ABSTRACT
DNA damage response is a fundamental mechanism to maintain genome stability. The ATR-WEE1 kinase module plays a central role in response to replication stress. Although the ATR-WEE1 pathway has been well studied in yeasts and animals, how ATR-WEE1 functions in plants remains unclear. Through a genetic screen for suppressors of the Arabidopsis atr mutant, we found that loss of function of PRL1, a core subunit of the evolutionarily conserved MAC complex involved in alternative splicing, suppresses the hypersensitivity of atr and wee1 to replication stress. Biochemical studies revealed that WEE1 directly interacts with and phosphorylates PRL1 at Serine 145, which promotes PRL1 ubiquitination and subsequent degradation. In line with the genetic and biochemical data, replication stress induces intron retention of cell cycle genes including CYCD1;1 and CYCD3;1, which is abolished in wee1 but restored in wee1 prl1. Remarkably, co-expressing the coding sequences of CYCD1;1 and CYCD3;1 partially restores the root length and HU response in wee1 prl1. These data suggested that the ATR-WEE1 module inhibits the MAC complex to regulate replication stress responses. Our study discovered PRL1 or the MAC complex as a key downstream regulator of the ATR-WEE1 module and revealed a novel cell cycle control mechanism.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/physiology , Ataxia Telangiectasia Mutated Proteins/genetics , Cell Cycle Proteins/genetics , DNA Damage , DNA Replication , Genes, cdc , Mutation , Nuclear Proteins/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , RNA Splicing , Stress, Physiological , Suppression, Genetic , UbiquitinationABSTRACT
Leaf senescence is an intrinsic biological process of plants. The phytohormones salicylic acid (SA) and ethylene (ET) are known to promote senescence. However, their relationship in this process is still unclear. We found that EIN3 and EIL1, two key transcription factors in ET signaling, are required for SA-induced leaf senescence in Arabidopsis. Furthermore, ET enhances the effect of SA in promoting senescence. Biochemical studies revealed that NPR1, the master regulator of SA signaling, interacts with EIN3 to promote its transcriptional activity. Our study suggests that SA and ET function coordinately in senescence, which is in contrast to their antagonistic crosstalk in other biological processes.
Subject(s)
Arabidopsis Proteins/metabolism , Ethylenes/metabolism , Salicylic Acid/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Promoter Regions, Genetic/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
Aim: This study explored whether chemotherapy combined with palliative surgery and/or radiotherapy is a possible treatment for metastatic gastric cancer. Materials & methods: Patients were divided into groups according to treatments. COX models were used to explore prognostic factors. Kaplan-Meier models and log-rank tests were used to analyze outcomes. Outcomes were analyzed before and after propensity score matching. Results: Chemotherapy combined with gastrectomy or metastasectomy prolongs the survival time compared with chemotherapy alone (p < 0.05). Chemotherapy combined with gastrectomy plus metastasectomy and/or radiation therapy also prolongs the survival time (p < 0.05). Conclusion: Chemotherapy combined with gastrectomy could be a more effective treatment for metastatic gastric cancer. Chemotherapy combined with gastrectomy plus metastasectomy and/or radiation therapy could also be a promising treatment.
Subject(s)
Stomach Neoplasms/drug therapy , Stomach Neoplasms/radiotherapy , Stomach Neoplasms/surgery , Adult , Aged , Disease-Free Survival , Female , Gastrectomy/methods , Humans , Kaplan-Meier Estimate , Male , Metastasectomy , Middle Aged , Neoplasm Staging , Palliative Care/methods , Proportional Hazards Models , SEER Program , Stomach Neoplasms/secondary , Treatment OutcomeABSTRACT
CH3NH3PbBr3 perovskite nanocrystals (NCs) suffer from poor stability because of their high sensitivity to environmental moisture and water. To solve this problem, previous works mainly focus on embedding perovskite NCs into water-resistant matrix to form large composites (size of microns or larger). As an alternative solution without serious changing of NC size, enhancing the stability of perovskite NCs themselves by ligand engineering is rarely reported. In this work, we used hyperbranched polyamidoamine (PAMAM) dendrimers with two different generations (G0 and G4) to synthesize CH3NH3PbBr3 perovskite NCs with high photoluminescence (PL) quantum yields (QY) above 70% and a new record stability. A novel dendrimers generation-dependent stability of perovskite NCs was observed. The water-resistance time is 18 h (27 h) for perovskite NCs capped by G0 (G4) generation of PAMAM, which is 7 times (11 times) longer than that of traditional oleic acid-capped NCs. Similar PAMAM generation-related stability is also observed in moisture-resistance tests. The stability time against moisture is 500 h (800 h) for G0 (G4) generation of PAMAM-capped perovskite NCs, which is a new record stability time against moisture for CH3NH3PbBr3 perovskite NCs. In addition, our results also indicate that PAMAM ligands outside perovskite NCs can dramatically slow down the speed of halide exchange. Even for the mixture of perovskite NCs with two different halide composition, the original luminescence properties of PAMAM-capped perovskite NCs can retain after mixing. In view of slow halide exchange speed, excellent water and moisture stability, PAMAM dendrimers-capped perovskite NCs and their mixture are available as color conversion single layer in fabrication of light-emitting diodes (LED).
