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We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojidmycin and A'-methyl-1-deoxynojidmycin [Hughes, R, C, Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544], Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [³H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean α-mannosidase. The data show that Glc3Man8GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2, Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosacchaddes to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases. [ABSTRACT FROM AUTHOR]

Five major sialyloligosaccharides and a sialylglycopeptide have been isolated from normal human urine by charcoal adsorption, gel filtration, ion-exchange chromatography, and paper chromatography. Structural studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, glycosidase treatments, and CrO3 oxidation indicated the Following structures for the compounds: 1, NcuAc(α2- 6)Gal(β1–4)GIe: 2. NeuAc(α2–6)Gal(β1–4)GIcNAc; 3. NeuAc(α2–3)Gal(β1–4)GIc; 4. NeuAc(α2–3)Gal(β1–4) GIcNAc: 5. NeuAc(α2–3)Gal(β1–3) [NeuAc(α2–6)]GalNAc; and 6. NeuAc(α2–3)Gal(β1–3) [NeuAc(α2–6)]GallNAc (α1-O)Ser. Compounds 4, 5, and 6 have not been described in a free form before. The presence of compound 5 in urine may suggest that it derives from glycoproteins through a catabolic pathway involving cleavage of the carbohydrate-peptide linkage by an endo-N-acetylgalactosaminidase. The predominating sialyloligosaccharides in urine were compounds 3 and 4. The predominance of the compounds with the sialyl(α2–3) linkage is of interest in view of the recent discovery of uropathogenic Escherchia coli strains with binding specificity for sialyl(α2–3)galactosides. [ABSTRACT FROM AUTHOR]

In this paper, a series of novel trans‐1, 4‐polyisoprene/isotactic polybutene (TPI/iPB) in‐reactor blends were synthesized by isoprene and butene sequential two‐stage polymerization technology with spherical TiCl4/MgCl2 type Ziegler‐Natta catalyst. The components, structures, and properties of the as‐obtained TPI/iPB reactor blends were characterized by gel permeation chromatography, Fourier transform Infrared spectroscopy, and differential scanning calorimetry. The active trans‐1, 4‐polyisoprene (TPI) particles obtained in the initial isoprene polymerization by the Z‐N catalyst can be acted as microreactors to initiate butene polymerization subsequently. The TPI/iPB reactor blends with varied components were in situ synthesized within the reactor. The preparative‐temperature rising elution fractionation (p‐TREF) technique was used to fractionate the TPI/iPB reactor blends based on the elution temperature ranged from −40°C to 90°C. The weight distribution and microstructure of each fraction were investigated. The reactor blends are composed of crystallizable high trans‐1, 4‐uint polyisoprene obtained from the first‐stage isoprene polymerization, high isotactic polybutene obtained from the second‐stage butene polymerization and TPI‐b‐iPB block copolymer with different sequence structure obtained from the initial time of the second stage. This work is expected to propose the possible polymerizations of a‐olefins and conjugated dienes by using heterogeneous Ziegler‐Natta catalyst and provide a kind of novel rubber/plastic reactor blend materials. Highlights: The first synthesis of the trans‐1, 4‐polyisoprene/isotactic polybutene reactor blends.p‐TREF technique was used to analyze the composition of TPI/iPB reactor blends.Detailed structure characterization of the fractions including GPC and NMRTPI‐b‐iPB block copolymer can be obtained through the sequential polymerization.Rubber/plastic reactor blend materials were fabricated by the Z‐N catalyst. [ABSTRACT FROM AUTHOR]

Vacuum residue (VR) is the most complex component of crude oil. Due to the special structure of heavy subfractions, physical aggregation and chemical coking reactions easily occur through molecular force, which affects the normal processing. Therefore, in‐depth study and analysis of their composition, structure and association behaviours are particularly important. In view of the shortcomings of the traditional separation method in terms of separation accuracy, mainly including the purification of asphaltenes and the poor separation of resins. In this paper, a reasonably improved separation method is adopted, and the multi‐stage asphaltene extraction and the multi‐stage silica gel coupling separation are carried out innovatively, which achieves a high yield of 99% while ensuring the separation accuracy. The samples were characterized by elemental analysis (EA), gel permeation chromatography (GPC), Fourier‐transform infrared spectroscopy (FT‐IR), hydrogen nuclear magnetic resonance spectroscopy (1H‐NMR), X‐ray photoelectron spectroscopy (XPS), X‐ray diffraction (XRD), and scanning electron microscope (SEM) to study their structural characteristics and association behaviours. The results show that the main forms of heteroatoms in asphaltene and resin surface are C‐O‐C, C‐OH, pyridine, pyrrole, and thiophene, and the content of these substances is higher in asphaltene. Compared with resins, asphaltenes contain a more peri‐condensed aromatic structure and shorter alkyl substituent side chains. By studying the hydrogen bond and acid–base interaction, it is found that asphaltene and resin mainly contain OH‐OH, OH‐π, and OH‐ether O, of which the content of OH‐OH is the highest. Asphaltene and resin have more neutral and basic substances. These hydrogen bonds and acid–base interactions caused by heteroatoms are the main forces for the association of the heavy subfractions of the VR. [ABSTRACT FROM AUTHOR]

In this paper, the boron‐modified polyferrocenylcarbosilanes (namely HBPFCS‐Bs) with hyperbranched structure, as single‐source‐precursors, were successfully prepared by using 9‐borabicyclo [3, 3, 1] nonane (9‐BBN) and polyferrocenylcarbosilanes (HBPFCSs) as starting materials. The obtained HBPFCS‐Bs were characterized using Fourier transform infrared spectroscopy (FT‐IR), nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC), providing that the boron element can be introduced into the HBPFCS chains by the hydroboration (B‐H/C = C) reaction. Then, the structural evolution of HBPFCS‐Bs and the boron‐containing SiFeC ceramics at different temperatures (1100–1500°C) was further investigated by thermogravimetric analysis, X‐ray diffraction, energy dispersive spectroscopy, scanning electron microscope, and transmission electron microscopy. The results show that the incorporation of a small amount of boron (1 wt%) into precursors significantly improves the properties of the final ceramics, including the significant increase of the ceramic yield, enhanced high‐temperature resistance, as well as the higher ceramic densification. Finally, the saturation magnetization of the HBPFCS‐Bs‐derived ceramics was also enhanced by the introduction of boron. [ABSTRACT FROM AUTHOR]

