Recombinant Components of the EcoRII Restriction–Modification System: Restriction Endonuclease Can Interact with DNA · RNA Duplexes

To obtain recombinant restriction endonuclease (R) and methylase (M) of the EcoRII restriction–modification system, bacterial strains overproducing their functional hexahistidine derivatives were constructed. Active full-length R·EcoRII was produced only in cells that also expressed M·EcoRII from a multicopy plasmid. Recombinant R·EcoRII bound with hybrid DNA·RNA duplexes.     

Purine and pyrimidine base and nucleoside concentrations in human cerebrospinal fluid and plasma

Purine and pyrimidine base and nucleoside levels were determined in adult human lumbar (CSF) and plasma by reversed-phase high performance liquid chromatography (HPLC). Guanine, thymine, cytosine and uracil were not detectable (<0.1 M) in human CSF or plasma. Adenine was detectable in plasma (0.3 M) but was not found in CSF (<0.2 M). Hypoxanthine and xanthine levels in CSF were each approximately 2.5 M. Plasma levels of hypoxanthine and xanthine were considerably lower (0.4–0.6 M). Purine and pyrimidine ribouncleosides in human CSF were less than or equal to 0.2 M with the exception of uridine which was present at concentrations of 2–3 M. Although low concentrations of thymidine and deoxyuridine (0.2 M) were present in human plasma, purine and pyrimidine deoxyribonucleosides were less than 0.1 M in human lumbar CSF.     

Relations between Computational and Experimental Engineering Approaches to Molecules from Molecular Fragments

Computational techniques have become important tools in the preliminary stages of the design of new molecules. The mutual arrangements of interacting molecular parts and the required accessibility of important reactive groups on the peripheral regions of possible new molecules can be tested by computational means before the expensive and often complex synthetic methods are used to the actual construction of these molecules. There are some common features involved in the computational representation of molecular fragments and the synthetic methodologies used in the process of incorporating actual molecular fragments in such engineered molecules. One trend that appears to link these two approaches is based on the following observation: the greater local autonomy is shown by the calculated electron density contribution of a given molecular fragment, the more likely that this fragment can be regarded as a suitable building block of the molecule, and it is also more likely that there are convenient synthetic methods for the delivery of this fragment to its desired target location in the new molecule to be synthesized. For a precise formulation of this statement, a new concept, the degree of molecular fragment autonomy is introduced, using the inherent properties of molecular electron densities.     

Introduction

The only concrete basis for the discontinuous and hierarchical organization of extant organisms lies in their genealogical (i. e. germ line) relationships. Individuals and populations of common descent are called sib or stirps. Ideally, systematic classification is based on the formulas: (1) sib + taxonomic category + name = taxon, and (2) divergent genealogy of sibs + hierarchy of taxonomic categories + names = taxonomic system (Fig. 1).Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

Physical and Epitope Analysis of a Recombinant Human T-Cell Receptor Vα/Vβ Construct Support the Similarity to Immunoglobulin

The genetic organization and protein structure of T-cell receptors (TCR) and immunoglobulins (Ig) are remarkably similar. Through recombinant, physical, and peptide-based immunological studies we demonstrated that rabbit antisera generated against a recombinant single-chain TCR (scTCR) react with defined peptide epitopes of their constituent TCR and chains. These antisera cross-react with the light-chain Mcg as well as with peptides duplicating its covalent structure. Conversely, rabbit antisera generated to human light chains cross-reacted with the recombinant scTCR. Rabbit anti- antibodies purified on an scTCR affinity column bound to T-cell lines and to T and B lymphocytes from peripheral blood. Circular dichroism analysis demonstrated plots characteristic of -sheets for both Mcg and recombinant scTCR. Antisera directed against TCR -chain synthetic peptides reacted with scTCR, Mcg light-chain protein, synthetic peptides from regions of sequence homology in -chains, and Mcg. Based upon this homology and the serological cross-reactions which reflect conformational determinants, we suggest that the V/V antigen-binding domain of this particular monoclonal scTCR construct is substantially similar to the conformational structure of light chains.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

High-affinity binding of fungal β-glucan elicitors to cell membranes of species of the plant family Fabaceae