ABSTRACT
A series of novel C-3-OH substituted gibberellin derivatives bearing an amide group were designed and synthesized from the natural product gibberellic acid (GA3). Their activities on the plant growth regulation of rice and Arabidopsis were evaluated in vivo. Among these compounds, 10d and 10f exhibited appreciable inhibitory activities on rice (48.6% at 100 µmol/L) and Arabidopsis (41.4% at 100 µmol/L), respectively. These results provide new insights into the design and synthesis of potential plant growth regulators.
Subject(s)
Arabidopsis/growth & development , Gibberellins/pharmacology , Oryza/growth & development , Plant Growth Regulators/pharmacology , Arabidopsis/drug effects , Crystallography, X-Ray , Gibberellins/chemical synthesis , Oryza/drug effectsABSTRACT
The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.
Subject(s)
Amino Acid Substitution , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Drug Compounding , Immunoglobulin G/chemistry , Point Mutation/genetics , Amino Acid Sequence , Antibodies, Monoclonal/genetics , Calorimetry, Differential Scanning , Complementarity Determining Regions , Humans , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Immunoglobulin G/genetics , Osmolar Concentration , Solubility , ViscosityABSTRACT
INTRODUCTION: The aim of this study was to investigate risk factors for nutritional risk in older adults with gastrointestinal tumours. MATERIALS AND METHODS: A total of 170 eligible hospitalised older adults with gastrointestinal tumours were included. Their clinical characteristics were collected, their nutritional risk was screened by NRS 2002, and then patients were divided into a nutritional risk group and a non-nutritional risk group. The observation indicators included body mass index (BMI), muscle mass, muscle strength, and calf circumference. The third lumbar skeletal muscle index (L3 SMI) was calculated using abdominal computed tomography (CT) scan results, and grip strength/muscle strength, 6-m walking speed and calf circumference were measured. Sarcopenia was diagnosed according to the criteria of the Asian Sarcopenia Working Group (AWGS). Finally, we analysed the relationship between nutritional risk and sarcopenia and other related factors (BMI, calf circumference, L3 SMI, grip strength/muscle strength, 6-m walking speed) in older adults with gastrointestinal tumours by multivariate logistic regression analysis. RESULTS: Older adults with gastrointestinal tumours who were at nutritional risk accounted for 51.8% of patients in this study. The differences between sex, tumour stage, age, BMI, calf circumference, L3 SMI, grip strength/muscle strength, 6-m walking speed, and prevalence of sarcopenia were statistically significant in two groups (all P < 0.05). Multivariate logistic regression analysis showed that age, BMI, grip strength/muscle strength, and sarcopenia were risk factors of nutritional risk in older adults with gastrointestinal tumours (all P < 0.05). DISCUSSION: Older adults with gastrointestinal cancer had a higher proportion of nutritional risk, and L3 SMI, grip strength/muscle strength were independent risk factors for nutritional risk. In clinical practice, attention to nutritional risk screening and sarcopenia development in older adults with gastrointestinal cancer is warranted.
