Ethanol Precipitation Procedure Research Articles

Purification and antioxidant and anti-Inflammatory activity of extracellular polysaccharopeptide from sanghuang mushroom, Sanghuangporus lonicericola.

Sanghuang mushrooms are medicinal fungi widely used in eastern Asia. In this study, the antioxidant and anti-inflammatory activity of a novel extracellular polysaccharopeptide, sanghuang extracellular polysaccharopeptide (SePSP) was investigated. The SePSP was purified from the submerged fermentation broth of a sanghuang mycelium, Sanghuangporus lonicericola strain CBS17, which was isolated from a wild sanghuang fruiting body. The SePSP was extracted using an ethanol precipitation procedure, followed by diethylaminoethanol (DEAE) anion-exchange and size-exclusion chromatography. The mass ratio of the polysaccharide and peptide components in the purified SePSP was approximately 4.87:1. By determining its free radical scavenging abilities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), the hydroxyl free radical, and the superoxide anion free radical, as well as its total reducing power, SePSP was shown to have strong concentration-dependent antioxidant activity in vitro. Further, SePSP effectively alleviated dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in mice. Administration of 200 mg kg-1 SePSP by gavage for 7 days prevented body weight loss; significantly reduced the mRNA levels of proinflammatory cytokines, including TNF-α and IL-1β; increased mRNA level of the anti-inflammatory cytokine IL-10 in the colon, and decreased the malondialdehyde concentration from 6.42 to 4.82 μmol L-1 in the blood in UC mice. The SePSP had strong concentration-dependent antioxidant activity in vitro and effectively alleviated DSS-induced UC in mice. The in vivo therapeutic efficacy in DSS-induced UC may be mediated by modulating the expression of inflammatory cytokines and inhibiting oxidative stress. The findings provide a scientific rationale for the use of bioactive nutraceuticals from sanghuang mushrooms to develop functional foods for the prevention and treatment of UC. © 2020 Society of Chemical Industry.

A simple preparative method for isolation and purification of polysaccharides from mulberry (Morus alba L.) leaves

SummaryAn effective method for preparative extraction and purification of the polysaccharides from mulberry leaves was studied. Orthogonal experiment was carried out to get the optimal conditions (T = 70 °C, pH = 6, t = 60 min, D = 0.4 g g−1 material) of activated carbon decolorisation. The staged, isocratic and multiple ethanol precipitation procedures were also investigated. Through the activated carbon decolorisation and 20% ethanol precipitation procedure, the polysaccharides yield reached 2.91%, and the content of neutral sugar and uronic acid were improved to 21.87% and 57.43% respectively. This process was superior to the macroporous resin adsorption and gel permeation chromatography (GPC) methods. The structure analysis indicated that the purified mulberry leaves polysaccharide (MLP) was composed of Man, GalA and Rha in the ratio of 1.0:7.2:20.0. The molecular weight measured using GPC was approximately 557 062 D. The IR and 1H NMR spectra indicated that the MLP was mainly pectin, which was classified as high‐methoxyl pectin.

Crystal Structure of Human Fibrinogen

A crystal structure of human fibrinogen has been determined at approximately 3.3 A resolution. The protein was purified from human blood plasma, first by a cold ethanol precipitation procedure and then by stepwise chromatography on DEAE-cellulose. A product was obtained that was homogeneous on SDS-polyacrylamide gels. Nonetheless, when individual crystals used for X-ray diffraction were examined by SDS gel electrophoresis after data collection, two species of alpha chain were present, indicating that some proteolysis had occurred during the course of operations. Amino-terminal sequencing on post-X-ray crystals showed mostly intact native alpha- and gamma-chain sequences (the native beta chain is blocked). The overall structure differs from that of a native fibrinogen from chicken blood and those reported for a partially proteolyzed bovine fibrinogen in the nature of twist in the coiled-coil regions, likely due to weak forces imparted by unique crystal packing. As such, the structure adds to the inventory of possible conformations that may occur in solution. Other features include a novel interface with an antiparallel arrangement of beta chains and a unique tangential association of coiled coils from neighboring molecules. The carbohydrate groups attached to beta chains are unusually prominent, the full sweep of 11 sugar residues being positioned. As was the case for native chicken fibrinogen, no resolvable electron density could be associated with alphaC domains.