Sialyl-glycopeptides containing an O-glycosidically linked tetrasaccharide chain were obtained from the urine of a patient suffering from mucolipidosis I. Isolation of these compounds was achieved by gel filtration, ion-exchange chromatography and preparative paper chromatography. Their structures were determined by a combination of carbohydrate and amino acid analysis, dansylation, periodate oxidation, methylation studies, enzymatic hydrolysis and ¹H-NMR spectroscopy, to be as follows: ... wherein R = peptide linked through -Thr-, Ser-, -Ser-Thr- or -Thr-Ser-. The finding of these glycopeptides in urine shows that mucolipidosis I is characterized by a general "glycoprotein-specific" sialidase deficiency. The possibility of the existence of a human endo-α-N-acetylgalactosaminidase is discussed. [ABSTRACT FROM AUTHOR]

The present paper is a commentary to 'Identification and characterization of hADSC‐derived exosome proteins from different isolation methods' (Huang et al. 2021; 10.1111/jcmm.16775). Given the enthusiasm for the potential of mesenchymal stromal cell‐derived extracellular vesicles (MSC‐EVs), some considerations deserve attention as they move through successive stages of research and application into humans. We herein remark the prerequisite of generating that evidence ensuring a high consistency in safety, composition and biological activity of the intended MSC‐EV preparations, and the suitability of disparate isolation techniques to produce efficacious EV preparations and fulfil requirements for standardized clinical‐grade biomanufacturing. [ABSTRACT FROM AUTHOR]

The next‐generation bioresorbable polymeric stents are undergoing continual research and development, especially how to control and optimize their degradation behaviors. The aim of this paper is to study the properties of a poly(l‐lactide) (PLLA) tube over accelerated degradation in vitro. Spherical nanoindentation was particularly adopted to measure the mechanical properties of PLLA tube; while differential scanning calorimetry and gel permeation chromatography were used for crystallinity and molecular weight measurements. Overall, the material degradation was captured by the local stress‐strain response obtained with spherical nanoindentation, and further confirmed with chemical analyses. The results are particularly useful for development of bioresorbable polymeric stents with improved performance. [ABSTRACT FROM AUTHOR]

The N-linked oligosacchafides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five tractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B-1), Man4GlcNAc2 (C-3) and one was the monoantennary structure Man3GlcNAc2 (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Manα6Manα6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reposed in mammals, plants and the parasitic protozoa. [ABSTRACT FROM AUTHOR]

Proteins are major effectors and regulators of biological processes that can elicit multiple functions depending on their interaction with other proteins. The organization of proteins into macromolecular complexes and their quantitative distribution across these complexes is, therefore, of great biological and clinical significance. In this paper, we describe an integrated experimental and computational technique to quantify hundreds of protein complexes in a single operation. The method consists of size exclusion chromatography (SEC) to fractionate native protein complexes, SWATH/DIA mass spectrometry to precisely quantify the proteins in each SEC fraction, and the computational framework CCprofiler to detect and quantify protein complexes by error‐controlled, complex‐centric analysis using prior information from generic protein interaction maps. Our analysis of the HEK293 cell line proteome delineates 462 complexes composed of 2,127 protein subunits. The technique identifies novel sub‐complexes and assembly intermediates of central regulatory complexes while assessing the quantitative subunit distribution across them. We make the toolset CCprofiler freely accessible and provide a web platform, SECexplorer, for custom exploration of the HEK293 proteome modularity. Synopsis: The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. The presented workflow is based on the concept of complex‐centric proteome profiling and combines size exclusion chromatography (SEC) with SWATH/DIA mass spectrometry.The implementation of the complex‐centric data analysis is supported by the computational framework CCprofiler.Application of the SEC‐SWATH‐MS workflow to HEK293 cells led the detection and quantification of subunit distribution of 462 distinct protein complexes containing 2,127 proteins and identified novel complex variants such as assembly intermediates.The interactive platform, SECexplorer is presented to support custom complex‐centric exploration of SEC‐SWATH‐MS datasets. The study presents an integrated framework for targeted, complex‐centric analysis based on size exclusion chromatography (SEC) and SWATH/DIA mass spectrometry. The workflow facilitates the parallel detection of hundreds of protein complexes and their variants at high selectivity and under error‐control. [ABSTRACT FROM AUTHOR]

Background: Exosomes are vesicles secreted by all types of mammalian cells. They are characterized by a double‐layered lipid membrane structure. They serve as carriers for a plethora of signal molecules, including DNA, RNA, proteins, and lipids. Their unique capability of effortlessly crossing the blood–brain barrier underscores their critical role in the progression of various neurological disorders. This includes, but is not limited to, diseases such as Alzheimer's, Parkinson's, and ischemic stroke. Establishing stable and mature methods for isolating exosomes is a prerequisite for the study of exosomes and their biomedical significance. The extraction technologies of exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer coprecipitation, immunoaffinity capture, microfluidic, and so forth. Each extraction technology has its own advantages and disadvantages, and the extraction standards of exosomes have not been unified internationally. Aims: This review aimed to showcase the recent advancements in exosome isolation techniques and thoroughly compare the advantages and disadvantages of different methods. Furthermore, the significant research progress made in using exosomes for diagnosing and treating central nervous system (CNS) diseases has been emphasized. Conclusion: The varying isolation methods result in differences in the concentration, purity, and size of exosomes. The efficient separation of exosomes facilitates their widespread application, particularly in the diagnosis and treatment of CNS diseases. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are cell derived membranous nanoparticles. EVs are important mediators of cell–cell communication via the transfer of bioactive content and as such they are being investigated for disease diagnostics as biomarkers and for potential therapeutic cargo delivery to recipient cells. However, existing methods for isolating EVs from biological samples suffer from challenges related to co‐isolation of unwanted materials such as proteins, nucleic acids, and lipoproteins. In the pursuit of improved EV isolation techniques, we introduce multimodal flowthrough chromatography (MFC) as a scalable alternative to size exclusion chromatography (SEC). The use of MFC offers significant advantages for purifying EVs, resulting in enhanced yields and increased purity with respect to protein and nucleic acid co‐isolates from conditioned 3D cell culture media. Compared to SEC, significantly higher EV yields with similar purity and preserved functionality were also obtained with MFC in 2D cell cultures. Additionally, MFC yielded EVs from serum with comparable purity to SEC and similar apolipoprotein B content. Overall, MFC presents an advancement in EV purification yielding EVs with high recovery, purity, and functionality, and offers an accessible improvement to researchers currently employing SEC. [ABSTRACT FROM AUTHOR]

