical condition, such as AMI and ethnicity. Methods: On-treatment platelet reactivity was measured with a VerifyNow P2Y12 assay (Accumetrics, San Diego, California) in 1,226 patients (824 men; age 65 ± 10 years), including 413 AMI cases, 12 to 24 h after PCI between March 2008 and March 2010. The prevalence of cardiovascular (CV) events defined as a composite of death from CV causes, nonfatal myocardial infarction, or stent thrombosis at 1-year follow-up were compared according to HTPR between patients with and without AMI. Results: The optimal cutoff for HTPR was 272 IU of the P2Y12 reaction unit (PRU) (area under the curve: 0.708; 95% confidence interval [CI]: 0.607 to 0.809, p = 0.03), which was the upper-tertile threshold. Among AMI patients, 1-year CV events occurred more frequently in patients with versus without HTPR (n = 14 [8.8%] vs. n = 1 [0.4%], p < 0.001), whereas there was no difference in the composite endpoint on the basis of HTPR in patients without AMI (n = 7 [2.8%] vs. n = 8 [1.4%], p = 0.193). Conclusions: Increased residual platelet reactivity is related to post-discharge CV events in subjects with AMI, whereas the prognostic significance of HTPR seems to be attenuated in patients with stable coronary disease after PCI. [ABSTRACT FROM AUTHOR]

Lipid-derived molecules produced by acylhydrolases play important roles in the regulation of diverse cellular functions in plants. In Arabidopsis, the DAD1-like phospholipase A1 family consists of 12 members, all of which possess a lipase 3 domain. In this study, the biochemical and cellular functions of AtDLAH, an Arabidopsis thaliana DAD1-like acylhydrolase, were examined. Bacterially expressed AtDLAH contained phospholipase A1 activity for catalysing the hydrolysis of phospholipids at the sn-1 position. However, AtDLAH displayed an even stronger preference for 1-lysophosphatidylcholine, 1-monodiacylglycerol, and phosphatidic acid, suggesting that AtDLAH is a sn-1-specific acylhydrolase. The AtDLAH gene was highly expressed in young seedlings, and its encoded protein was exclusively localized to the mitochondria. AtDLAH-overexpressing transgenic seeds (35S:AtDLAH) were markedly tolerant to accelerated-ageing treatment and thus had higher germination percentages than wild-type seeds. In contrast, the atdlah loss-of-function knockout mutant seeds were hypersusceptible to accelerated-ageing conditions. The 35S:AtDLAH seeds, as opposed to the atdlah seeds, exhibited a dark red staining pattern following tetrazolium treatment under both normal and accelerated-ageing conditions, suggesting that AtDLAH expression is positively correlated with seed viability. The enhanced viability of 35S:AtDLAH seeds was accompanied by more densely populated epidermal cells, lower levels of accumulated lipid hydroperoxides, and higher levels of polar lipids as compared with wild-type and atdlah mutant seeds. These results suggest that AtDLAH, a mitochondrial-localized sn-1-specific acylhydrolase, plays an important role in Arabidopsis seed viability. [ABSTRACT FROM PUBLISHER]

The plant U-box (PUB) protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the class-II PUB family. Self-ubiquitination assays showed that bacterially expressed MBP-OsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling-lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of HO and oxidized proteins were higher in the knock-out mutant compared with the wild type. OsPUB15 transcript levels were increased upon HO, salt and drought stresses; plants overexpressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces reactive oxygen species (ROS) stress and cell death. [ABSTRACT FROM AUTHOR]

Telomeres are nucleoprotein complexes essential for the integrity of eukaryotic chromosomes. Cellular roles of single-stranded telomeric DNA binding proteins have been extensively described in yeast and animals, but our knowledge about plant single-strand telomeric factors is rudimentary. Here, we investigated Nicotiana tabacum G-strand-specific single-stranded telomere binding proteins (GTBPs), homologs of a human heterogeneous nuclear ribonucleoprotein. GTBPs bound specifically to the plant single-stranded (TTTAGGG)4 telomeric repeat element in vitro and were associated with telomeric sequences in tobacco BY-2 suspension cells. Transgenic plants (35S:RNAi-GTBP1), in which GTBP1 was suppressed, exhibited severe developmental anomalies. In addition, the chromosomes of 35S:RNAi-GTBP1 cells displayed elongated telomeres, frequent formation of extrachromosomal telomeric circles, and numerous abnormal anaphase bridges, indicating that GTBP1 knockdown tobacco plants experienced genome instability. GTBP1 inhibited strand invasion, an initial step in interchromosomal homologous recombination. We propose that GTBP1 plays a critical role in telomere structure and function by preventing aberrant interchromosomal telomeric homologous recombination in tobacco. [ABSTRACT FROM AUTHOR]