Subject(s)
Gastrointestinal Neoplasms , Sarcopenia , Humans , Aged , Sarcopenia/epidemiology , Sarcopenia/diagnosis , Muscle Strength , Muscle, Skeletal/diagnostic imaging , Hand Strength , Risk Factors , Gastrointestinal Neoplasms/complications , Gastrointestinal Neoplasms/epidemiologyABSTRACT
Osteoporotic vertebral fracture (OVF) is a risk factor for morbidity and mortality in elderly population, and accurate diagnosis is important for improving treatment outcomes. OVF diagnosis suffers from high misdiagnosis and underdiagnosis rates, as well as high workload. Deep learning methods applied to plain radiographs, a simple, fast, and inexpensive examination, might solve this problem. We developed and validated a deep-learning-based vertebral fracture diagnostic system using area loss ratio, which assisted a multitasking network to perform skeletal position detection and segmentation and identify and grade vertebral fractures. As the training set and internal validation set, we used 11,397 plain radiographs from six community centers in Shanghai. For the external validation set, 1276 participants were recruited from the outpatient clinic of the Shanghai Sixth People's Hospital (1276 plain radiographs). Radiologists performed all X-ray images and used the Genant semiquantitative tool for fracture diagnosis and grading as the ground truth data. Accuracy, sensitivity, specificity, positive predictive value, and negative predictive value were used to evaluate diagnostic performance. The AI_OVF_SH system demonstrated high accuracy and computational speed in skeletal position detection and segmentation. In the internal validation set, the accuracy, sensitivity, and specificity with the AI_OVF_SH model were 97.41%, 84.08%, and 97.25%, respectively, for all fractures. The sensitivity and specificity for moderate fractures were 88.55% and 99.74%, respectively, and for severe fractures, they were 92.30% and 99.92%. In the external validation set, the accuracy, sensitivity, and specificity for all fractures were 96.85%, 83.35%, and 94.70%, respectively. For moderate fractures, the sensitivity and specificity were 85.61% and 99.85%, respectively, and 93.46% and 99.92% for severe fractures. Therefore, the AI_OVF_SH system is an efficient tool to assist radiologists and clinicians to improve the diagnosing of vertebral fractures. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Osteoporotic Fractures , Spinal Fractures , Aged , Humans , Spinal Fractures/etiology , Artificial Intelligence , China , Osteoporotic Fractures/diagnostic imaging , Osteoporotic Fractures/complications , SpineABSTRACT
We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg²âº-dependent cleavage of the 5' leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5-RPP30 and RPP21-RPP29. Nuclear magnetic resonance studies of the Pyrococcus furiosus RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (ΔC(p)), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess ΔC(p). We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess ΔC(p). These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Protein Folding , Pyrococcus furiosus/enzymology , Ribonuclease P/chemistry , Ribonuclease P/metabolism , Thermodynamics , Amino Acid Substitution , Archaeal Proteins/genetics , Calorimetry , Databases, Protein , Hydrogen-Ion Concentration , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Osmolar Concentration , Point Mutation , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Pyrococcus furiosus/metabolism , Ribonuclease P/genetics , Temperature , TitrimetryABSTRACT
Lignans are widely present in traditional medicinal plants. Many natural arylnaphthalene lactone lignans (NALLs) isolated from the genera Justicia, Haplophyllum, and Phyllanthus possess interesting biological activities. Herein, we report a general strategy for the total synthesis of this kind of lignans. Features of this new approach are an aryl-alkyl Suzuki cross-coupling to introduce the dioxinone unit, a cation-induced cyclization to construct the aryl dihydronaphthalene, and base-mediated oxidative aromatization to furnish the arylnaphthalene core. By incorporating these key transformations, the total syntheses of justicidins B and E and taiwanin C covered type I and type II NALLs were accomplished.
ABSTRACT
PURPOSE: To compare ischemic core volume (ICV) and penumbra volume (PV) measured by MIStar, F-STROKE, and Syngo.via with that measured by RAPID in acute ischemic stroke (AIS), and their concordance in selecting patients for endovascular thrombectomy (EVT). METHODS: Computed tomography perfusion (CTP) data were processed with four software packages. Bland-Altman analysis and intraclass correlation coefficient (ICC) were performed to evaluate their agreement in quantifying ICV and PV. Kappa test was conducted to assess consistency in the selection of EVT candidates. The correlation between predicted ICV and segmented final infarct volume (FIV) on follow-up images was investigated. RESULTS: A total of 91 patients were retrospectively included. F-STROKE had the best consistency with RAPID (ICV: ICC = 0.97; PV: ICC = 0.84) and Syngo.via had the worst consistency (ICV: ICC = 0.77; PV: ICC = 0.66). F-STROKE had the narrowest limits of agreements both in ICV (-27.02, 24.40 mL) and PV (-85.59, 101.80 mL). When selecting EVT candidates, MIStar (kappa = 0.71-0.88) and F-STROKE (kappa = 0.84-0.90) had good to excellent consistency with RAPID, while Syngo.via had poor consistency (kappa = 0.20-0.41). ICV predicted by MIStar was correlated strongest with FIV (r = 0.77). CONCLUSIONS: F-STROKE is most consistent with RAPID in quantitative ICV and PV. F-STROKE and MIStar exhibit similar EVT candidate selection to RAPID. Syngo.via, for its part, seems to have overestimated ICV and underestimated PV, leading to an overly restrictive selection of EVT candidates.
