Identification of some major cytokinins in Phaseolus vulgaris and their distribution

The determination of the various endogenous cylokinins and their distribution among organs is important in understanding their role in growth and development in the intact plant. Cytokinins in young plants of Phaseolus vulgaris were purified by immunoaffinity chromatography and HPLC, and characterised by UV spectra. Zeatin nucleotide (zeatin riboside-5'-monophosphate) and isopentenyladenine nucleotide (isopentenyladenosne-5'-monopnosphate) were the most abundant cytokinins in all organs. Their identities were confirmed by GC-MS. The levels of zeatin, zeatin riboside, isopentenyladenine and isopentenyladenosine never exceeded 5% of the nucleotides, as assessed by a methodology that preserves cytokinin nucleotides. Three extraction methods were compared with qualitatively similar results, though differing in their suppression of nucleotidase activity. Cytokinin nucleotide levels were greater in the stems and petioles than in the roots and leaves on a per gram fresh weight basis, and were greater in the stems than in the other organs on a per plant basis. Levels of the zeatin and isopentenyladenine nucleotides were about equal in the stems and leaves, but in the petioles the zeatin nucleotide levels were about twice the level of isopentenyladenine nucleotide, while in the roots they were about half the isopentenyladenine nucleotide level. The importance of considering the cytokinin form is emphasised.     

Antimicrobial activity of lipoprotein particles containing apolipoprotein Al

Human plasmain vitro inhibits the growth of coagulase negative staphylococci,S. epidermidis, which may be pathogenic in the immunocompromised host. To determine the antimicrobial components, serum was fractionated by column chromatography, which revealed that elution areas where lipoproteins can be yielded had high antimicrobial activity againstS. epidermidis. Therefore, lipoprotein fractions, including very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL), were separated by ultracentrifugation and incubated withS. epidermidis. All 3 lipoprotein fractions suppressed bacterial growth within the first 3 h but VLDL enhanced bacterial growth after 9 h of incubation compared with the control. HDL, however, inhibited bacterial growth throughout 21 h of incubation.To confirm these results, serum from healthy volunteers was separated by ion exchange column chromatography and again by HPLC to purify the antimicrobial fraction. In the protein analysis with gradient polyacrylamide-SDS gel, apolipoprotein Al (apo Al), which is a major apolipoprotein of HDL, was detected in the antimicrobial fraction. Therefore, this fraction was loaded onto an immunoaffinity column coupled with the anti-apo Al monoclonal antibody (Mab). Unbound fraction had no antimicrobial activity, but anti-S. epidermidis activity was recovered from the bound fraction which consisted mainly of apo Al, All and apo C in protein composition.These results indicated that the antimicrobial activity was associated with the apo Al-containing lipoprotein particles (HDL). This property of HDL may directly affect bacterial growth and promote the self-defense mechanisms of normal and immunocompromised individuals.     

Effect of matrices on affinity purification of protein C

One of the critical problems in scale-up of affinity chromatography is the mechanical strength of the support matrix against pressure. Because the costs of both the gel matrix and the ligand for the affinity chromatography are very high, the reusability of gel matrices is directly related to the total production cost. In certain cases, where the source material is viscous (e.g., blood plasma), irreversible deformation of gel matrices can readily occur, necessitating severe constraints in the flow rate. Consequently, productivity is low.We have characterized the system parameters and investigated the performance of various matrices that are commercially available. The experimental system used for this study was the immunoaffinity purification of protein C (an anticoagulant protein) from human blood plasma. The support matrices studied were cross-linked agarose, polymethyl acrylic, cellulose, and polyvinyl alcohol polymers. The major system parameters studied were pressure tolerance, coupling efficiency, adsorption efficiency, and batch adsorption/desorption kinetics of protein C to/from the monoclonal antibody (MAb)-Matrix complex. In addition, the apparent equilibrium constant and bandwidth of the product concentration profile in the eluate were characterized by performing pulse tests.A methodology was developed for evaluating the immunoaffinity colum performance for the separation of protein C. By utilizing the experimentally measured parameters, the flow rate limitation for each purification step was computed. Then, the purification performance of the matrices were evaluated in terms of productivity per unit time. Among the matrices tested, cellulose was superior in overall performance for the immunoaffinity purification of protein C using a 10 cm x 10 cm column.     

舟山眼镜蛇毒神经生长因子的亲和层析分离

用层析和制备SDS-PAGE法纯化舟山眼镜蛇(Najanajaatra Cantor)毒神经生长因子(NGF),免疫家兔获得抗血清。用辛酸-硫酸铵沉淀法初步纯化IgG,蛋白A-Sepharose亲和层析进一步纯化IgG,并与CNBr活化的Sepharose4B偶联,采用亲和层析法对舟山眼镜蛇毒神经生长因子进行分离纯化。产物经过SDS-PAGE检测呈一条带,并显示了良好的生物学活性。纯化NGF最大比活性为5.0×104U/mg蛋白,亲和常数为4.35×108L/mol,亲和层析分离NGF得率比传统分离方法得率提高35.2%,该方法为NGF的大量提取提供了技术支持。     

Phosphoproteome Profiling of the Receptor Tyrosine Kinase MuSK Identifies Tyrosine Phosphorylation of Rab GTPases

Muscle-specific receptor tyrosine kinase (MuSK) agonist antibodies were developed 2 decades ago to explore the benefits of receptor activation at the neuromuscular junction. Unlike agrin, the endogenous agonist of MuSK, agonist antibodies function independently of its coreceptor low-density lipoprotein receptor–related protein 4 to delay the onset of muscle denervation in mouse models of ALS. Here, we performed dose–response and time-course experiments on myotubes to systematically compare site-specific phosphorylation downstream of each agonist. Remarkably, both agonists elicited similar intracellular responses at known and newly identified MuSK signaling components. Among these was inducible tyrosine phosphorylation of multiple Rab GTPases that was blocked by MuSK inhibition. Importantly, mutation of this site in Rab10 disrupts association with its effector proteins, molecule interacting with CasL 1/3. Together, these data provide in-depth characterization of MuSK signaling, describe two novel MuSK inhibitors, and expose phosphorylation of Rab GTPases downstream of receptor tyrosine kinase activation in myotubes.     

A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

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Highlights

• •Liver Mallory-Denk-Body inducers elicited an IκBα-loss and NF-κB-activation.
• •IκBα-loss was due to its sequestration into insoluble cytoplasmic aggregates.
• •Four proteomic approaches identified 10 IκBα-interacting/aggregating proteins.
• •Nup153/RanBP2-aggregation prevented IκBα nuclear entry for ending NF-κB-activation.

     

A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high‐resolution mass spectrometry, a total of 147 symmetric dimethyl‐arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH‐type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography

Abstract

The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400?SEC, we obtained highly pure, i.e.,?>?90%, rHBsAg virus-like particles (VLP).