Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures

Summary Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Cullinget al. (1976), have been devised for the histochemical identificantion of side-chainO-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiffborohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chainvicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chainO-acylated sialic acids.     

Histochemical detection of sialic acid residues using periodate oxidation

Synopsis The use of low concentrations of periodate for the detection of sialic acid residues in tissue sections has been investigated. Oxidation of aqueous solutions of sugar glycosides with 0.4mm periodate revealed that sialic acid was oxidized more rapidly than other sugars found in glycoproteins. Sequential treatment of tissue sections with 0.4mm periodate for 30 min followed by Schiff's reagent stained sialic acid residues but other sugar components were not stained under these conditions.     

Glycoengineering the N-acyl side chain of sialic acid of human erythropoietin affects its resistance to sialidase

Abstract During the last years, the use of therapeutic glycoproteins has increased strikingly. Glycosylation of recombinant glycoproteins is of major importance in biotechnology, as the glycan composition of recombinant glycoproteins impacts their pharmacological properties. The terminal position of N-linked complex glycans in mammals is typically occupied by sialic acid. The presence of sialic acid is crucial for functionality and affects the half-life of glycoproteins. However, glycoproteins in the bloodstream become desialylated over time and are recognized by the asialoglycoprotein receptors via the exposed galactose and targeted for degradation. Non-natural sialic acid precursors can be used to engineer the glycosylation side chains by biochemically introducing new non-natural terminal sialic acids. Previously, we demonstrated that the physiological precursor of sialic acid (i.e., N-acetylmannosamine) can be substituted by the non-natural precursors N-propanoylmannosamine (ManNProp) or N-pentanoylmannosamine (ManNPent) by their simple application to the cell culture medium. Here, we analyzed the glycosylation of erythropoietin (EPO). By feeding cells with ManNProp or ManNPent, we were able to incorporate N-propanoyl or N-pentanoyl sialic acid in significant amounts into EPO. Using a degradation assay with sialidase, we observed a higher resistance of EPO to sialidase after incorporation of N-propanoyl or N-pentanoyl sialic acid.     

A new histochemical method for the identification and visualization of both side chain acylated and nonacylated sialic acids.

A new histochemical method is described for the differentiation of mucins that utilizes two different Schiff reagents and allows single section identification of side chain O-acylated, and nonacylated, sialic acids in contrasting colors. In the event of mucins containing only one type of sialic acid, it may allow their specific identification (e.g., C7 or C8 side chain O-acylated). It has been shown to be useful in the identification of some metastases from adenocarcinomas of colon (where the primary is potassium hydroxide/periodic acid-Schiff positive) and should prove of great value in the investigation of diseases of the gastrointestinal tract and particularly those of the colon. It should also be valuable in the general field of epithelial mucin histochemistry, particularly for those mucins of the salivary and parotid glands, etc.     

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

Histochemical investigations on sialic acid containing compounds in the CNS of teleosts (author's transl)]

By means of histochemical experiments the qualitative (microphotographs) and quantitative (cytophotometry) distribution of sialic acid containing compounds was investigated in the mesencephalon and cerebellum of Carassius carassius. 1. The perikarya of nerve cells are especially stained by means of Azur A. 2. Nerve fibres, on the other hand, showed positive reactions with colloidal ironhydroxyd (CIH). 3. In different structures of the CNS the intensity of CIH-staining is decreased up to about 55% following treatment with neuraminidase. 4. There are no differences in the reactions of 7 degrees C as compared to 20 degrees C adapted animals.     

全细胞高通量筛选a-氨基酸酯水解酶突变体的方法

【目的】建立高效敏感的高通量筛选方法,用于筛选头孢克洛合成活性提高或热稳定性提高的a-氨基酸酯水解酶。【方法】根据头孢克洛在碱性条件下水解生成的衍生物在340 nm处有特征吸收峰的原理,制作出标准曲线。采用全细胞96孔板紫外分光光度法高通量测定a-氨基酸酯水解酶突变体的头孢克洛合成活性。【结果】头孢克洛含量与△A340?405在0.1?0.6×10?3 mol/L浓度范围内有良好的线性关系, 服从朗伯-比尔定律, 平均回收率为99.8%?101.3％。一轮定点饱和突变产生的2 300个克隆经该方法的筛选, 获得3株kcat提高40%以上, 4株半失活温度较野生型提高5 °C以上的突变体酶。【结论】该方法准确可靠,每天筛选量可达到2 000个反应, 达到高通量筛选要求。     