Microsomal preparations of six species of the plant family Fabaceae were screened for high-affinity binding of branched (1 3), (1 6)--glucans. Oligoglucosides of this type are specific elicitors of phytoalexin accumulation in soybean (Glycine max L.), a member of this family. The species studied were alfalfa (Medicago sativa L.), broadbean (Vicia faba L.), chickpea (Cicer arietinum L.), french bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and white lupin (Lupinus albus L.). A ¹²⁵I-labeled 4-(2-aminophenyl)ethylamine conjugate of a (1 3), (1 6)--glucan fraction with an average degree of polymerization (DP) of 18, obtained from mycelial walls of Phytophthora sojae, was used as radioligand for initial screening. The structural complexity of this fraction allowed the identification of binding sites with affinities for isomeric structures other than the (1 3), (1 6) hepta--glucoside for which soybean binding sites display highest affinity. Radioligand competition experiments against unlabeled fungal -glucan resulted in the identification of high-affinity binding in alfalfa, bean, lupin, and pea. Half-maximal competition concentrations (IC₅₀) for fungal -glucan in these species were between 5 and 30 nM. Pseudoheterologous radioligand competition by unlabeled hepta--glucoside showed that for alfalfa, lupin and pea the IC₅₀ values for this structure (4 to 16 nM) were similar to those of soybean (7.7 nM). Bean microsomes, however, displayed an IC₅₀ significantly higher than soybean (68 nM) suggesting that the structural motif recognized by its binding sites is not identical to that of soybean or the other three species. Radioligand saturation assays with alfalfa, lupin and pea microsomes using an ₁₂₅I-labeled aminophenylethylamine hepta--glucoside conjugate gave dissociation constants (K_d) of 5.3, 3.7, and 1.8 nM, respectively. The affinity of these sites for hepta- glucoside was in the same range as that of soybean (K_d 1–3 nM), whereas the affinity of the binding sites of bean for the same ligand was significantly lower (K_d = 33 nM). Good correlation was found between the presence of high-affinity binding and the accumulation of isoflavonoid phytoalexins in roots of alfalfa, bean, chickpea and pea seedlings after exposure to fungal -glucan. Lupin displayed a strong wound-induced accumulation of prenylated isoflavones which was independent of the presence of -glucan, making it impossible to determine phytoalexin induction in response to elicitor. No specific binding or phytoalexin accumulation in response to glucans was observed in broadbean. This is the first report on the existence of possibly homologous elicitor-binding sites within a plant taxonomic family and may provide preliminary evidence for putative evolutionary relationships in pathogen perception mechanisms in plants.Abbreviations DP degree of polymerization - EC₅₀ concentration of elicitor necessary to obtain a half-maximal biological response - HG synthetic (1 3), (1 6)-hepta--glucoside phytoalexin elicitor - HG-APEA 1-[4-(2-aminophenyl)ethylamino-1-hexaglucosyl]deoxyglucitol - IC₅₀ ligand concentration necessary to obtain half-maximal displacement of radioligand in competition binding assays - K_d dissociation constant - OS branched (1 3), (1 6)--glucan obtained by hydrolysis of mycelial walls of Phytophthora sojae - OS-APEA 1-[4-(2-armnophenyl)ethylamino-1-oligoglucosyl]deoxyglucitol conjugate of OS This work was supported by the Comision Interministerial de Ciencia y Tecnologia grant BI091-0366 (E.G.C.), the Volkswagen-Stiftung (E.G.C. and J.E.), the Deutsche Forschungsgemeinschaft, SFB-369 (J.E.), the Bundesministerium fiir Bildung, Wissenschaft, Forschung und Technologie (J.E.), Fonds der Chemischen Industrie (J.E.) and the EU Human Capital and Mobility Program (J.E. and E.G.C.).     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

Hemoglobins S and C in Upper Volta

Summary We have studied the incidence of hemoglobinopathies in 1059 individuals in Upper Volta. We have found that this population has a high frequency of HbS and HbC and -thalassemia. The gene frequency of HbS was high (0.1 for the ^S gene) in the arid Sahel portion of Upper Volta accompanied by a lower frequency for HbC (0.05 for the ^c gene). The reverse was true in the humid Savanna region of this country (0.03 for the ^S gene and 0.14 for the ^c gene). There was no age dependency of the HbS gene frequency, but -thalassemia, detectable in HbS heterozygotes, showed a statistically significant decrease with age. No homozygote for HbS was detected after the age of 1 year, and SC and CC genotypes were found at a lower incidence than expected. The environmental and medical conditions in Upper Volta preclude the survival of SS individuals and decrease the survival of SC and CC genotypes.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Toxic responses of germinating pollen and soybeans to aflatoxins