Here, we investigate the recovery and reuse of polyvinylidene fluoride (PVDF) binders from both homemade and commercial cathode films in Li ion batteries. We find that PVDF solubility depends on whether the polymer is an isolated powder or cast into a composite film. A mixture of tetrahydrofuran:N-methyl-2- pyrrolidone (THF :NMP, 50:50 v/v) at 90°C delaminates composite cathodes from Al current collectors and yields pure PVDF as characterized by 1 H nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), wide-angle X-ray scattering (WAXS), and scanning electron microscopy (SEM). PVDF recovered from Li ion cells post-cycling exhibits similar performance to pristine PVDF. These data suggest that PVDF can be extracted and reused during Li ion battery recycling while simultaneously eliminating the formation of HF etchants, providing an incentive for use in direct cathode recycling. [ABSTRACT FROM AUTHOR]

Describes the circumstances guiding the Sephadex group of products for gel filtration with reference to the importance of electrophoresis. Description of dextran-based gels; Information on Jerker Porath's experience from zone electrophoresis; Details on Porath's research of electrophoresis.

The disulphide linkages of the 16 half-cystine residues in the sweet-tasting protein thaumatin have been investigated by enzymatic hydrolysis of the intact molecule. The peptides obtained after proteolytic cleavage with trypsin and pepsin, and in one case with chymotrypsin have been purified by gel filtration, high-performance liquid chromatography and peptide mapping by paper high-voltage electrophoresis in one direction and paper chromatography in the second dimension. Disulphide bonds appeared to be formed by cysteine residues in positions 9–204, 56–66, 71–77, 121–193, 126–177, 134–149, 145–158 and 159–164. The labile disulphide bond responsible for the enzymatic properties of the sweet tasting protein thaumatin appeared to be between Cys-145 and Cys-158. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase C dominates quantitatively among the isoperoxidases of horseradish root and has an isoelectric point close to 9. It consists of a heroin prosthetic group, 2 Ca2+ and 308 amino acid residues, including 4 disulfide bridges, in a single polypeptide chain that carries 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890. Assuming an average carbohydrate composition of (GlcNAc)2, Man3. Fuc, Xyl for each carbohydrate chain, the molecular weight of native horseradish peroxidase C is close to 44000. Cyanogen bromide fragments of reduced and carboxymethylated apo-peroxidase were purified by a combination of gel filtration and isoelectric focusing in urea, and cystine-containing tryptic fragments of apo-peroxidase were purified by gel filtration followed by disulfide cleavage and rechromatography at the initial conditions. The present paper discusses (a) isoelectric points and charge distribution within the native protein, the apoprotein and the cyanogen bromide fragments, (b) a buried pyrrolidonecarboxylyl amino terminus, (c) heterogeneity at the carboxyl terminus, and (d) a possible domain structure, likely from partial tryptic digestion. [ABSTRACT FROM AUTHOR]

The composition of lipopolysaccharide and polysaccharides isolated after acidic or alkaline degradation of lipopolysaccharide of Salmonella toucra has been investigated. The following analytical methods were used in this study: gel-filtration and ion-exchange techniques, paper and gas-liquid chromatography as well as spectrophotometric analysis. The products of the lipopolysaccharide degradation were fractionated on the Sephadex G-25 and Sephadex G-50 columns. Lipopolysaccharide and products of its degradation besides glucosamine, galactose, glucose, heptose(s) and 3-deoxy-D-manno-octulosonate described as ‘basal’ sugars, also contained N-acetylneuraminic acid and an unidentified amino sugar. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

In healthy individuals, physical exercise improves cardiovascular health and muscle strength, alleviates fatigue and reduces the risk of chronic diseases. Although exercise is suggested as a lifestyle intervention to manage various chronic illnesses, it negatively affects people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), who suffer from exercise intolerance. We hypothesized that altered extracellular vesicle (EV) signalling in ME/CFS patients after an exercise challenge may contribute to their prolonged and exacerbated negative response to exertion (post‐exertional malaise). EVs were isolated by size exclusion chromatography from the plasma of 18 female ME/CFS patients and 17 age‐ and BMI‐matched female sedentary controls at three time points: before, 15 min, and 24 h after a maximal cardiopulmonary exercise test. EVs were characterized using nanoparticle tracking analysis and their protein cargo was quantified using Tandem Mass Tag‐based (TMT) proteomics. The results show that exercise affects the EV proteome in ME/CFS patients differently than in healthy individuals and that changes in EV proteins after exercise are strongly correlated with symptom severity in ME/CFS. Differentially abundant proteins in ME/CFS patients versus controls were involved in many pathways and systems, including coagulation processes, muscle contraction (both smooth and skeletal muscle), cytoskeletal proteins, the immune system and brain signalling. [ABSTRACT FROM AUTHOR]