Histochemical analysis of sialic acids in the epididymis of the rat

 A variety of sialic acids contained in the rat epididymis were histochemically examined by means of lectin and pre-lectin methods by light microscopy. Epididymides from adult male Sprague-Dawley rats were fixed in Bouin’s fluid and routinely embedded in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Limax flavus, Sambucus nigra, Sambucus sieboldiana or Maackia amurensis lectins or to the selective periodate oxidation–phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The present results revealed that principal cells in the initial segment and caput contain sialic acid linked to α2,6-galactose/N-acetylgalactosamine, whereas those in the corpus and cauda include the sialic acidα2,3-galactose sequence. Narrow and clear cells involve all the types of sialic acids examined. Basal and halo cells mainly contain sialic acidα2,3-galactose. 8- And/or 9-O-acetylated sialic acids were predominantly distributed in principal cells of the initial segment and proximal caput. These findings are taken to indicate that various sialic acids in the epididymis could participate in different physiological functions characteristic of the regions in this organ. Accepted: 10 November 1997     

In vitro selection of sialic acid specific RNA aptamer and its application to the rapid sensing of sialic acid modified sugars

Sialic acids (SAs) are located on the terminal positions of glycan on a cell surface, which play important role in the spread and metastasis of cancer cells and infection of pathogen. For their detection and diagnosis, the finding of SA specific ligand is an essential prerequisite. Here, RNA aptamer for N‐acetylneuraminic acid (Neu5Ac), a representative of SAs, with the high affinity of 1.35 nM and the selectivity was screened by in vitro selection method. The strong binding of the screened aptamer was enough to protect the hydrolysis of Neu5Ac by neuraminidase with the stoichiometry of 1:1 molar ratio. For the rapid detection of SAs, the RNA aptamer was further engineered to the aptazyme sensor by conjugating with a ribozyme following the characterization of selected aptamer by RNase footprinting assay. Without additional desialylation, modification, or/and purification processes, the aptazyme indicated high catalytic activities in the presence of Neu5Ac over 20 µM in several minutes. Also, we observed that the aptazyme sensor shows high sensitivities to Neu5Ac‐conjugated sugars as well as Neu5Ac monomer, but not in non‐Neu5Ac modified sugars. The aptamer for Neu5Ac can support valuable tools in a wide range of bioanalytical applications as well as biosensors. Biotechnol. Bioeng. 2013; 110: 905–913. © 2012 Wiley Periodicals, Inc.     

Cholesteryl ester storage disease and Wolman disease: phenotypic variants of lysosomal acid cholesteryl ester hydrolase deficiency

The lysosomal enzyme responsible for cholesteryl ester hydrolysis, acid cholesteryl ester hydrolase, or acid lipase (E.C.3.1.1.13) plays an important role in cellular cholesterol metabolism. Loss of the activity of this enzyme in tissues of individuals with both Wolman disease and cholesteryl ester storage disease is believed to play a causal role in these conditions. The objectives of our studies were not only to directly compare and contrast the clinical features of Wolman disease and cholesteryl ester storage disease but also to determine the reasons(s) for the varied phenotype expression of acid cholesteryl ester hydrolase deficiency. Although both diseases manifest a type II hyperlipoproteinemic phenotype and hepatomegaly secondary to lipid accumulation, a more malignant clinical course with more significant hepatic and adrenal manifestations was observed in the patient with Wolman disease. However, the acid cholesteryl ester hydrolase activity in cultured fibroblasts in both diseases was virtually absent. In addition, fibroblasts from both Wolman disease and cholesteryl ester storage disease were able to utilize exogenously supplied enzyme, suggesting that neither disease was due to defective enzyme delivery by the mannose-6-phosphate receptor pathway. Coculture and cell fusion of fibroblasts from Wolman disease and cholesteryl ester storage disease subjects did not lead to correction of the enzyme deficiency, indicating that these disorders are allelic. However, the activities of the hepatic acid and neutral lipase in these two clinical variants were quite different. Hepatic acid lipase activity was only 4% normal in Wolman disease, but the activity was 23% normal in cholesteryl ester storage disease. The hepatic neutral lipase activity was normal in Wolman disease but increased more than twofold in cholesteryl ester storage disease. These combined results indicate that the clinical heterogeneity in acid cholesteryl ester hydrolase deficiency can be explained by a varied hepatic metabolic response to an allelic mutation.