In an effort to elucidate biological functions of transcription factors of Brassica rapa L. (ssp. pekinensis), an NGATHA homolog, BrNGA1, that belongs to the B3-type transcription factor superfamily was identified and expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants overexpressing BrNGA1, named BrNGA1ox, displayed markedly reduced organ growth compared with the wild type: lateral organs, such as leaves, flowers and cotyledons, were small and distinctively narrow, and their root growth was also severely retarded. Reduced sizes of BrNGA1ox organs were mainly due to reduction in cell numbers. Kinematic analysis of leaf growth revealed that both the rate and duration of cell proliferation declined during organogenesis, which was consistent with the reduced expression of cyclin genes. Reduction in organ growth was strongly correlated with the small size of meristematic cell pools in the shoot and root meristems. Taken together, these data indicate that BrNGA1 acts as a negative regulator of cell proliferation and may do so, in part, by regulating the size of the meristematic cell pool. [ABSTRACT FROM PUBLISHER]

Retinoblastoma protein (Rb) plays a key role in cell cycle control, cell differentiation, and apoptosis in animals. In this study, we used virus-induced gene silencing (VIGS) to investigate the cellular functions of Rb in higher plants. VIGS ofNbRBR1, which encodes theNicotiana benthamianaRb homolog, resulted in growth retardation and abnormal organ development. At the cellular level, Rb suppression caused prolonged cell proliferation in tissues that are normally differentiated, which indicates that Rb is a negative regulator of plant cell division. Furthermore, differentiation of the epidermal pavement cells and trichomes was partially retarded, and stomatal clusters formed in the epidermis, likely due to uncontrolled cell division of stomata precursor cells. Rb suppression also caused extra DNA replication in endoreduplicating leaf cells, suggesting a role of Rb in the endocycle. These Rb phenotypes were accompanied by stimulated transcription ofE2Fand E2F-regulated S-phase genes. Thus, disruption of Rb function in plants leads to ectopic cell division in major organs that correlates with a delay in cell differentiation as well as increased endoreduplication, which indicates that Rb coordinates these processes in plant organ development. [ABSTRACT FROM AUTHOR]

Abstract: Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression. [Copyright &y& Elsevier]

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (Mr=67.4 kDa) is reminiscent of the δ-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed. [Copyright &y& Elsevier]

Two cDNA clones, pOS-ACO2 and pOS-ACO3, encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase were isolated from rice seedling cDNA library. pOS-ACO3 is a 1,299 bp full-length clone encoding 321 amino acids (Mr=35.9 kDa), while pOS-ACO2 is 1,072 bp long and is a partial cDNA clone encoding 314 amino acids. These two deduced amino acid sequences share 70% identity, and display a high degree of sequence identity (72-92%) with previously isolated pOS-ACOl of deepwater rice. The chromosomal location studies show that OS-ACO2 is positioned on the long arm of chromosome 9, while OSACO3 on the long arm of chromosome 2 of rice genome. A marked increase in the level of OS-ACO2 transcript was observed in IAA-treated etiolated rice seedlings, whereas the OS-ACO3 mRNA was greatly accumulated by ethylene treatment. Results of ethylene inhibitor studies indicated that auxin promotion of the OS-ACO2 transcription was not mediated through the action of auxin-induced ethylene. Thus, it appears that there are two groups of ACC oxidase transcripts in rice plants, either auxin-induced or ethylene-induced. The auxin-induced OS-ACO2 expression was partially inhibited by ethylene, while ethylene induction of OS-ACO3 transcription was completely blocked by auxin. These results indicate that the expression of ACC oxidase genes is regulated by complex hormonal networks in a gene specific manner in rice seedlings. Okadaic acid, a potent inhibitor of protein phosphatase, effectively suppressed the IAA induction of OS-ACO2 expression, suggesting that protein dephosphorylation plays a role in the induction of ACC oxidase by auxin. A scheme of the multiple regulatory pathways for the expression of ACC oxidase gene family by auxin, ethylene and protein phosphatase is presented. [ABSTRACT FROM AUTHOR]

