A method for RNA isolation from marine macro-algae

Sulfated, carboxylic polysaccharides and polyphenols found in marine macro-algae interfere with RNA isolation from these plants and inhibit RNA activities in vitro. Methods based on differential precipitation of RNA or carbohydrates in high salts were used to eliminate the acidic carbohydrates. To protect RNA from inactivation by oxidized polyphenols, strong reducing reagents were used to prevent polyphenol oxidation. RNA was successfully isolated from Macro-cystis pyrifera (brown alga), Porphyra schizophylla (red alga), and Enteromorpha intestinalis (green alga). mRNA isolated from the total RNA was shown to be translationally active.     

Effect of thiol reagents on extractability of protein from yeast.

The effect of soluble thiol reagents on the extractability of protein from yeast cells was studied. The incubation of yeast cells with dithiothreitol, 2-mercaptoethanol, or monothioglycerol markedly stimulated the release of soluble carbohydrates into the medium. There was a concomitant improvement (over twofold) in the extractability of protein from the yeast cells. The thiol reagents activated the proteolytic enzymes of the yeast cells. Unless inactivated, these enzymes hydrolyze the extracted protein.     

Carbohydrate preferences of mammalian cells.

Carbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety-three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.     

Fluorometric determination of carbohydrate with 2-aminothiophenol

The 2-aminothiophenol-based fluorometric assay of Nakano et al. (1973, J. Pharm. Soc. Jpn. 93, 350-353) for monosaccharides has been modified to improve the speed, applicability, and sensitivity of the method. The improved assay is applicable to complex carbohydrates as well as to monosaccharides. Less than 50 ng of carbohydrate in a final volume of 2 ml can be quantitatively measured within 30 min. The assay is reasonably compatible with the presence of a variety of reagents commonly used in aqueous buffer solutions. The assay is especially useful for monitoring column eluents during the purification of small quantities of carbohydrates or their conjugates.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Ultrastructural and cytochemical studies on the developing adhesive disc of Boston Ivy tendrils

Summary Ultrastructural observations of the immature adhesive disc from tendrils of Boston Ivy showed that the peripheral cells, which are the presumptive contact layer, contain vacuoles of varied sizes which are filled with electron-dense aggregates. In small vacuoles, these deposits were appressed to the tonoplast and fusion of these small vacuoles with the large vacuoles apparently occurs. Cells from the central zone were largely parenchymatous. The vacuoles of many of these parenchyma cells contained electron-dense spheres and hemispheres of material either appressed to the tonoplast or within the vacuole lumen. In these cells, the vacuole-cytoplasm interface was characterized by a filiform network of interconnected membranes. Positive reactions with reagents for the identification of polyphenols indicate that the vacuoles of nearly all the peripheral cells and scattered cells of the central zone contain tanniniferous substances. Insoluble carbohydrates also occur in the vacuoles of the peripheral cells, but they contain little or no protein or lipid.     

The Use of Leuco Triphenylmethanes as Reagents for Bacterial Polysaccharide S. (Preliminary Report)

The present investigation was initiated because of the need of a reagent for detecting and estimating bacterial polysaccharides without having to hydrolyze them before testing. It was found that acidulated solutions of leuco triphenylmethanes and reduced bases of triphenylmethanes are either precipitated or oxidized to colored compounds by bacterial polysaccharides. Nonbacterial polysaccharides and simpler carbohydrates gave negative reactions. The wide variety of reactions obtainable by using different compounds of this group of intermediates makes it possible to apply them to many different polysaccharide problems. An outline is given of the reactions obtained by typical reagents.     

Respiratory activity of Acholeplasma laidlawii cells and its role in the active transport of carbohydrates]

The respiratory activity of the Acholeplasma laidlawii cells was studied in order to elucidate a possible mechanism of coupling of transport with energy. The respiration of the cells is stimulated by ethanol, glucose, NADH, lactate, and pyruvate. The substrates of the Krebs cycle have no effect on the respiration. The respiratory activity, stimulated by ethanol and glucose, is inhibited by the inhibitors of the respiratory chain, SH reagents, and the inhibitors of glycolysis. The results of experiments with inhibitors suggest that the respiratory chain in the A. laidlawii cells is reduced and terminated by flavoprotein. This is confirmed by the results of spectroscopic analysis of cytochromes. Respiration coupled with phosphorylation did not play any important role in the active transport of carbohydrates. Probably, the energy, necessary for the transport of carbohydrates, is supplied by the substrate phosphorylation. This explains the activation of respiration by glucose, which is so sensitive to arsenate. The respiration of the A. laidlawii cells is not stimulated by some carbohydrates (fructose, 3-O-methyl-D-glucose).     