Cyclin-dependent Kinase 9 Is Required for Tumor Necrosis Factor-α-stimulated Matrix Metalloproteinase-9 Expression in Human Lung Adenocarcinoma Cells

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-alpha-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-alpha-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-alpha-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-beta1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-alpha-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-beta1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-alpha-stimulated human MMP-9 promoter activity. TNF-alpha did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-alpha stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-alpha-induced MMP-9 transcription. Disruption of TNF-alpha signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.

Glutathione as a matrix for the synthesis of CdS nanocrystallites

GSH-capped CdS nanocrystallites were synthesized by reacting Cd(II)-GSH with aqueous sodium sulfide using specific initial sulfide/Cd(II) ratios. Spectroscopic analyses of fractions obtained from a size exclusion column showed varying absorption spectra indicating a significant dispersion in size-distribution of nanocrystallites at lower sulfide/Cd(II) ratios. However, size distribution of the nanocrystallites was narrower at initial sulfide/Cd(II) ratios that exceeded 1.0. An ethanol precipitation procedure was used to remove free Cd(II)-GSH complexes and selectively isolate GSH-capped nanocrystallites in a very narrow size range. Size exclusion chromatography indicated similar chemical compositions and overlapping spectral profiles of ethanol-precipitated samples suggesting apparent uniformity in both the size and the cap content. All of the GSH-capped CdS nanocrystallites with varying cap contents degraded p-nitrophenol upon irradiation at 366 nm. However, photocatalytic degradation of p-nitrophenol was significantly higher in samples with higher sulfide/Cd ratio and less capping material. The addition of H 2O 2 enhanced levels of photo-oxidation of p-nitrophenol.

Cysteine-mediated synthesis of CdS bionanocrystallites

The synthesis of CdS bionanocrystallites has been studied by reacting Cd(II)-cysteine complexes with inorganic sulfide. Cysteine-mediated synthesis of CdS bionanocrystallites proceeded slightly faster at 45°C than at room temperature. The sizes of these CdS bionanocrystallites, as determined by optical spectroscopy, increased with increasing initial sulfide/Cd(II) ratios. Analyses of size-distribution showed significant heterogeneity in sizes only at sulfide/Cd(II) ratios of 0.25. An ethanol precipitation procedure was developed to remove unreacted Cd(II)-cysteine complexes. This procedure also resulted in the isolation of CdS bionanocrystallites (bioNCs) that appeared uniform in size and chemical composition. Cysteine behaved like glutathione insofar as the size-range of CdS particles was concerned. However, cysteine-mediated synthesis of CdS bioNCs resulted in uniformly sized-particles as has been observed previously with phytochelatins. Cysteine-capped CdS particles exhibited pH-dependent changes in their properties. pH-induced changes were more pronounced in emission than in absorption spectra. Photocatalytic activities of these bioNCs were indicated by reduction of three dyes. Irradiation of methylviologen, basic fuchsin and naphthol blue black at 366 nm in the presence of cysteine-capped CdS bioNCs caused reduction of these dyes. Samples exhibiting higher luminescence were also more active in the photocatalysis experiments.

Cysteine-capped ZnS nanocrystallites: Preparation and characterization

Cysteine-capped ZnS nanocrystalline semiconductors (NCs) were prepared by titrating sodium sulfide into preformed Zn–cysteine complexes. Only a maximum of ∼40% of Zn(II) in Zn–cysteine complex was converted into ZnS NCs when the reaction was carried out at room temperature for 30 min. However, incubation of the reaction mixture at 45°C for 60 min significantly enhanced the production of ZnS NCs as a maximum of ∼75% of Zn(II) was converted into NCs. Cysteine capping produced NCs that were smaller than those capped by glutathione. Furthermore, cysteine-capped ZnS exhibited a narrow range of size distribution as suggested by UV/VIS spectroscopy of the NCs separated on a size-fractionation column. Unreacted Zn–cysteine complex could be removed from ZnS NC preparations by selective precipitation with ethanol. The precipitation procedure also led to the isolation of ZnS NCs that appeared more uniform by gel-filtration analysis. Ethanol precipitation procedures allowed preparation of large quantities of powdered NCs which retained their colloidal nature upon resuspension in water or buffers. pH titration experiments indicate that the average size of the particles was smallest in the pH range 7–10, but the size increased as the samples were made acidic or alkaline. Cysteine-capped NCs were capable of causing photoreduction of methylviologen, basic fuchsin and naphthol blue black.

