Suppression of the ER-localized AAA ATPase NgCDC48 inhibits tobacco growth and development

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57–83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.     

Caffeic acid phenethyl ester accumulates β-catenin through GSK-3β and participates in proliferation through mTOR in C2C12 cells

AimThe aim of this study is to characterize the roles of caffeic acid phenethyl ester (CAPE) in the skeletal muscle cells.Main methodsWe performed immunoblotting assay using various phosphorylation specific antibodies.Key findingsWe found that CAPE induces rapid and transient phosphorylation of glycogen synthase kinase (GSK)-3β in a phosphoinositide 3-kinase (PI3K)-dependent manner. CAPE also decreases phosphorylation of β-catenin, ultimately leading to β-catenin accumulation. In addition, we demonstrated that CAPE activated the mammalian target of rapamycin (mTOR)-p70 S6 ribosomal kinase (S6K) and also stimulated extracellular signal-regulated kinase (ERK). The inhibition of mTOR blocked CAPE-induced ERK phosphorylation.SignificanceOur results suggest that CAPE may act through β-catenin accumulation via stimulation of GSK-3β and may also participate in cellular proliferation through the mTOR–ERK pathway.     

Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Generation of red fluorescent protein transgenic dogs

Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene‐expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP‐fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009. © 2009 Wiley‐Liss, Inc.     

AtDSEL, an Arabidopsis cytosolic DAD1-like acylhydrolase, is involved in negative regulation of storage oil mobilization during seedling establishment

Mobilization of seed storage reserves is essential for seed germination and seedling establishment. Here, we report that AtDSEL, an Arabidopsis thalianaDAD1-like Seedling Establishment-related Lipase, is involved in the mobilization of storage oils for early seedling establishment. AtDSEL is a cytosolic member of the DAD1-like acylhydrolase family encoded by At4g18550. Bacterially expressed AtDSEL preferentially hydrolyzed 1,3-diacylglycerol and 1-monoacylglycerol, suggesting that AtDSEL is an sn-1-specific lipase. AtDSEL-overexpressing transgenic Arabidopsis plants (35S:AtDSEL) were defective in post-germinative seedling growth in medium without an exogenous carbon source. This phenotype was rescued by the addition of sucrose to the growth medium. In contrast, loss-of-function mutant plants (atdsel-1 and atdsel-2) had a mildly fast-growing phenotype regardless of the presence of an exogenous carbon source. Electron microscopy revealed that 5-day-old 35S:AtDSEL cotyledons retained numerous peroxisomes and oil bodies, which were exhausted in wild-type and mutant cotyledons. The impaired seedling establishment of 35S:AtDSEL was not rescued by the addition of an exogenous fatty acid source, and 35S:AtDSEL seedling growth was insensitive to 2,4-dichlorophenoxybutyric acid, indicating that β-oxidation was blocked in AtDSEL-overexpressers. These results suggest that AtDSEL is involved in the negative regulation of seedling establishment by inhibiting the breakdown of storage oils.     

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.     

Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11?±?0.004 h^?1 and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.     

Overexpression of CaDSR6 increases tolerance to drought and salt stresses in transgenic Arabidopsis plants

The partial CaDSR6 (Capsicum annuum Drought Stress Responsive 6) cDNA was previously identified as a drought-induced gene in hot pepper root tissues. However, the cellular role of CaDSR6 with regard to drought stress tolerance was unknown. In this report, full-length CaDSR6 cDNA was isolated. The deduced CaDSR6 protein was composed of 234 amino acids and contained an approximately 30 amino acid-long Asp-rich domain in its central region. This Asp-rich domain was highly conserved in all plant DSR6 homologs identified and shared a sequence identity with the N-terminal regions of yeast p23^fyp and human hTCTP, which contain Rab protein binding sites. Transgenic Arabidopsis plants overexpressing CaDSR6 (35S:CaDSR6-sGFP) were tolerant to high salinity, as identified by more vigorous root growth and higher levels of total chlorophyll than wild type plants. CaDSR6-overexpressors were also more tolerant to drought stress compared to wild type plants. The 35S:CaDSR6-sGFP leaves retained their water content and chlorophyll more efficiently than wild type leaves in response to dehydration stress. The expression of drought-induced marker genes, such as RD20, RD22, RD26, RD29A, RD29B, RAB18, KIN2, ABF3, and ABI5, was markedly increased in CaDSR6-overexpressing plants relative to wild type plants under both normal and drought conditions. These results suggest that overexpression of CaDSR6 is associated with increased levels of stress-induced genes, which, in turn, conferred a drought tolerant phenotype in transgenic Arabidopsis plants. Overall, our data suggest that CaDSR6 plays a positive role in the response to drought and salt stresses.     

Translational Validation of Personalized Treatment Strategy Based on Genetic Characteristics of Glioblastoma

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient''s surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.     

Engineering an aglycosylated Fc variant for enhanced FcγRI engagement and pH-dependent human FcRn binding

The clinical use of therapeutic antibodies has increased sharply because of their many advantages over conventional small molecule drugs, particularly with respect to their affinity, specificity, and serum stability. Tumor or infected cells are removed by the binding of antibody Fc regions to Fc gamma receptors (FcγRs), which stimulate the activation of immune effector cells. Aglycosylated full-length IgG antibodies expressed in bacteria have different Fc conformations compared to their glycosylated counterparts produced in mammalian cells. As a result, they are unable to bind FcγRs, resulting in little to no activation of immune effector cells. In this study, we created a combinatorial library randomized at the upper CH2 loops of an aglycosylated Fc variant (Fc5: E382V/M428) and used a high-throughput flow cytometry library screening method, combined with bacterial display of homodimeric Fc domains for enhanced FcγR binding affinity. The trastuzumab Fc variant containing the identified mutations (Q295R, L328W, A330V, P331A, I332Y, E382V, M428I) not only exhibited over 120 fold higher affinity of specific binding to FcγRI than wild type aglycosylated Fc, but also retained pH-dependent FcRn binding. These results show that an aglycosylated antibody expressed in bacteria can be evolved for novel FcγR affinity and specificity.