Owing to the unfavorable impact on the environment of mineral oil-based lubricants, there has been a steady increase in the demand for biodegradable, environment-friendly lubricants. However, development of a biodegradable base fluid that could replace or partially substitute conventional mineral oil is a big challenge. Vegetable oils are recognized as rapidly biodegradable and are thus promising candidates as base fluids in environment-friendly lubricants. Vegetable oils have excellent lubricity, but poor oxidation and low-temperature stability. This paper presents a series of structural modifications of vegetable oils using anhydrides of different chain lengths. The reaction was monitored and products were confirmed by NMR, FTIR, gel permeation chromatography, and thermogravimetric analysis (TGA). Experimental conditions were optimized for research quantity and for laboratory scale-up (up to 4 lb = 1.8 kg). The thermo-oxidation stability of these new lubricant base fluids was tested using pressure differential scanning calorimetry and TGA. The chemically modified base fluids exhibit superior oxidation stability in comparison with unmodified vegetable oils. These base fluids in combination with suitable additives exhibit equivalent oxidation stability compared with mineral oil-based formulations. [ABSTRACT FROM AUTHOR]

The use of biodegradable lubricants in diverse applications continues to increase. Vegetable oils (e.g., soybean oil) are the main biodegradable lubricant base stocks used worldwide. However, there are concerns about their oxidative stability and low-temperature performance. Improvements in oxidative stability can be made through chemical or genetic modifications. This work compared the effects of oils with different chemical compositions. Soybean oil, high-oleic soybean oil, and epoxidized soybean oil were compared in laboratory bench tests. The tests conducted include the Penn State sequential four-ball wear test and the Penn State micro-oxidation test. Oxidation products from the micro-oxidation test were analyzed by FTIR and gel permeation chromatography. In this paper, all oils were evaluated neat, without additives. [ABSTRACT FROM AUTHOR]

In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non‐native ones. In this study, Norway spruce (Picea abies) tissue‐cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin–carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two‐dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology. [ABSTRACT FROM AUTHOR]

Three phosphate-containing sialyloligosaccharides were isolated from normal human urine using charcoal adsorption, gel-filtration chromatography, ion-exchange chromatography and paper chromatography. Studies including gas-liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, phosphate determination, ion-exchange chromatography and glycosidase and phosphatase treatments indicated the following three structures for the compounds isolated: NeuAc(α2-6)Gal(β1-4)GlcNAc(α)-p; NeuAc(α2-3)Gal(β1-4)GlcNAc(α)-P; NeuAc(α2-3)Gal(β1-3)GalNAc(α)-P. These sialyloligosaccharide 1-phosphates represent a novel class of oligosaccharides. Their oligosaccharide chains are identical with the common sialyloligosaccharide end groups of glycoproteins and glycolipids. The excretion of these compounds in normal human urine may indicate the existence of a novel, as yet unrevealed pathway in the metabolism of complex carbohydrates. [ABSTRACT FROM AUTHOR]

Rabbit reticulocyte lysates were passed through 2'5'ADP-Sepharose columns under conditions in which the gel- filtration effect was negligible and low-molecular-weight compounds were retained in the flow-through lysate. Glucose-6-phosphate dehydrogenase. 6-phosphogluconate dehydrogenase and glutathione reductase were quantitatively adsorbed by the column and removed from the lysate.. but isocitrate dehydrogenase and thioredoxin reductase were retained in the flow-through lysate. The initial rate of protein synthesis in lysates treated in this way was normal, but synthesis stopped after about 20 mm of incubation. This shut-off could be prevented by the addition of dithiothreitol or by providing a means of NADPH generation, which could be achieved either by adding isocitrate or glucose-6-phosphate dehydrogenase. Further experiments used lysates which were first gel-filtered to remove low- molecular-weight metabolites and then passed through 2'5'ADP-Sepharose columns. Under these conditions thioredoxin reductase was efficiently adsorbed by the affinity column, in addition to the three enzymes already listed. The maintenance of full protein synthesis activity in these lysates required the addition of both a sugarphosphate and a reducing agent. The sugar phosphate requirement could be satisfied by glucose 6-phosphate. or 2-deoxyglucose 6- phosphate, or fructose 1,6-bisphosphate, but not by 6-phosphogluconic acid. The requirement for reducing agent could be met by the addition of dithiothreitol, or by an NADPH-generating system together with rabbit thioredoxin ceductase. Purified thioredoxin reductase from Escherichia coli was also effective provided E. coli thioredoxin was also added, but the addition of glutathione with glutathione reductase did not activate protein synthesis. It is concluded that there is a dual requirement for the maintenance of high rates of protein synthesis in reticulocyte lysates: certain sugar phosphates must be present. in addition to an NADPI-I-generating system and a functional thioredoxin/thioredoxin reductase system. [ABSTRACT FROM AUTHOR]

The interaction of actin with various cations has been studied at pH 7.6, at 20 °C, by means of gel filtration and centrifugation techniques. In addition to the single high-affinity site, five low-affinity sites with apparent association constants of 5-6 x 10³ M-1 for Ca2+, Mn2+ and Sr2+ and of 1.6 x 104 M-1 for Ni2+ have been found in the actin molecule. Unlike the other cations, Zn2+ appears to have three high-affinity sites from which it is not removed by Dowex 50 treatment, and eight low-affinity sites with an apparent association constant of 1.3 x 104 M -1. Competition experiments suggest that Ca2+, Sr2+, Mn2+ and Ni2+ are bound to the same low-affinity sites, some of which are also available for Zn2+. Zn2+ also binds to additional sites which are not available for the other cations. Binding of 4 mol of either Ca2+, Mn2+, Sr2+, Ni2+ or Zn2+ per mol of actin monomer at the low-affinity sites characterized above causes a complete transformation of the monomeric actin into polymers. The nonidentity of some of the 2+-binding sites with those for the other cations seems to explain structural differences between the polymers formed in the presence of this cation and those produced upon binding of Mn2+, Ni2+ or alkaline earth metals, described in the preceding paper in this journal. The results suggest that formation of the double-stranded F-actin filaments requires the neutralization of negative charge at specific sites in the monomer molecules. The aggregation of F-actin filaments into paracrystals observed at pH 7.6 in the presence of Ca2+, Mg2+, Sr2+ or Mn2+ at millimolar concentrations seems to involve the binding of these cations to a third class of binding sites with apparent association constants of the order of 10² M-1. The binding parameters to this class of sites could not be determined by the methods used in this work. [ABSTRACT FROM AUTHOR]