The separation of biomolecules using capillary electrochromatography

The unique properties of capillary electrochromatography such as high performance, high selectivity, minimum consumption of both reagents and samples, and good compatibility with mass spectrometry make this technique an attractive one for the analysis of biomolecules including peptides, proteins, carbohydrates, nucleosides and nucleotides. Irreversible adsorption between the biomolecules and the charged packing surface leads to a lack of reproducibility and serious peak tailing, so various approaches have been taken to overcome this and to improve the technique for future challenges.     

Some alternative coupling chemistries for affinity chromatography

Three different coupling chemistries that have been tried and tested for use in affinity chromatography are described. These methods are particularly recommended for use by workers who do not have access to, or do not wish to use, complex organic chemical synthetic procedures. They have been demonstrated repeatedly to be reliable, efficient, low cost, and easily scaleable up or down in size. The periodate oxidation method works best with Sephacryl type gels and uses only low toxicity reagents and couples well to proteins with both high efficiency and high capacity. The vinyl sulfone method is more reactive and couples both carbohydrates and proteins. The bis-epoxide method, although less reactive, can be used under more extreme conditions of pH to couple otherwise unreactive molecules, such as synthetic polymers, drugs, and so forth.     

Mucins: structure, function, and associations with malignancy.

Mucins are a family of high molecular weight, highly glycosylated glycoproteins found in the apical cell membrane of human epithelial cells from the mammary gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus and rete testis. Increased synthesis of the core protein and alterations in the carbohydrates attached to these glycoproteins are believed to play important roles in the function and proliferation of tumour cells. Aberrant glycosylation leads not only to the production of novel carbohydrate structures, but also to the exposure of the core peptide. These novel epitopes may be candidates for diagnosis or therapy, by using either synthetic mucin fragments as vaccines, or monoclonal antibody-based reagents which detect these structures.     

Ultrastructural Cytochemistry of Secretory Granules of Esophageal Glands of Meloidogyne incognita

Ultrastructural cytochemical tests for several enzymes, proteins, carbohydrates, and nucleic acids were conducted on secretory granules o£ dorsal and subventral esophageal glands of preparasitic second-stage juveniles and the dorsal gland of adult females of Meloidogyne incognita. Secretory granules in the subventral glands of juveniles stained positive for acid phosphatase. Peroxidase, DNase, RNase, cellulase, and nucleic acids were not detected in these granules. Secretory granules in the dorsal gland of adult females stained positive for peroxidase (pH 7.6) in < 50% of the tests, Acid phosphatase, β-glucuronidase, DNase, RNase, polyphenoloxidase, cellulase, and carbohydrates were not detected in dorsal gland granules in adult females. Positive staining with cobalt thiocyanate, a stain for amino groups of basic proteins, occurred in secretory granules in the dorsal gland, ribosomes, and chromatin in adult females. Ribosomes, nuclei, and secretory granules of the dorsal gland of adult females intensely stained when incubated in three reagents specific for nucleic acid.     

Preparation of 6-aminohexyl d-aldopyranosides

6-Aminohexyl glycosides covalently linked to solid matrices are effective reagents for the isolation of proteins that bind to carbohydrates [Schnaar and Lee, Biochemistry, 14 (1975) 1535–1541], and for the study of interactions between intact cells and immobilized carbohydrates [Weigel et al., J. Biol. Chem., 253 (1978) 330–333]. The preparation of the 6-aminohexyl glycosides of the following D-pyranoses is now described: β-glucose, β-galactose, 2-acetamido-2-deoxy-β-glucose, α-mannose, β-maltose, β-melibiose, β-lactose, and β-cellobiose. These glycosides were prepared by glycosylation of 6-(trifluoroacetamido)hexanol with the appropriate acetylated glycosyl halide in 1:1 (v/v) benzene-nitromethane, with mercuric cyanide as the catalyst. Deacylation of the glycosides was achieved in two steps: use of sodium methoxide for O-deacetylation, and of an anion-exchange resin for N-de(trifluoroacetyl)ation.     