Summary Marchantia polymorpha, occupying a basal position in the monophyletic assemblage of land plants, displays a notable expansion of plant U‐box (PUB) proteins compared with those in animals. We elucidated the roles of MpPUB9 in regulating salt stress tolerance in M. polymorpha. MpPUB9 expression was rapidly induced by high salinity and dehydration. MpPUB9 possessed an intact U‐box domain in the N‐terminus. MpPUB9‐Citrine localized to punctate structures and was peripherally associated with microsomal membranes. Phenotypic analyses demonstrate that the hyponastic and epinastic thallus growth phenotypes, which were induced by the overexpression and suppression of MpPUB9, may provoke salt stress‐resistant and ‐susceptible phenotypes, respectively. MpPUB9 was also found to directly interact with the exocyst protein MpEXO70.1, leading to its ubiquitination. Under high‐salinity conditions, though the stability of MpPUB9 was dramatically increased, MpEXO70.1 showed slightly faster turnover rates. Transcriptome analyses showed that salt treatment and the overexpression of MpPUB9 co‐upregulated the genes related to the modulation of H2O2 and cell wall organization. Overall, our results suggest that MpPUB9 plays a crucial role in the positive regulation of salt stress tolerance, resulting from its interaction with MpEXO70.1 and modulating turnover of the protein under high‐salt conditions via the coordination of UPS with autophagy. [ABSTRACT FROM AUTHOR]

Three full length 1-aminocyclopropane-l-carboxylate (ACC) oxidase cDNA clones (pNG-ACOl, 1,254 bp; pNG-ACO2,1,198 bp; and pNG-ACO3,1,053 bp) were isolated from the TMV-treated leaf cDNA library of Nicotiana glutinosa plant. They share a high degree of sequence identity (78–81%) throughout the coding regions but are divergent within the 3′-untranslated regions. The gene-specific probes were prepared using these regions to investigate the differential expression of the ACC oxidase gene family in various organs and in response to a multitude of biotic and abiotic stresses in N. glutinosa plants. All three genes were transcriptionally active displaying unique patterns of expression. Both the pNG-ACOl and pNG-ACO3 transcripts highly accumulated during the senescence of leaves, while the pNG-ACO2 mRNA was constitutively present. In addition, the NG-ACO1 and NG-ACO3 transcripts were predominantly found in roots whereas the NG-ACO2 mRNA was mainly in stems. Upon TMV infection, both NG-ACO1 and NG-ACO3 were markedly induced, but in mock treatment which has an effect of mild wounding, only the NG-ACO3 gene was induced. Furthermore, salicylic acid and CuSO4 treatments of leaves increased the level of NG-ACO1 and NG-ACO3 transcripts, while they did not affect the NG-ACO2 gene expression. Results showed that both the NG-ACO1 and NG-ACO3 genes were highly inducible by ethylene and methyl jasmonate treatments, with NG-ACO3 being more responsive. By contrast, NG-ACO2 did not respond to these growth regulators. Thus, it appears that there are two groups of ACC oxidase transcripts expressed in leaf tissue of N. glutinosa, either stress-induced or constitutive. The possible molecular mechanism of differential regulation of ACC oxidase gene expression and its physiological significance are discussed. [ABSTRACT FROM AUTHOR]

SUMMARY: The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite‐induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U‐box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite‐insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo‐proteasome complex, resulting in intracellular accumulation of UbG76V‐GFP, an artificial substrate of the proteasome complex, and insoluble poly‐ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite‐induced proteotoxic stress response via negative regulation of 26S proteasome integrity. [ABSTRACT FROM AUTHOR]

Background: Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley. Results: In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses. Conclusion: This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley. [ABSTRACT FROM AUTHOR]

The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae. [ABSTRACT FROM AUTHOR]