1. From 90 1 erythrocytes 200 mg glutathione reductase was purified in four steps with an overall yield of 40%. The specific activity rose from 3.71 U glutathione reductase per g hemoglobin in the erythrocytes to 235 U/mg protein in the isolated enzyme. The purification procedure which was completed in 5 days, included (a) denaturation of hemoglobin by a mixture of butan-1-oI and chloroform [Scott. E. M. (1976) Prep. Biochem. 6, 147-152]. (b) precipitation of glutathione reductase with acetone, (c) extraction of the precipitate and fractionation of the extract with ammonium sulfate, (d) affinity chromatography on adenosine-2',5'-bisphosphate -Sepharose 4B. 2. The 280/460 nm absorbance ratio was found to be 5.9 for the purified enzyme; all other studies on physical and chemical properties of the enzyme confirmed the results of Worthington and Rosemeyer [Eur. J. Biochem. 67, 231-238 (1976) and earlier papers]. 3. The new purification procedure provided crystals suitable for X-ray diffraction analysis at high resolution. Furthermore sufficient amounts for sequencing the enzyme were isolated. As a first step in the determination of the protein's primary structure, CNBr-produced fragments of the carboxymethyiated enzyme were fractionated. 4. A chain segment containing the catalytic disulfide of the native protein was isolated. Its sequence was determined to be Leu-Gly-Gly-Thr-Cys-Val-Asn-Val-Gly-Cys-Val-Pro-Lys. An identical sequence is present in yeast glutathione reductase, and lipoamide dehydrogenases contain an active site peptide with very similar sequence [Williams, C. H. Jr (1976) The Enzymes 13, 89 - 173]. Thus the long-recognized mechanistic similarities between glutathione reductases and lipoamide dehydrogenases are reinforced by sequence similarities around the two redox-active cysteine residues. The sequenced regions are, however, too short to allow speculations on phylogenetic relationships within this category of proteins. [ABSTRACT FROM AUTHOR]

Three toxins (CM-8, CM-11, and CM-13 a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose, Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 6t residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-tt resemble the corresponding properties of the cytotoxin group, Tile sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group, [ABSTRACT FROM AUTHOR]

Incubation of UDP-[14C]galactose with membranes of Bacitillus coagulans led to the formation of a radioactive glycolipid, which was tentatively characterized as β-galactosyl phosphorylpolyprenol (Gal-P-prenol) on the basis of its chromatographic behavior and data from structural analysis of its sugar 1-phosphate moiety. The sugar moiety of [14C]Gal-P-prenol was shown to be incorporated into a membrane-bound polymer, which coincided with the diacyl form of lipoteichoic acid in its chromatographic behavior on columns of Sephacryl S-300, DEAE-Sephacel and octyl-Sepharose. Hydrogen fluoride hydrolysis of the polymer afforded an α-galactoside identical with Gal(α1 → 2)Gro obtained from lipoteichoic acids. The incorporation of galactose residues from [14C]Gal-P-prenol into the polymer was greatly enhanced by exogenous lipoteichoic acids, especially of the diacyl and monoacyl forms. The optimal pH and metal concentration for the Gal-P-prenol formation, respectively, were found to be 8.4 and 10 mM (MgCl2), whereas those for the transfer of galactose from this lipid intermediate to polymer were 4.5 and 16 mM (CaCl2). The above results lead to the conclusion that Gal-P-prenol serves as the direct galactosyl donor in the synthesis of lipoteichoic acids in B. coagulans. [ABSTRACT FROM AUTHOR]

Control of the density and uniformity of semiconducting single-walled carbon nanotube (SWNT) networks is crucial for their application in field-effect transistors (FETs) and critically depends on the surface of the substrate. Here, the concept of self-assembled monolayer (SAM) dielectrics on aluminum/aluminum oxide bottom-gate electrodes is used to create low-voltage (<2 V) FETs with SWNT networks. The influence of different phosphonic acid SAMs is investigated on the final network density and charge transport properties of solution-deposited, polyfluorene-sorted large-diameter semiconducting SWNT. Positively charged, imidazolium-terminated SAMs and those with high dipole moments result in the highest network densities for both spin-coating and dip-coating. However, FETs with moderate SWNT coverage on SAMs with long alkyl chains and amine or hydroxyl end-groups yield the best combination of high on-conductances and high hole mobilities (up to 20 cm2 V−1 s−1) with low trap densities and short critical channel lengths (<2 μm). These parameters are suitable for future short channel, flexible, and high-frequency FETs. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) have shown promise as biological delivery vehicles, but therapeutic applications require efficient cargo loading. Here, we developed new methods for CRISPR/Cas9 loading into EVs through reversible heterodimerization of Cas9‐fusions with EV sorting partners. Cas9‐loaded EVs were collected from engineered Expi293F cells using standard methodology, characterized using nanoparticle tracking analysis, western blotting, and transmission electron microscopy and analysed for CRISPR/Cas9‐mediated functional gene editing in a Cre‐reporter cellular assay. Light‐induced dimerization using Cryptochrome 2 combined with CD9 or a Myristoylation‐Palmitoylation‐Palmitoylation lipid modification resulted in efficient loading with approximately 25 Cas9 molecules per EV and high functional delivery with 51% gene editing of the Cre reporter cassette in HEK293 and 25% in HepG2 cells, respectively. This approach was also effective for targeting knock‐down of the therapeutically relevant PCSK9 gene with 6% indel efficiency in HEK293. Cas9 transfer was detergent‐sensitive and associated with the EV fractions after size exclusion chromatography, indicative of EV‐mediated transfer. Considering the advantages of EVs over other delivery vectors we envision that this study will prove useful for a range of therapeutic applications, including CRISPR/Cas9 mediated genome editing. [ABSTRACT FROM AUTHOR]