ABSTRACT
Graphene is often used to improve interlaminar fracture toughness of carbon fiber/epoxy resin (CF/EP) composites. It is still a challenge to improve the toughness while maintaining the in-layer properties. In this study, 2D graphene oxide carbon fiber reinforced epoxy resin matrix (2D CF-GO/EP) composites were prepared by a vacuum infiltration hot-press forming experimental system (VIHPS), and three-point flexural and end notch flexural (ENF) tests were carried out. With the increase of the fiber mass fraction in the composites, the mode II interlaminar fracture toughness (GIIC) layers decrease gradually, and the bond property between the fiber and matrix interface layer becomes worse, because the accumulation of dense fiber bundles reduces the matrix penetration ability of cracks. However, the flexural properties increased first and then decreased, and reached the best flexural properties at 64.9%. When the fiber mass fraction is too high, the interlamellar bonding properties will decrease, and the fiber bundles will compress and affect each other. The delamination phenomenon will occur between the layers of the composites, which affects the overall bearing strength and stress limit of the composites. The results of the study show that the composites prepared by VIHPS have excellent mechanical properties, and the content of carbon fiber plays an important role in the influencing factors of the interlaminar and in-layer properties of composites.
ABSTRACT
PURPOSE: Esophageal cancer is one of the most common malignancies worldwide. Ubiquitin-dependent degradation of regulatory proteins reportedly plays a central role in diverse cellular processes. This study investigated the expression levels of ubiquitin in esophageal squamous cell carcinoma tissues and the functions of ubiquitin in the context of esophageal squamous cell carcinoma progression. METHODS: The expression of ubiquitin in esophageal squamous cell carcinoma and normal esophageal samples was determined via immunohistochemistry. Serum ubiquitin levels were determined by enzyme-linked immunosorbent assay. The association between serum ubiquitin level and clinicopathological factors was analyzed. Real-time PCR analysis was employed to measure the mRNA levels of the ubiquitin coding genes ubiquitin B and ubiquitin C. Proliferation assays, colony formation assays, and Transwell-based assays were used to determine the influence of ubiquitin on cell growth and cell invasion. Proteomic analysis was performed to identify the proteins associated with ubiquitin. RESULTS: Ubiquitin expression in esophageal squamous cell carcinoma tissues was markedly higher than that in normal and tumor adjacent tissues. The levels of ubiquitin in esophageal squamous cell carcinoma serum samples were significantly higher than those in healthy controls. Serum ubiquitin levels were correlated with tumor stage and lymph node metastasis. To silence the expression of ubiquitin, we knocked down the ubiquitin coding genes ubiquitin B and ubiquitin C in TE-1 and Eca-109 cells. Silencing ubiquitin resulted in the suppression of cell growth, chemoresistance, colony formation and cell migration in esophageal squamous cell carcinoma cells. Proteomic analysis in esophageal squamous cell carcinoma cells showed that knockdown of ubiquitin coding genes deregulated the expression of 159 proteins (92 were upregulated and 67 were downregulated) involved in multiple pathways. These proteins included ferritin light chain, ferritin heavy chain, cellular retinoic acid-binding protein 2, and DNA replication factor 1. CONCLUSION: Ubiquitin expression is upregulated in esophageal squamous cell carcinoma tissues and serum samples. Serum ubiquitin levels were correlated with tumor stage and lymph node metastasis. Downregulation of ubiquitin suppresses the aggressive phenotypes of esophageal squamous cell carcinoma cells by complex mechanisms; ubiquitin may represent a novel target for the treatment of esophageal squamous cell carcinoma.
Subject(s)
Esophageal Squamous Cell Carcinoma/metabolism , Esophageal Squamous Cell Carcinoma/pathology , Ubiquitin/metabolism , Aged , Aged, 80 and over , Cell Line, Tumor , Cell Movement , Cell Proliferation/genetics , Chromatography, Liquid , Disease Progression , Down-Regulation , Esophageal Squamous Cell Carcinoma/diagnostic imaging , Esophageal Squamous Cell Carcinoma/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , Middle Aged , Phenotype , Proteome , Proteomics/methods , Tandem Mass Spectrometry , Tomography, X-Ray Computed , Ubiquitin/genetics , UltrasonographyABSTRACT
RNase P is the ubiquitous ribonucleoprotein metalloenzyme responsible for cleaving the 5'-leader sequence of precursor tRNAs during their maturation. While the RNA subunit is catalytically active on its own at high monovalent and divalent ion concentrations, four protein subunits are associated with archaeal RNase P activity in vivo: RPP21, RPP29, RPP30, and POP5. These proteins have been shown to function in pairs: RPP21-RPP29 and POP5-RPP30. We have determined the solution structure of RPP21 from the hyperthermophilic archaeon Pyrococcus furiosus ( Pfu) using conventional and paramagnetic NMR techniques. Pfu RPP21 in solution consists of an unstructured N-terminus, two alpha-helices, a zinc binding motif, and an unstructured C-terminus. Moreover, we have used chemical shift perturbations to characterize the interaction of RPP21 with RPP29. The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step toward understanding structure-function relationships of the archaeal RNase P holoenzyme.