Cancer therapy using immune checkpoint inhibitor antibodies has markedly shifted the paradigm of cancer treatment. However, methods completely eliminating the effector function of these signal-regulating antibodies is urgently required. The heterogeneity of glycan chains in antibodies limits their use as therapeutic agents due to their variability; thus, the development of uniform glycan chains is necessary. Here, we subjected the anti-programmed cell death protein (PD)-1 antibody nivolumab, a representative immune checkpoint inhibitor, to GlycoDelete (GD) engineering to remove the antibody-dependent cellular cytotoxicity (ADCC) of the antibody, leaving only one glycan in the Fc. Glyco-engineered CHO cells were prepared by overexpressing endo-β-N-acetyl-glucosaminidase (Endo T) in CHO cells, in which N-acetyl-glucosaminyl-transferase I was knocked out using Cas9. GD IgG1 nivolumab and GD IgG4 nivolumab were produced using GD CHO cells, and glycan removal was confirmed using mass spectrometry. Target binding and PD-1 inhibition was not altered; however, ADCC decreased. Furthermore, the IgG4 form, determined to be the most suitable form of GD nivolumab, was produced in a plant GD system. The plant GD nivolumab also reduced ADCC without affecting PD-1 inhibitory function. Thus, CHO and plant GD platforms can be used to improve signal-regulating antibodies by reducing their effector function. [ABSTRACT FROM AUTHOR]

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization. [ABSTRACT FROM AUTHOR]

One of the major regulatory pathways that permits plants to convert an external stimulus into an internal cellular response within a short period of time is the ubiquitination pathway. In this study, OsATL38 was identified as a low temperature-induced gene that encodes a rice homolog of Arabidopsis Tóxicos en Levadura RING-type E3 ubiquitin (Ub) ligase, which was predominantly localized to the plasma membrane. OsATL38 -overexpressing transgenic rice plants exhibited decreased tolerance to cold stress as compared with wild-type rice plants. In contrast, RNAi-mediated OsATL38 knockdown transgenic progeny exhibited markedly increased tolerance to cold stress relative to that of wild-type plants, which indicated a negative role of OsATL38 in response to cold stress. Yeast two-hybrid, in vitro pull-down, and co-immunoprecipitation assays revealed that OsATL38 physically interacted with OsGF14d, a rice 14-3-3 protein. An in vivo target ubiquitination assay indicated that OsGF14d was mono-ubiquitinated by OsATL38. osgf14d knockout mutant plants were more sensitive to cold stress than wild-type rice plants, indicating that OsGF14d is a positive factor in the response to cold stress. These results provide evidence that the RING E3 Ub ligase OsATL38 negatively regulates the cold stress response in rice via mono-ubiquitination of OsGF14d 14-3-3 protein. [ABSTRACT FROM AUTHOR]

In this study, we investigated the RDE (real driving emissions) test with a diesel passenger vehicle and two RDE driving routes. We then experimentally analyzed the implications for fuel consumption of a 2.2L diesel-fueled vehicle. First, we found that the weighting factor, which is the product of the carbon dioxide average value from the MAW (moving average window) analysis, results in real-road fuel consumption measurement that are closer to the WLTC (worldwide harmonized light vehicles test cycles) measurement results. Therefore, we ignored the weighting factor of the WLTC and used a new weighting factor of distance instead. In order to determine the influence of each factor on the fuel efficiency, we analyzed the carbon-dioxide emission for the operation of additional equipment. And OBD values were used to develop correction equations to compensate for vehicle driving regions where GPS data was not measured in the test route. When using the weighting factor based on the WLTC standard, the weighting factor based on distance was introduced to calculate the fuel consumption in this study. [ABSTRACT FROM AUTHOR]

SUMMARY: Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H2O2 after ABA treatment. These findings indicated that JUL1 acts downstream of H2O2 and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H2O2, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress. Significance Statement: Ubiquitination is a universal post‐translational protein modification in eukaryotic organisms. We show that Arabidopsis RING E3 ubiquitin ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress. The present study demonstrates that the ubiquitin‐mediated microtubule structure in guard cell is functionally correlated with ABA‐dependent drought stress response in Arabidopsis. [ABSTRACT FROM AUTHOR]