Objectives: To restore tissue growth without increasing the risk for cancer during aging, there is a need to identify small molecule drugs that can increase cell growth without increasing cell proliferation. While there have been numerous high‐throughput drug screens for cell proliferation, there have been few screens for post‐mitotic anabolic growth. Materials and Methods: A machine learning (ML)‐based phenotypic screening strategy was used to discover metabolites that boost muscle growth. Western blot, qRT‐PCR and immunofluorescence staining were used to evaluate myotube hypertrophy/maturation or protein synthesis. Mass spectrometry (MS)‐based thermal proteome profiling‐temperature range (TPP‐TR) technology was used to identify the protein targets that bind the metabolites. Ribo‐MEGA size exclusion chromatography (SEC) analysis was used to verify whether the ribosome proteins bound to calcitriol. Results: We discovered both the inactive cholecalciferol and the bioactive calcitriol are amongst the top hits that boost post‐mitotic growth. A large number of ribosomal proteins' melting curves were affected by calcitriol treatment, suggesting that calcitriol binds to the ribosome complex directly. Purified ribosomes directly bound to pure calcitriol. Moreover, we found that calcitriol could increase myosin heavy chain (MHC) protein translation and overall nascent protein synthesis in a cycloheximide‐sensitive manner, indicating that calcitriol can directly bind and enhance ribosomal activity to boost muscle growth. Conclusion: Through the combined strategy of ML‐based phenotypic screening and MS‐based omics, we have fortuitously discovered a new class of metabolite small molecules that can directly activate ribosomes to promote post‐mitotic growth. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection. [ABSTRACT FROM AUTHOR]

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta. [ABSTRACT FROM AUTHOR]

The method for producing polyvinyl alcohol (PVA) by the solution polymerization of vinyl acetate first and then alcoholysis strategy is industrial. Where solvent plays a vital role in control over stereoregularity, molecular weight and its distribution, crystallinity and so on. In this report, when methyl acetate was used as a solvent in the polymerization of vinyl acetate instead of methanol, prepared PVA was characterized by Fourier‐transform infrared spectroscopy (FT‐IR), nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). The results showed that PVA obtained in methyl acetate has a higher content of syndiotactic structure, crystallinity melting temperature and enhanced thermal stability compared with PVA obtained in methanol due to the formation of Π ‐ Π complex between methyl acetate and vinyl acetate and the enhanced steric hindrance effect during polymerization. Additionally, methyl acetate has a smaller chain transfer constant (1.31 × 10−4), which benefits to decrease the branching degree and molecular weight distribution index of PVA. This research provides more ideas for the choice of solvents in the industrial production of polyvinyl alcohol. [ABSTRACT FROM AUTHOR]

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While −80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of −80°C storage and freeze‐thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent‐tagged EVs populations (GFP and mCherry), mixed before preservation. We found that −80°C storage reduces EVs concentration and sample purity in a time‐dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size‐charge plots. None of the tested conditions prevented the observed effects. Freeze‐thaw cycles lead to an EVs reduction after the first cycle and to a cycle‐dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double‐positive EVs (GFP+‐mCherry+). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non‐archival, samples in majority of cases. [ABSTRACT FROM AUTHOR]

Previous study has suggested the colonic nontoxicity and obesity inhibition of food‐grade κ‐carrageenan in obese mice. Further study using transcriptome is important to provide further understanding on the gene expressions of inflammation and obesity. Here, the obese mice without any treatment (HFD) or with 5% food‐grade κ‐carrageenan diet intervention (H5%) were used to perform colonic transcriptome sequencing. The results showed that genes involved in the inflammatory pathways or tight junction protein encoding were not significantly dysregulated by 5% carrageenan. However, the expression of lipid metabolism genes meaningfully changed as evidenced by the decreased gene levels of adipocytokines, lipogenesis, lipid absorption and transport, and the increased adipolysis and oxidation. In addition, the carrageenan metabolism experiments by toluidine blue (TB) staining of colon and high‐performance size exclusion chromatography (HPSEC) of feces supernatant showed that the food‐grade κ‐carrageenan was not absorbed or significantly degraded in the digestive tract of obese mice. Hence, the fact that food‐grade κ‐carrageenan was not significantly metabolized by the organism and did not cause obvious dysregulation of colonic inflammatory genes provided evidences for its noncolonic toxicity in obese mice. An anti‐obesity potential of food‐grade κ‐carrageenan was probably mediated by the regulation of lipids metabolism‐related genes. [ABSTRACT FROM AUTHOR]

A novel polysaccharide was extracted from Sipunculus nudus (SNP). The molecular weight (MW) of SNP was determined to be 9223 Da by high‐performance gel permeation chromatography analyses, and the structure of the SNP repeat units was determined to be →3,4‐β‐D‐GlcpNAC (1→ and →4) ‐α‐D‐Glcp (1→ in the ratio of 15:1; →2) ‐α ‐D‐Galp ‐ (1→ as a side chain; and β‐D‐Galp‐(1→ and α‐ D‐Glcp ‐ (1→ as end groups by GC‐MS analysis and NMR assays. The effect of SNP on hepatoma HepG2‐bearing mice was analysed to verify its potential in the clinical treatment of liver cancer. A total of 90 male athymic nu/nu mice were divided into therapeutic and preventive groups and fed with different amounts of SNP. The antitumour effect of SNP on HepG2‐bearing mice and mechanism of such were studied by analysing the tumour size, spleen index, thymus index, immune factors in the blood, tumour apoptosis factors, etc. The results suggest that SNP not only increased the index of immune organs in the body, but also enhanced the secretion of immune factors, including interleukin‐2, interferon gamma and tumour necrosis factor‐alpha in the serum. SNP induced the apoptosis of tumour cells via the mitochondrial apoptosis pathway, which upregulated caspase‐3, caspase‐8, caspase‐9 and BCL2‐associated X, but downregulated B‐cell lymphoma‐2 and vascular endothelial growth factor protein expression. In conclusion, SNP inhibited tumour growth by enhancing immune function and inducing tumour cell apoptosis in HepG2‐bearing mice. Therefore, SNP may be further investigated as a promising candidate for future antitumour drugs. [ABSTRACT FROM AUTHOR]

