Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts
Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood.
Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems’ antenna complexes.
Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes.
In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.
Outer membrane compositions enhance the rate of extracellular electron transport via cell-surface MtrC protein in Shewanella oneidensis MR-1
While cell membrane composition is critical for the function of membrane proteins, membrane modification has not been used to control the rate of extracellular electron transfer (EET) via the outer membrane protein complexes. Here, the rate of electron flow via the cell-surface redox protein, MtrC, was highly enhanced upon change in the outer membrane composition in Shewanella oneidensis MR-1.
The MR-1 strain was pre-cultured at 4 °C and 30 °C to initiate differentiation of membrane composition. The whole-cell difference electrochemical assay of wild-type and mutant strains lacking MtrC suggested that the rate of EET via MtrC increased approximately 18 times at 4 °C than 30 °C.
Circular dichroism spectroscopy showed that the molar exciton coupling coefficient for inter-heme interaction in MtrC increased in MR-1 at 4 °C than 30 °C. Results suggest that membrane modification may be a novel strategy for improving the efficiency of EET-based technologies.
Leucine-rich alpha-2 glycoprotein (LRG): A novel acute phase protein expressed in stage 3 grade C periodontitis before and after periodontal therapy
Background: Leucine-rich alpha-2 glycoprotein (LRG) is a novel acute phase protein involved in inflammation-associated diseases and that considered to be induced by multiple proinflammatory cytokines.
This study aimed to investigate gingival crevicular fluid (GCF) and serum levels of LRG, interleukin (IL)-6 and tumor necrosis factor (TNF)-α in patients with stage 3 periodontitis before and after non-surgical periodontal treatment.
Methods: Twenty-five stage 3 periodontitis and twenty-five periodontally healthy individuals were enrolled in the study. Clinical periodontal measurements were recorded; periodontitis patients received non-surgical periodontal treatment, and GCF and serum samples were obtained at baseline and at 6 weeks after treatment. LRG, IL-6 and TNF-α were determined by ELISA.
Results: GCF and serum LRG, IL-6 and TNF-α were significantly higher in periodontitis group than healthy controls (P < 0.001). A significant decrease in GCF and serum LRG, IL-6 and TNF-α was detected after periodontal treatment compared to baseline values of periodontitis patients (P < 0.001).
Conclusion: Our findings revealed that LRG expression was increased in stage 3 periodontitis both locally and systemically, and non-surgical periodontal therapy was effective in reducing LRG levels in GCF and serum of these patients. This article is protected by copyright. All rights reserved.
Aqueous Two-Phase-Assisted Precipitation of Proteins: A Platform for Isolation of Process-Related Impurities from Therapeutic Proteins
Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components.
Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Tyrosyl DNA Phosphodiesterase 1 (TDP1) in serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.
Human Tyrosyl DNA Phosphodiesterase 1 (TDP1) ELISA Kit
Description: This is Double-antibody Sandwich Enzyme-linked immunosorbent assay for detection of Human Tyrosyl DNA Phosphodiesterase 1 (TDP1) in serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids.
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In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids.
This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250-500 to 10-15 ppm) and of nucleic acids (from 15-20 to 5-15 ng/mL) were observed.
Affinity Tags in Protein Purification and Peptide Enrichment: An Overview
The reversible interaction between an affinity ligand and a complementary receptor has been widely explored in purification systems for several biomolecules. The development of tailored affinity ligands highly specific toward particular target biomolecules is one of the options in affinity purification systems.
However, both genetic and chemical modifications in proteins and peptides widen the application of affinity ligand-tag receptors pairs toward universal capture and purification strategies.
In particular, this chapter will focus on two case studies highly relevant for biotechnology and biomedical areas, namely the affinity tags and receptors employed on the production of recombinant fusion proteins, and the chemical modification of phosphate groups on proteins and peptides and the subsequent specific capture and enrichment, a mandatory step before further proteomic analysis.
Synthetic Ligand Affinity Chromatography Purification of Human Serum Albumin and Related Fusion Proteins
Synthetic ligand affinity adsorbents offer an efficient means for purification of biopharmaceuticals. Single-isomer textile dye C.I. Reactive Blue and newer ligands developed by rational design and screening of chemical combinatorial libraries based on a triazine scaffold are routinely used for the capture and purification of these proteins from engineered recombinant expression systems.
Here, we describe methods for the purification of recombinant human serum albumin and related fusion proteins using synthetic ligand affinity adsorbents.
Redox homeostasis and cell cycle activation mediate beta-cell mass expansion in aged, diabetes-prone mice under metabolic stress conditions: Role of thioredoxin-interacting protein (TXNIP)
Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated.
Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age.
Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion.
Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation.
These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice.