Development of an immunomagnetic antigen capture system for detecting leptospires in bovine urine

A magnetic bead antigen capture system which combined the use of two evolving techniques - immunomagnetic separation (IMS) and time-resolved fluoroimmunoassay (TR-FIA) - was developed to detect Leptospira borgpetersenii serovar hardjo in bovine urine. The assay utilised monoclonal antibody coated magnetic beads to capture leptospiral antigen which was in turn detected using another monoclonal antibody (Indicator) labelled with biotin. Signal was generated by the binding of europium labelled streptavidin to indicator antibody. The sensitivity of the assay was improved from 10 3 to 10 2 leptospires per ml by using an ethanol precipitation procedure to treat each sample. The assay detected only 31 of 56 (55 per cent) urine specimens culture-positive for hardjo, but seven of 24 urine samples culture-negative for hardjo were identified as positive by the assay. These seven samples were from animals which were culture positive on at least one other occasion. These results suggest that this system should be further investigated as a complementary test to culture for the identification of hardjo carrier animals.

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes

1. 1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161–167) detected a single DNA fragment after digestion with Barn H1, Hind III or Pst 1. 4. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.

A high-capacity hydrophobic adsorbent for human serum albumin

A simple method, based on salting out hydrophobic interaction chromatography, for the efficient removal of trace amounts of serum albumin from partially purified protein preparations is described. The method is also successfully applied for the purification of albumin from Cohn fraction IV, a by-product obtained from the commercial fractionation of human serum proteins by the ethanol precipitation procedure. About 70% of the adsorbed albumin can be eluted by buffer of low ionic strength and can thus be lyophilized directly, if required. The adsorbent can be used for several cycles of adsorption and desorption without affecting its selectivity or capacity. Its adsorption properties and capacity for serum albumin are compared with those of the commercially available adsorbent Blue Sepharose CL-6B.

Ethanol Fraction of Lacustrine Colloidal Fibrils

Ethanol fractionation of colloidal organic fibrils can provide a highly purified fraction in appropriate quantities for standard chemical analyses of fibril structure and function. Using a sequential ethanol precipitation procedure, applied to lacustrine fibrils isolated and concentrated by physical separation techniques, we obtained a fibril fraction (approximately 60% yield). Its characteristics were as follows: (1) absence of any major category of colloidal impurity detectable by transmission electron microscopy, (2) relative freedom from fibrils of atypical morphology, (3) relative freedom from a class of small granules, typically associated with lacustrine fibrils and with some other kinds of polyanionic fibrillar colloids found in nature, (4) freedom from derivatization reactions, and (5) freedom from an undesirable exposure to caustic reagents. Our objective was to arrive at a nondegradative fractionation procedure that yielded a fraction relatively free of other colloidal components found in lake water. This fraction would then be appropriate for assays of heavy metal binding and bioavailability.

Capsular material and morphology of some ampicillin sensitive and resistant strains of Escherichia coli K12.

In a previous genetic study of a highly ampicillin resistant mutant of Escherichia coli it was observed that certain classes of recombinants always showed smooth colony morphology. The present work was undertaken in order to investigate a possible correlation between ampicillin resistance and capsule production, giving smooth colony morphology. Two methods for preparing capsular material were compared: ethanol precipitation and negative pressure concentration. Chromatographic examination of these preparations indicated that the negative pressure concentration method gives a more native product with a larger molecular weight than was obtained by the ethanol precipitation procedure. We have compared the amount of capsular material produced by seven different strains, two rough and five smooth. Large variations were observed in the amount of capsule produced but there was no obvious correlation between capsule production and ampicillin resistance. The surface structure of five of the strains was studied by electron microscopy. The smooth strains showed two types of structures closely adhering to the outermost layer of the wall, small vesicles of the order of 40–150 mμ in diameter and minute lamellated units measuring from 80 to 400 Å in length. The rough strains showed very few of these structures. When capsule producing strains were grown at room temperature more capsular material was produced and more of these extra‐surface structures were observed. It is suggested that the small vesicles and minute lamellated units may represent cell wall material produced in excess amounts.