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose‐derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC‐derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size‐based isolation, polymer precipitation and immuno‐affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick‐TC precipitation and ExoQuick‐TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D‐LC‐MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC‐derived exosomes. We proved that these proteins were potential hADSC‐derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC‐derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC‐derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications. [ABSTRACT FROM AUTHOR]

In this study, poly(p‐phenylenediamine) (PPDA) was synthesized by an enzymatic polymerization method using p‐phenylenediamine as the monomer in the water phase. The PPDA structure was characterized by infrared (IR), gel permeation chromatography, and elemental analysis. The antioxidation behavior of the as‐synthesized PPDA in biodegradable castor oil was investigated using a rotary oxygen bomb test and pressurized differential scanning calorimetry. A theoretical calculation was conducted to investigate its antioxidation mechanism. PPDA was found to exhibit excellent oxidation resistance in castor oil at 150 °C, which effectively prolonged the oxidation induction time of the oil. The results obtained herein suggest that as with phenolic antioxidants, PPDA with a hindered structure effectively improves the antioxidation performance of the oil by enhancing the stability of nitrogen free radicals. [ABSTRACT FROM AUTHOR]

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer‐based precipitation and size exclusion chromatography (Pre‐SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early‐ and late‐eluting EV fractions, we performed a quantitative proteomic analysis of MDA‐MB‐468‐derived EVs. We identified 286 exclusive proteins in early‐eluting fractions and 148 proteins with a differential concentration between early‐ and late‐eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies. [ABSTRACT FROM AUTHOR]

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single‐particle interferometric reflectance imaging sensing (SP‐IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP‐IRIS events could not be used to estimate particle concentrations. For sizing, SP‐IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP‐IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP‐IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single‐particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies. [ABSTRACT FROM AUTHOR]

The article presents a study which aims to analyse data on sorption kinetics on the penetration of a range of silicone rubbers by a linear and cyclic polydimethylsiloxane (PDMS) elastomers. It says that the molar mass and architecture of the silicone liquids were measured by gas chromatography and size exclusion chromatography. Results show that the diffusivity activation energy was constant for linear PDMS liquids and solubility increased with the decrease in penetrant molar mass.

Embryonic and adult chicken liver galactosyltransferases behave differently on DEAE-Sepharose chromatography. After solubilization, two embryonic activities (one transfers galactose in a β1&-4 linkage to asialo-agalacto-α1-acid glycoprotein and to N-acetylglucosamine; the other transfers galactose in a β-3 linkage to asialo-ovine submaxillary mucin) elute after the bulk of the protein, and after free glucose. The same two enzymes in adults elute more rapidly, almost coincident with the bulk of the protein, and before free glucose. The difference in elution patterns occurs when the column buffer contains 0.1 M NaCl. Without salt, both embryonic and adult transferases bind to the column, but with 0.5 M NaCl, the embryonic and adult transferases elute identically, and with the bulk of the protein. After treatment with neuraminidase, the embryonic transferase activities elute significantly earlier on a DEAE-Sepharose column in the presence of 0.1 M NaCl. The embryonic forms migrate more rapidly than do the adult forms on cellulose acetate electrophoresis, but neuraminidase treatment renders both enzyme forms immobile in this system. Neuraminidase treatment also inhibits the binding of the embryonic transferases to a wheat-germ-agglutinin-Sepharose column. Kinetically, the embryonic and adult transferases are indistinguishable. [ABSTRACT FROM AUTHOR]

A pyroglutamate aminopeptidase activity, distinct from that of cytoplasm, was released from a synaptosomal membrane preparation of guinea-pig brain by papain treatment. This activity was further purified 3560-fold relative to the homogenate with a yield of 17% by a procedure involving gel filtration chromatography, calcium phosphate cellulose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The purified synaptosomal pyroglutamate aminopeptidase hydrolysed only thyroliberin, acid-thyroliberin, the luliberin N-terminal tripeptide (Glp-His-Trp) and, only slightly, Glp-His-Gly. No hydrolysis was observed with dipeptides containing N-terminal pyroglutamic acid (Glp) or with pyroglutamyl peptides containing more than three amino acids. AKm value of 40 μM was recorded when thyroliberin was used as substrate; however, luliberin was found to inhibit the hydrolysis of thyroliberin competitively with a Ki value of 20 μM. [ABSTRACT FROM AUTHOR]

Seryl-tRNA synthetase was purified 1800-fold from bovine liver extract by ultracentrifugation at 150000 x g, chromatography on DEAE-cellulose, fractional precipitation with ammonium sulfate, gel chromatography on Sephacryl S-300, adsorption chromatography on hydroxyapatite, affinity chromatography on blue-Sepharose and finally on M&asline;trex gel red A. The relative molecular mass, Mr, in the denatured state was estimated as 87000 by sodium dodecyl sulfate disc gel electrophoresis; in the active state the Mr was estimated as 170000 for the dimeric native enzyme (α2 type) by chromatography on Sephacryi S-300. The amino acid composition of the enzyme was determined. The Km values for ATP and serine were 0.49 mM and 30 μM, respectively. The Km values for tNAIGASer and tRNAcmCASer were 1.40 μM and 1.25 μM, respectively. Sequences common to the two isoaccepting tRNASer molecules are discussed in relation to the recognition mechanism of the purified seryl-tRNA synthetase. [ABSTRACT FROM AUTHOR]

1. Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAEcellulose, blue-dextran-Sepharose and Sephadex G150. 2. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. 3. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. 4. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. 5. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was n good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. 6. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity. [ABSTRACT FROM AUTHOR]

