O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei

1987 ◽  
Vol 33 (8) ◽  
pp. 698-703 ◽  
Author(s):  
C. P. Kubicek ◽  
T. Panda ◽  
G. Schreferl-kunar ◽  
F. Gruber ◽  
R. Messner
Keyword(s):  
Trichoderma Reesei ◽  
Protein Glycosylation ◽  
The Other ◽  
Detectable Effect ◽  
Secreted Protein ◽  
Intracellular Protein ◽  
Molecular Weights ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses

The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.

scholarly journals Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays)

1996 ◽  
Vol 315 (3) ◽  
pp. 965-970 ◽  
Author(s):  
Philip S. SHELDON ◽  
Michael A. VENIS
Keyword(s):  
Cyclosporin A ◽  
Sequence Similarity ◽  
Terminal Sequence ◽  
Purification And Characterization ◽  
Cyclophilin B ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses ◽  
High Level

Methods for the purification and separation of peptidyl prolyl cis–trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appear as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).

scholarly journals Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Emiko Kinoshita-Kikuta ◽  
Toshihiko Utsumi ◽  
Aya Miyazaki ◽  
Chiharu Tokumoto ◽  
Kyosuke Doi ◽  
...  
Keyword(s):  
Casein Kinase ◽  
Src Family Kinases ◽  
The Other ◽  
Hek293 Cells ◽  
Intracellular Protein ◽  
Protein Traffic ◽  
Phosphorylation Level ◽  
Sds Page ◽  
Membrane Phosphorylation ◽  
Palmitoylation Site

Abstract Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.

2D Color-Intensity Representation of Ultrasonic Thickness Gauge Measurements

2020 ◽  
pp. 1192-1198
Author(s):  
M.S. Mohammad ◽  
Tibebe Tesfaye ◽  
Kim Ki-Seong
Keyword(s):  
Cost Effective ◽  
The Other ◽  
Thickness Gauge ◽  
Color Intensity ◽  
Digital Readout ◽  
Flat Surfaces ◽  
Video Cameras ◽  
Wide Range ◽  
Cost Effective Alternative ◽  
Ultrasonic Thickness

Ultrasonic thickness gauges are easy to operate and reliable, and can be used to measure a wide range of thicknesses and inspect all engineering materials. Supplementing the simple ultrasonic thickness gauges that present results in either a digital readout or as an A-scan with systems that enable correlating the measured values to their positions on the inspected surface to produce a two-dimensional (2D) thickness representation can extend their benefits and provide a cost-effective alternative to expensive advanced C-scan machines. In previous work, the authors introduced a system for the positioning and mapping of the values measured by the ultrasonic thickness gauges and flaw detectors (Tesfaye et al. 2019). The system is an alternative to the systems that use mechanical scanners, encoders, and sophisticated UT machines. It used a camera to record the probe’s movement and a projected laser grid obtained by a laser pattern generator to locate the probe on the inspected surface. In this paper, a novel system is proposed to be applied to flat surfaces, in addition to overcoming the other limitations posed due to the use of the laser projection. The proposed system uses two video cameras, one to monitor the probe’s movement on the inspected surface and the other to capture the corresponding digital readout of the thickness gauge. The acquired images of the probe’s position and thickness gauge readout are processed to plot the measured data in a 2D color-coded map. The system is meant to be simpler and more effective than the previous development.

p-Sulfonic acid calix[n]arene catalyzed synthesis of bioactive heterocycles: A review

2020 ◽  
Vol 24 ◽  
Author(s):  
Bubun Banerjee ◽  
Gurpreet Kaur ◽  
Navdeep Kaur
Keyword(s):  
Supramolecular Chemistry ◽  
Sulfonic Acid ◽  
Catalytic Efficiency ◽  
The Other ◽  
Reaction Conditions ◽  
Metal Free ◽  
Bioactive Scaffolds ◽  
Wide Range ◽  
Efficient Alternative ◽  
Bioactive Heterocycles