Carbohydrate content of bovine collagen preparations.

Collagen preparations from bovine tissues were analysed for their carbohydrate content. Crude preparations of tropocollagen and polymeric collagen were found to be contaminated with considerable amounts of mannose, fucose and hexosamine, sugars known to be present in the mucoprotein of the interfibrillar material with which collagen is associated in vivo. A pure preparation of tropocollagen obtained by ethanol precipitation procedures contained only galactose and glucose in the approximate ratio of 7:3 residues/3000 amino acid residues. Purification of crude polymeric collagen by EDTA extraction or by crude bacterial amylase extraction considerably decreased the mucoprotein contamination, particularly in the enzymic treatment, which yielded a preparation containing predominantly galactose and glucose in the ratio of 4:2 residues/3000 amino acid residues. The results confirm previous work that demonstrated the purity of these collagen preparations as inferred by amino acid analysis. The results also indicate the suitability of the pure tropocollagen and the amylase-extracted polymeric collagen for studies on the role of the carbohydrate residues in intramolecular and intermolecular cross-linking in collagen.

A method for preparing plasma factor 8 (antihemophilic globulin) concentrate.

Treatment of patients with hemophilia A is based on the administration of exogenous Factor VIII (antihemophilic globulin). Usually this is accomplished by infusing fresh, fresh-frozen, or reconstituted freeze-dried normal human plasma. The large volume of plasma needed to achieve and maintain hemostasis may be sufficient to cause circulatory overload, and thereby represents a critical limiting factor in therapy. During recent years, several therapeutically effective concentrated Factor VIII preparations have become available. These products are similar to Cohn Fraction I, which is prepared from whole plasma by ethanol extraction. Unfortunately, the difficulties encountered in procuring large volumes of fresh plasma to use as raw material, and other problems related to the lability of Factor VIII, the complexity of necessary equipment and methods, and the need for rigid bacteriologic control, have limited production and raised the cost of these products. Attempts to produce an inexpensive concentrate have recently met with success. Two developments have permitted formulation of a method for preparation of Factor VIII concentrate that may be used in any blood bank. The first important observation was that of Pool and Robinson, who found that Factor VIII precipitates with fibrinogen in the cold, just as it does in salting-out and ethanol precipitation procedures. The second contributing factor was the definition of plasmapheresis as an alternate form of blood donation which permits collection of large

Immunochemical studies on blood groups. XVIII. Fractionation of hog gastric mucin and individual hog stomach linings

1. 1. Blood-group substances were prepared from pooled hog gastric mucin, and from individual hog stomach linings after autolysis and peptic digestion, by the phenol extraction and fractional ethanol precipitation method of Aminoff, Morgan, and Watkins. The analytical and immunochemical properties of these fractions were compared with the properties of substances purified by the earlier phenol-ethanol method of Morgan and King. No significant separation of the A and O(H) substances in pooled hog gastric mucin was achieved by the fractional ethanol precipitation procedure. The use of either method to prepare blood-group A substance from single A hog stomachs devoid of O(H) activity resulted in products which contained similar amounts of blood-group A antigen, as judged by quantitative immunochemical methods. In the fractionation of single hog stomach linings, whether A or O(H), the dispersion of active material in the phenol-ethanol system appeared to depend on the completeness of peptic digestion. 2. 2. An acid-soluble and an acid-insoluble fraction prepared from hog gastric mucin by ethanol precipitation and electrodecantation by Holzman and Niemann differed in no important respect from fractions of gastric mucin purified by either phenol-ethanol procedure.