Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

A Simple Preparative Method for the Isolation of Amylose and Amylopectin from Potato Starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8, 0.1 M) ± urea (2 M) ± 1-butanol (8.5 %, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and β -amylolysis limit.     

A simple preparative method for the isolation of amylose and amylopectin from potato starch

The defatted starch was dispersed in NaOH (1 M) and neutralized with HCl (1 M). The amylose 1-butanol complex is adsorbed on defatted cellulose powder in the solvent system containing acetate buffer (pH 4.8,0.1 M) + urea (2 M) + 1-butanol (8.5%, v/v). The complex adsorbed on cellulose powder is separated by centrifugation (2418 g). The sediment is washed with the solvent system-I to obtain the intermediate fraction. The adsorbed amylose is eluted with urea (2 M) in acetate buffer (pH 4.8, 0.1 M). The amylose, intermediate fraction and amylopectin were precipitated with ethanol, washed free of urea and air dried. They were characterized by determining their blue value and beta -amylolysis limit.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

Glycosaminoglycans and acidic glycoproteins in rabbit uterus under estrogenic conditions.

Uterine slices obtained from the estrogen-treated rabbits were digested with pronase. Glycosaminoglycans and acidic glycopeptides were then isolated by Dowex 1 column chromatography and preparative electrophoresis on cellulose acetate membrane (Separax), in succession. Each subfraction thus obtained was identified by the mobility on Separax electrophoresis and the digestibility with mucopolysaccharidases (Streptomyces hyaluronidase, testicular hyaluronidase, chondroitinase AC, chondroitinase ABC and heparinase). The resulting data showed that each complex saccharide (hyaluronic acid, heparan sulfate, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, sulfated glycopeptide and sialoglycopeptide) was separated into 2-5 fractions, indicating charge and/or molecular heterogeneity of each complex saccharide.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

A Toluidine Blue-Membrane Filter Method for the Quantitative Staining of Bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

A toluidine blue-membrane filter method for the quantitative staining of bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

Dual staining of some sulfated mucopolysaccharides with alcian blue (pH 1.0) and ruthenium red (pH 2.5)

Summary A dual staining technique has been presented for the histochemical characterization of some sulfated mucopolysaccharides. It is a combined alcian blue (pH 1.0)-ruthenium red (pH 2.5) staining method which colors most sulfated mucopolysaccharides tested purple or purplish blue. A series of histochemical experiments using histological sections and casein films containing acidic polysaccharides of known chemical structure indicate that reactive sulfate and carboxyl groupings of polysaccharides are responsible, to an appreciable degree, for the alcianophilia and affinity towards ruthenium red of the substances respectively. A hypothesis is advanced as to the mechanism whereby ruthenium red binds anionic groupings of mucopolysaccharides.This investigation was supported by a Grant-in-Aid from the Japanese Ministry of Education (1968).     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

Strongyloides ratti and Trichinella spiralis: net charge of epicuticle

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.     

PRESENCE OF A SULFATED POLYSACCHARIDE IN THE EXTRACELLULAR MATRIX OF PLATYDORINA CAUDATA (VOLVOCALES,CHLOROPHYTA)1

Evidence for the presence of a sulfated polysaccharide component within the extracellular matrix of Platydorina caudata Kofoid is presented. In situ staining with alcian blue and toluidine blue O indicates accumulation of a sulfated polysaccharide in the matrix. The entire matrix was readily solubilized by a hot aqueous extraction and a sulfated proteoglycan complex was isolated. Thin-layer chromatography of hydrolysates and infrared analysis and chemical desulfation of the intact molecule indicate that the polysaccharide component is principally an arabinogalactan with ester-linked sulfate groups. Protease treatment of the extract revealed two distinct bands separable on cellulose acetate electrophoresis. The slower moving component was a sulfated glycoprotein while the faster moving component was a sulfated mucopolysaccharide essentially free of protein. This is the first report of specific chemical analyses and electrophoretic separation of a sulfated polysaccharide within the matrix of a member of the Volvocales. The cytochemistry and electrophoretic patterns of the P. caudata preparation are compared with the same type of extract made from Chlamydomonas reinhardtii Dang. The possible evolutionary significance of the electrophoretic patterns is presented.     

Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0–5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R ² = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41–47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (μ _max) were 0.42 and 0.32 h⁻¹ for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.     

Histochemistry of carbohydrates in the epithelium lining the bulbourethral gland of the rat

The histochemistry of carbohydrates has been studied in the epithelium lining the bulbourethral gland of the rat by means of light- and electron-microscopic methods. The results obtained show that the cytoplasms of the epithelial cells contain neutral and acidic carbohydrates. The neutral carbohydrates exhibited positive reactions with periodic acid-Schiff and periodic acid-thiosemicarbazide-silver proteinate, whereas the acidic carbohydrates reacted positively with alcian blue (AB; pH 1.0 and 2.5) and dialyzed iron. Most neutral carbohydrates were found to be glycoproteins localized within the secretory granules. The acidic carbohydrates consist of at least two types, AB (pH 1.0)-reactive sulfated and AB (pH 2.5)-reactive nonsulfated carbohydrates; most nonsulfated carbohydrates were determined to be sialic acid. The acidic carbohydrates were also localized within the secretory granules.     