: Metal-free organocatalysts are becoming an important tool for the sustainable developments of various bioactive heterocycles. On the other hand, during last two decades, calix[n]arenes have been gaining considerable attention due to their wide range of applicability in the field of supramolecular chemistry. Recently, sulfonic acid functionalized calix[n] arenes are being employed as an efficient alternative catalyst for the synthesis of various bioactive scaffolds. In this review we have summarized the catalytic efficiency of p-sulfonic acid calix[n]arenes for the synthesis of diverse biologically promising scaffolds under various reaction conditions. There is no such review available in the literature showing the catalytic applicability of p-sulfonic acid calix[n]arenes. Therefore, we strongly believe that this review will surely attract those researchers who are interested about this fascinating organocatalyst.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1244
Author(s):  
Malik Adil Nawaz ◽  
Tanoj Kumar Singh ◽  
Regine Stockmann ◽  
Hema Jegasothy ◽  
Roman Buckow
Keyword(s):  
Physicochemical Properties ◽  
Faba Bean ◽  
Protein Concentration ◽  
High Concentration ◽  
Molecular Weights ◽  
Oil In Water ◽  
Sds Page ◽  
Lower Protein ◽  
7S Globulin ◽  
Bean Protein

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~β-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5–8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5–6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7–8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

scholarly journals Density Guarantee on Finding Multiple Subgraphs and Subtensors

2021 ◽  
Vol 15 (5) ◽  
pp. 1-32
Author(s):  
Quang-huy Duong ◽  
Heri Ramampiaro ◽  
Kjetil Nørvåg ◽  
Thu-lan Dam
Keyword(s):  
Lower Bound ◽  
State Of The Art ◽  
The State ◽  
The Other ◽  
Exact Methods ◽  
Practical Solution ◽  
Novel Approach ◽  
Wide Range ◽  
Real World Datasets ◽  
Tensor Data

Dense subregion (subgraph & subtensor) detection is a well-studied area, with a wide range of applications, and numerous efficient approaches and algorithms have been proposed. Approximation approaches are commonly used for detecting dense subregions due to the complexity of the exact methods. Existing algorithms are generally efficient for dense subtensor and subgraph detection, and can perform well in many applications. However, most of the existing works utilize the state-or-the-art greedy 2-approximation algorithm to capably provide solutions with a loose theoretical density guarantee. The main drawback of most of these algorithms is that they can estimate only one subtensor, or subgraph, at a time, with a low guarantee on its density. While some methods can, on the other hand, estimate multiple subtensors, they can give a guarantee on the density with respect to the input tensor for the first estimated subsensor only. We address these drawbacks by providing both theoretical and practical solution for estimating multiple dense subtensors in tensor data and giving a higher lower bound of the density. In particular, we guarantee and prove a higher bound of the lower-bound density of the estimated subgraph and subtensors. We also propose a novel approach to show that there are multiple dense subtensors with a guarantee on its density that is greater than the lower bound used in the state-of-the-art algorithms. We evaluate our approach with extensive experiments on several real-world datasets, which demonstrates its efficiency and feasibility.

scholarly journals Recent Advancements in Polymer/Liposome Assembly for Drug Delivery: From Surface Modifications to Hybrid Vesicles

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1027
Author(s):  
Vincenzo De Leo ◽  
Francesco Milano ◽  
Angela Agostiano ◽  
Lucia Catucci
Keyword(s):  
Drug Delivery ◽  
First Generation ◽  
Phospholipid Bilayer ◽  
Bioactive Molecules ◽  
Systemic Delivery ◽  
Molecular Weights ◽  
Liposome Preparation ◽  
Wide Range ◽  
Liposome Surface

Liposomes are consolidated and attractive biomimetic nanocarriers widely used in the field of drug delivery. The structural versatility of liposomes has been exploited for the development of various carriers for the topical or systemic delivery of drugs and bioactive molecules, with the possibility of increasing their bioavailability and stability, and modulating and directing their release, while limiting the side effects at the same time. Nevertheless, first-generation vesicles suffer from some limitations including physical instability, short in vivo circulation lifetime, reduced payload, uncontrolled release properties, and low targeting abilities. Therefore, liposome preparation technology soon took advantage of the possibility of improving vesicle performance using both natural and synthetic polymers. Polymers can easily be synthesized in a controlled manner over a wide range of molecular weights and in a low dispersity range. Their properties are widely tunable and therefore allow the low chemical versatility typical of lipids to be overcome. Moreover, depending on their structure, polymers can be used to create a simple covering on the liposome surface or to intercalate in the phospholipid bilayer to give rise to real hybrid structures. This review illustrates the main strategies implemented in the field of polymer/liposome assembly for drug delivery, with a look at the most recent publications without neglecting basic concepts for a simple and complete understanding by the reader.

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