Protein phosphatase-2B was purified from extracts of rabbit skeletal muscle by a procedure that involved fractionation with ammonium sulphate, chromatography on DEAE-Sepharose, fractionation with poly(ethylene glycol), gel filtration on Sephadex 0-200 (Mr = 98000 ± 4000). chromatography on Affi-Gel Blue and affinity chromatography on calmodulin-Sepharose. The enzyme was purified 3500-fold in seven days with an overall yield of 0.5%. The α-subunit of phosphorylase kinase, protein phosphatase inhibitor-i and the myosin P-Light chain from rabbit skeletal muscle were dephosphorylated by protein phosphatase-2B with similar kinetic constants. The &alpha-subunit of phosphorylase kinase was dephosphorylated at least 100-fold more rapidly than the β-subunit, while glycogen phosphorylase, glycogen synthase, histones H1 and H2B. ATP-citrate lyase, acetyl-CoA carboxylase, L-pyruvate kinase and protein synthesis initiation factor elF-2 were not dephosphorylated at significant rates. Protein phosphatase-2B became activated 10-fold by calmodulin (A0.5 = 6 nM) after chromatography on DEAE-Sepharose and this degree of activation was maintained throughout the remainder of the purification. Calmodulin increased the Vmax of the reaction without altering the Km for inhibitor-I. The activity of protein phosphatase-2B was completely dependent on Ca2+ in the presence or absence of calmodulin. Half-maximal activation was observed at 1.0 μM Ca2 + in the absence, and at 0.5 μM Ca2+ in the presence, of 0.03 μM calmodulin. Protein phosphatase-2B was inhibited completely by trifluoperazine; half- maximal inhibition occurred at 45 μM in the absence and 35 μM in the presence of 0.03 μM calmodulin. The metabolic rote of protein phosphatase-2B in vivo is discussed in the light of the observation that this enzyme is probably identical to a major calmodulinabinding protein of neural tissue termed calcineurin or CaM-BP80 (Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P. (1982) FEBS Let!. 137, 80-84]. [ABSTRACT FROM AUTHOR]

The light-harvesting bacteriochlorophyll-carotenoid-protein complex B800–850 has been isolated from membranes of the phototroph-negative mutant strain Y5 of Rhodopseudomonas capsulata. The three polypeptides of the complex have been found to be soluble in chloroform-methanol (1:1, v/v) in the presence of 0.1 M ammonium acetate. They were extracted from the complex and separated by gel filtration on Sephadex LH-60 in the same solvent mixture. Minimum molecular weights based on amino acid composition are 12000, 9300, and 5100. Values previously determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are 14000, 10000 and 8000. The two smaller polypeptides (polarities 31% and 39%) are completely soluble in chloroform/methanol/ammonium acetate while the largest and most polar (41%) polypeptide is only partially soluble. The largest polypeptide contains no tryptophan. The middle polypeptide contains no cysteine and arginine, while the small polypeptide lacks cysteine. Methionine is shown to be the amino terminus for the small and middle polypeptides by two independent methods (Edman degradation and dansylation). Both methods also indicated that the N terminus of the 14000 polypeptide seems to be blocked. Partial N-terminal amino acid sequences were obtained for the two smaller polypeptides. No homology between the two proteins was observed. [ABSTRACT FROM AUTHOR]

The Macrotyloma axillare plant, belonging to the Leguminosae family, is a perennial climbing or trailing herb 0.2–3.5 m long. The plant is indigenous to South Africa and it occurs in the warm dry northern parts of the Transvaal. It has been introduced into Australia, where the seed are used as animal food. Two protease inhibitors, DE-3 and DE4, were purified from Macrotyloma axillare seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DEAE- cellulose. They each comprise 76 amino acid residues including 14 half-cystine residues. The complete primary structures of the two protease inhibitors have been elucidated and their sequences are 67% identical. The inhibitor specificities, the sequences, the invariant amino acid residues and the reactive inhibitor sites of protease inhibitors DE-3 and DE-4 resemble the corresponding properties of the Bowman­Birk double-headed protease inhibitor group. The cysteine residues are in similar locations to those in protease inhibitors of known structure so they are presumed to rink similarly. [ABSTRACT FROM AUTHOR]

Glycogen synthase I was purified from human polymorphonuclear leukocytes by a procedure involving affinity chromatography of the glycogen-enzyme complex, digestion of endogenous glycogen by amylase, starch chromatography and gel filtration. The purified enzyme had a specific activity of 7–11 U/mg protein, or 4–5 U when expressed per mg of residual glycogen. Further purification to 21 U/mg protein could be achieved. The enzyme was inactive in the absence of added glycogen. A subunit molecular weight of 85000 was determined by polyacrylamide electrophoresis in sodium dodecylsulfate. The molecular weight of the native enzyme was estimated to be 390000 (13.2 S) by sucrose gradient centrifugation and 410000 by gel filtration indicating that the native enzyme is a tetramer. The gel filtration behavior was not affected by enzyme concentration, temperature, or the presence of ligands. The energy of activation was estimated to 13500cal/mol (56.5 kJ/mol), corresponding to a Q10 of 2.2. In the presence of glucose 6-phosphate or Na2SO4, the enzyme showed a broad pH optimum between pH 6.8-9.2. In the absence of these ligands and in particularly in the presence of Mg2+, the enzyme is sensitive to small changes of pH in the interval pH 7.4- 8.4. During purification, synthase I requires protection by 0.6 mM dithiothreitol, while high concentrations of mercaptoethanol or dithiothreitol inactivates the enzyme, particularly during freezing. During 24-h incubations, synthase I undergoes a spontaneous, temperature-dependent inactivation which is not due to proteolysis, but presumably is caused by irreversible conformational changes. These can be prevented by high concentrations of glucose 6-phosphate, Na2SO4, inorganic phosphate, UDP and glycogen. Mg2+ and traces of ethanol inactivates the enzyme. The lyophilized enzyme is stable for years. [ABSTRACT FROM AUTHOR]