FORMATION AND CHARACTERIZATION OF THREE TYPES OF YEAST INVERTASE

Three types of invertase (invertase I, II and III) are separatedfrom the soluble and insoluble fractions (4,500xg, 10 min supernatantand pellets of the homogenate, respectively) of baker's yeastby a DEAE cellulose column chromatography. The invertases Iand II are eluted with 0.1 M sodium acetate buffer (pH 3.9)and with 0.1 M sodium acetate buffer (pH 6.2) containing 0.1M NaCl from DEAE cellulose respectively, whereas the invertase-IIIremains adsorbed on the cellulose under these conditions. Theyare present in proportions of 2.5: 1 : 0.06 in the soluble fractionand 1.4: 1 : 0.12 in the insoluble fraction of the fresh baker'syeast cells. While in-vertase-II remains at a constant level,invertases I and III in the soluble fraction increase upon incubationof cells for the formation of invertase under the continuoussupply of sucrose. Invertases I and II differ from each other considerably in theoptimum pH and slightly in the response to (activation and inactivationby) crude papain and are identical with respect to the heatstability and probably to the affinity for sucrose. ¹Present address: Chemical Laboratory, Nippon Medical School,Konodai, Ichikawa-shi, Chiba-ken.     

An optimized method for extraction and detection of Coconut cadang-cadang viroid(CCCVd) from oil palm

Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Stability of glucose oxidase and catalase adsorbed on variously activated 13X zeolite

The use of 13X zeolite (0.1-0.4-mm granules), treated with 2N and 0.01N HCI, 0.01M citric acid, 0.1M citric-phosphate buffer (pH 3.6), and in untreated form to adsorb glucose oxidase of fungal origin and microbial catalase was examined. Physicochemical analysis of the support demonstrated that its crystalline structure, greatly altered by the HCl and buffer, could be partially maintained with citric acid. The specific adsorption of the enzymes increased with decreasing pH and proved to be considerable for all the supports. The stability with storage at 25 degrees C is strictly correlated with the titrable acidity of the activated zeolite expressed as meq NaOH/g and with pH value of the activation solution. It proved to be lower than 55 h for both enzymes if adsorbed on zeolite treated with 2N HCl, and 15-fold and 30-fold higher for glucose oxidase and catalase adsorbed, respectively, on zeolite treated with the 0.1M citric-phosphate buffer and 0.01M citric acid. The specific adsorption of glucose oxidase and catalase was, respectively, 1840 U/g at pH 3.0 and 6910 U/g at pH 5.0. Their half-life at 25 degrees C with storage at pH 3.5 for the former and at pH 5.0 for the latter was 800 and 1560 h vs. 40 and 110 h for the corresponding free enzymes.     

Protease-catalyzed tripeptide (RGD) synthesis

The tripeptide Bz-Arg-Gly-Asp(-OMe)-OH was synthesized by enzymatic method. Bz-Arg-Gly-OEt was synthesized by trypsin in ethanol containing 0.1 M Tris/HCl buffer (pH 8.0), and then H-Asp(-OMe)(2) was incorporated into the Bz-Arg-Gly-OEt using chymopapain in 0.25M CHES/NaOH buffer (pH = 9.0, EDTA 10 mM). The yield of Bz-Arg-Gly-OEt and Bz-Arg-Gly-Asp(-OMe)-OH were 80% and 70% using 1M Bz-Arg-OEt and 0.5M Bz-Arg-Gly-OEt, respectively. For Bz-Arg-Gly-OEt synthesis reaction at high concentrations of the substrates, the buffer content in ethanol was a key factor to determine the optimal reaction condition. In Bz-Arg-Gly-Asp(-OMe)-OH synthesis reaction, the yield was low in organic solvent due to various side products such as Bz-Arg-OH, Bz-Arg-Gly-OH, and Bz-Arg-Gly-Asp(-OMe)-Asp(-OMe)-OH, suggesting that chymopapain has a very broad substrate specificity of the S(1) site. The Bz-Arg-Gly-Asp(-OMe)-OH synthesis rate and its yield were dramatically elevated and the side reactions were reduced using only the CHES/NaOH buffer (pH = 9.0, EDTA 10 mM) as a reaction media. The final product Bz-Arg-Gly-Asp(-OMe)-OH was identified to be formed via C-terminal hydrolysis of Bz-Arg-Gly-Asp(-OMe)(2) after the nucleophile, H-Asp(-OMe)(2), was added.     

Simple method for the preparation of haptoglobin]

The present paper deals with a method permitting the isolation of haptoglobin 2-2 from human serum or plasma. The haptoglobin is adsorbed to DEAE-cellulose at pH 5.1 by batching. The loaded cellulose is given into a column and the haptoglobin eluted by a 0.1 M to 0.15 M acetate buffer. The last step is a gel-chromatography on Sephadex G-200. Disc- and immunoelectrophoresis were used to test the purity. As by-product acid alpha1-glycoprotein can be obtained.