Enzymatic and chemical inactivation of partially purified prothoracicotropic (brain) hormone of the silkworm,Bombyx mori

The effects of various enzymatic and chemical treatments on the biological activity of the partially purified PTTH are described. PTTH was inactivated by pepsin, trypsin and α-chymotrypsin. Leucine aminopeptidase and carboxypeptidase A did not affect the biological activity of PTTH, suggesting that N- and C-terminus are blocked. Treatments with chemical reagents suggest that the tryptophan residue and disulfide bond are essential for the activity, whereas the sulfhydryl group is not. Tyrosinase exerted no effects. Glycosidases, neuraminidase, and periodate oxidation did not affect the activity, suggesting that carbohydrates are not essential for the biological activity of PTTH.     

Nonantibody-based recognition: alternative molecules for detection of pathogens

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.     

Influence of alternansucrase-derived oligosaccharides and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis.

AIMS: To determine the influence of alternansucrase-derived oligosaccharides (AOS) and other carbohydrates on alpha-galactosidase and alpha-glucosidase activity in Bifidobacterium adolescentis. METHODS AND RESULTS: Activities for alpha-galactosidase and alpha-glucosidase were determined from cell extracts of B. adolescentis grown on 18 test carbohydrates including AOS. alpha-galactosidase activity was enhanced on a variety of alpha-linked or beta-linked carbohydrates regardless of a galactoside or glucoside. alpha-glucosidase, however, was enhanced only on alpha-linked carbohydrates. AOS significantly enhanced enzyme activity compared with most of the carbohydrates tested. Most of the AOS showed significant increases in activity for both enzymes over that displayed by their corresponding acceptor carbohydrates. CONCLUSIONS: alpha-galactosidase may serve as a biomarker for microbial metabolic activity within the large intestine for potential prebiotics composed of alpha-linked or beta-linked oligosaccharides whereas alpha-glucosidase activity may be restricted to assessing the influence of only alpha-linked carbohydrates. The AOS synthesis process provided a value-added component to carbohydrates by increasing metabolic activity (via alpha-galactosidase and alpha-glucosidase) over certain acceptor carbohydrates. SIGNIFICANCE AND IMPACT OF THE STUDY: Fundamental knowledge of enzyme activity in Bifidobacterium may aid in the design of more effective prebiotics and may also help identify enzyme indicators of metabolic activity when assessing influence within the intestine.     

Cellular metabolic control by chemical modification of cell membrane.

Various reagents used in the chemical modification of amino- and carboxy-groups of proteins, and of carbohydrates of glycoproteins and glycolipids, inhibit respiration in ascites tumor cells concomitant with release of potassium ion from those cells. The respiratory activity of washed ascites tumor cells is increased by exogenous addition of potassium ion. The lowered respiratory control index as well as oxidative phosphorylation of aged mitochondria are restored upon increasing the potassium concentration of the incubation mixture in the presence of respiratory substrates. The data suggest that the potassium ion level of cells is changed by modifying physicochemical properties of membrane components and that cellular energy metabolism is regulated by intracellular potassium ion concentration.     

Integrity of glycoprotein complex sugars is required for homing but not for several other membrane-mediated functions

In order to correlate the biochemistry of cell surface carbohydrates with cell function, we have treated cells with swainsonine and followed the biochemical and functional modifications induced by this compound. After treatment with swainsonine, surface glycoproteins had a lower apparent molecular weight and a higher isoelectric point. This is compatible with the replacement of complex carbohydrates by hybrid or high-mannose carbohydrates. Several functional tests were unaffected. However, swainsonine-treated cells displayed an altered pattern of in vivo homing, suggesting that carbohydrates play a role in this process.     

Amino acids in nectar enhance longevity of female Culex quinquefasciatus mosquitoes

Culex mosquitoes feed on a wide range of nectars consisting of mostly carbohydrates and amino acids, however, little is known about the utilization and effects of these different carbohydrates and their accompanying amino acids on longevity. Culex quinquefasciatus larvae were reared on low- and high-quantity food diets to produce adults that were nutritionally representative of wild-caught and laboratory-reared mosquitoes. Emerging adults reared on low- or high-quantity food diets as larvae were then provided Lantana camara nectar mimics containing mixtures of carbohydrates and amino acids to evaluate effects of nectar amino acids on longevity. Carbohydrates (with or without amino acids) were a critical component of the adult diet, and in their absence, adult mosquitoes died within 3-5 days. The nectar mimic that contained both carbohydrates and amino acids did not increase adult longevity of males originating from either poorly or well-fed larvae. However, females receiving adult diets containing both carbohydrates and amino acids lived 5% longer than females fed adult diets with only sugar.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.