Subject(s)
Archaeal Proteins/chemistry , Pyrococcus furiosus/enzymology , Ribonuclease P/chemistry , Amino Acid Sequence , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Ribonuclease P/genetics , Ribonuclease P/metabolism , Sequence Homology, Amino AcidABSTRACT
Synthesis of the 6-deoxy-talose (6-dTal) containing tetrasaccharide, naturally found in Franconibacter helveticus LMG23732T, has been described. The synthetic method utilized an allyloxyethylidene group for protecting the 1-OH and 2-OH groups of rhamnopyranose and a redox reaction for synthesizing 6-deoxy talose, which eventually formed a disaccharide containing α-Glcp-(1â2)-6dTalp configured glycosidic bonds using a [2 + 2] synthetic strategy.
Subject(s)
Enterobacteriaceae/chemistry , Lipopolysaccharides/chemistry , Polysaccharides, Bacterial/chemistryABSTRACT
Aeromonas bestiarum 207 is a bacterial pathogen with severe impact on aquaculture. In a recent study, the structure of OPS antigens from Aeromonas bestiarum was identified as pentasaccharide repeating units. Synthesis of the pentasaccharide repeating unit and its derivative are reported. Stereo- and regio-specific synthesis was achieved under Schmidt glycosylation conditions employing appropriately protected L-rhamopyranosyl and D-glucopyranosylamine building blocks. The pentasaccharide synthesis was achieved using a [3 + 2] strategy with an overall yield of 5.2% through 11 linear steps from the monosaccharide building blocks 10 and 14.
Subject(s)
Aeromonas/chemistry , O Antigens/chemistry , Polysaccharides, Bacterial/chemistry , Carbohydrate Sequence , Glycosylation , Magnetic Resonance SpectroscopyABSTRACT
The class of Omega glutathione transferases is newly identified with novel structural and functional characteristics. Human GSTO 1-1 (glutathione S-transferase Omega 1) is the first member of the GST Omega class. It was found to play a role in apoptosis and be in association with age-at-onset of AD and PD. In order to improve the understanding of the properties of other Omega class members, we screened a human fetal brain cDNA library and obtained the human GSTO2 (glutathione S-transferase Omega 2) cDNA. The full-length cDNA of human GSTO2 is 1179 bp long and encodes a protein of 243 amino acid residues. Expression pattern analysis revealed that GSTO2 was ubiquitously expressed at a low level, with a higher expression in pancreas and prostate. Enzyme assays showed that GSTO2 protein had activities similar to Omega class GSTs. It has detectable glutathione-dependent thiol transferase activity and glutathione-dependent dehydroascorbate reductase activity. But different from GSTO1-1, GSTO2 exhibits a high catalytic activity with CDNB. Subcellular localization analysis of GSTO2-EGFP fusion protein revealed that GSTO2 distributed to cytoplasm of COS-7 cells and both cytoplasm and nucleus of L-02, QGY-7703 and SMMC-7721 cells. Overexpression of GSTO2 induced apoptosis of L-02 cells detected by Annexin V-PE staining. The results suggest that GSTO2 may play an important role in cellular signaling.
Subject(s)
Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Amino Acid Sequence , Animals , Apoptosis , Base Sequence , Cell Line , Cell Survival , Chlorocebus aethiops , Cloning, Molecular , Dinitrochlorobenzene/pharmacology , Gene Expression , Gene Expression Profiling , Glutathione/metabolism , Glutathione Transferase/chemistry , Glutathione Transferase/isolation & purification , Humans , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , TemperatureABSTRACT
Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering CH3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.
Subject(s)
Antibodies, Bispecific/biosynthesis , Gene Expression , Single-Chain Antibodies/biosynthesis , Animals , Antibodies, Bispecific/genetics , CHO Cells , Cell-Free System/metabolism , Cricetinae , Cricetulus , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Single-Chain Antibodies/geneticsABSTRACT
Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.