Abstract The plant protein production system is a platform that can not only reduce production costs but also produce monoclonal antibodies that do not have the risk of residual proteins from the host. However, due to the difference between post-translational processes in plants and animals, there may be a modification in the Fab region of the monoclonal antibody produced in the plant; thus, it is necessary to compare the antigen affinity of this antibody with that of the prototype. In this study, ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody used for its non-cross resistance to rituximab, was expressed in Nicotiana benthamiana , and its affinities and efficacies were compared with those of native ofatumumab produced from CHO cells. Two forms of plant ofatumumab (with or without HDEL-tag) were generated and their production yields were compared. The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL-tag. The specificity of the target recognition of plant-derived ofatumumab was confirmed by mCherry-CD20-expressing HEK cells via immuno-staining, and the capping of CD20 after ofatumumab binding was also confirmed using Ramos B cells. In the functional equivalence tests, the binding affinities and complement-dependent cell cytotoxicity efficacy of plant-ofatumumab-HDEL and plant-ofatumumab without HDEL were significantly reduced compared to those of CHO-derived ofatumumab. Therefore, we suggest that although ofatumumab is not a good candidate as a template for plant-derived monoclonal antibodies because of its decreased affinity when produced in plants, it is an interesting target to study the differences between post-translational modifications in mammals and plants. Highlights • Ofatumumab, a fully human anti-CD20 IgG1κ monoclonal antibody, was successfully expressed in Nicotiana benthamiana leaves. • The HDEL-tagged ofatumumab was more expressed in plants than the form without HDEL tag. • Both plant-ofatumumabs retained the antigen binding specificities. • Both plant-ofatumumabs showed reduced binding affinities to the target and reduced Complement dependent cell death efficacies than CHO cell expressed ofatumumab. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Mycoplasma pneumoniae (M. pneumoniae) is a common causative agent of pneumonia in children. The aim of this study is to determine whether there is any difference in selected cytokine or chemokines response in asthmatic children compared to non-asthmatic children during acute M. pneumoniae pneumonia. Methods: Seventy-five children, 6–12years of age, admitted with M. pneumoniae pneumonia were enrolled. Two patient groups were defined: the children with known asthma (N =40) and non-asthmatic children (N =35). Interleukin (IL)-18 and selected chemokines, IL-8, CXCL9, CXCL10, and regulation upon activation normal T-cell expressed and secreted (RANTES) were measured by means of ELISA in the plasma samples of the patients collected on admission. We investigated the values of these mediators in relation to the asthma status and symptom severity of the patients. Twenty age-matched, non-infected controls were also studied. Results: Plasma levels of IL-18 and the chemokines increased significantly in the patients with M. pneumoniae pneumonia compared to non-infected, age-matched controls (P <0.01). However, the asthmatic patients showed significantly reduced IL-18 and CXCL10 responses (P <0.01, <0.05, respectively) and had more severe pneumonia symptoms (P <0.01) compared to non-asthmatic patients. IL-18 was significantly lower in severe pneumonia group than in non-severe group (P <0.05). Conclusions: Our study suggests that IL-18 and the chemokines are importantly involved in the pathogenesis of M. pneumoniae pneumonia. It also indicates that some asthmatic children have deficient IL-18 response when affected by M. pneumoniae pneumonia, which might be associated with more severe pneumonia observed in this group of patients. [Copyright &y& Elsevier]

Abstract: Homogentisate phytyltransferase (HPT) is an important enzyme in the biosynthesis of tocopherols (vitamin E). Herein, an HPT homolog (MdHPT1) was isolated from apple (Malus domestica Borkh. cv. Fuji) fruits, whose gene expression level gradually decreased during fruit ripening, reaching a background level in ripened apple fruits. The amounts of α- and γ-tocopherols, two major tocopherols in plant organs, were 5- to 14-fold lower in the fruits than in the leaves and flowers of apple plants. Transgenic tomato plants (Solanum lycopersicum cv. Micro-Tom) overexpressing MdHPT1 were next constructed. Transgenic independent T1 leaves contained ∼1.8- to 3.6-fold and ∼1.6- to 2.9-fold higher levels of α-tocopherol and γ-tocopherol, respectively, than those in control plants. In addition, the levels of α-tocopherol and γ-tocopherol in 35S:MdHPT1 T1 fruits increased up to 1.7-fold and 3.1-fold, respectively, as compared to the control fruits, indicating that an increase in α-tocopherol in fruits (maximal 1.7-fold) was less evident than that in leaves (maximal 3.6-fold). This finding suggests that the apple MdHPT1 plays a role in tocopherol production in transgenic tomatoes. [Copyright &y& Elsevier]