Aflatoxin producing strains of Aspergillus grow on soybeans thereby contaminating the latter through secretion of the toxin. Investigations dealing with either soybean seed germination or intact seedling growth responses to aflatoxin (B₁) are lacking. Similarly, a possible interaction of aflatoxins with phosphate in the germination and elongation of both soybeans and pollen as well as roots of the former and tubes of the latter has not been examined. Imbibition of Glycine max, cv. Essex seeds for 18 hours in solutions containing 0.38, 2.90, 5.80 or 11.60 g/ml (AFB₁) yielded% germination inhibitions of 5, 20, 40 and 80, respectively. By 36 hours these were 6, 4, 13 and 19 % for the same toxin concentration series. At 140 hours attached root elongation was inhibited 26, 35 and 50 % for 2.90, 5.80 and 11.60 g/ml AFB₁. No effect was noted at 0.38 g/ml AFB₁. Incubation of excised roots in medium containing 3.0 mM KH₂PO₄ stimulated their elongation 3.2 fold. Addition of 33.28 g/ml mixed aflatoxins together with KH₂PO₄ resulted in only a 1.5 fold stimulation. When KH₂PO₄ was added to a culture medium lacking AFB₁, Lilium longiflorum, cv. Ace pollen germination was enhanced 50%. Withholding KH₂PO₂ but supplying AFB₁ did not markedly affect germination. However, supplementing the medium with KH₂PO₄ while simultaneously adding AFB₁ did not inhibit germination at 5 and 10 g/ml but caused 27.3 and 45.1 % declines at 25 and 30 g/ml. In the absence of KH₂PO₄ AFB₁ stimulated pollen tube elongation 7.5, 14.3, 16.5 and 13.2 % at 5, 10, 15 and 20 g/ml but 30 g/ml inhibited it 11.1%. In contrast, tube elongation was suppressed at all AFB₁ concentrations (maximum 36.1% at 30 g/ml) tested upon KH₂PO₄ addition. Results derived from germinating pollen in medium supplemented with KH₂PO₄ or NaH₂PO₄ indicate that the phosphate anion does not preferentially promote aflatoxin-induced inhibition of tube elongation.Aided by grant IN-127 from the American Cancer Society to W.V. Dashek and funds from the Departments of Biology, West Virginia University and Virginia Commonwealth University and the West Virginia University Foundation.     

Polygonal inequalities as a key to neuronic equations

Neuronic or decision equations, first proposed as a mathematical model of neural activity, have shown, after their exact, compact solution was found, typical behaviours that make them natural tools for General Systems studies. It is shown here that their mathematical investigation is remarkably furthered by generalizing the triangular inequality to polygonal ones. These permit the immediate computation of the tensorial expansion of linearly separable boolean functions, and exhibit clearly the connection between their continuous and discontinuous aspects.     

Signaling Pathways That Control the Growth and Survival of Prostate Carcinoma Cells in the Absence of Androgens

Androgen-dependent human prostate adenocarcinoma cell line LNCaP was used to study the effect of androgen deprivation on the cell response to TNF-related cytokines. Several signaling pathways were implicated in cell survival in the absence of androgens. In androgen-deprived LNCaP cells, TNF- and TRAIL stimulated the cell growth and activated the mitogenic and antiapoptotic signaling pathways involving NF-B, STAT3, PI3K, and -catenin. The results suggested a role of cytokines in the survival of prostate adenocarcinoma cells deprived of androgens in vitro.     

Primary structure of hemoglobin β-chain fromColumba livia (gray wild pigeon)

Primary structure of -chain of pigeon is presented. It was determined by amino acid sequence analysis of intact -chain and its peptides obtained by the enzymatic and chemical cleavage. Comparison of amino acid sequence of the chain with other available data shows 14 Ile, 61 Lys, and 113 Ile as residues specific to pigeon. One important replacement at ₁₁ contact is 55 MetSer.     

We Do Not Dream of the 3 R's: Implications for the Nature of Dreaming Mentation

This report examines the extent to which dream recall involves the 3 R's (reading, writing, and arithmetic). Two separate studies were done. In the first study, two scorers rated, on a blind basis, a total of 456 written dream reports, available from five previous studies. There was perfect agreement between the two scorers. They agreed that there were no instances of reading, no instances of writing, and one instance of probable calculating in the 456 dreams. The second study was a questionnaire survey. Complete responses were obtained from 240 frequent dreamers (who reported remembering a mean of seven dreams per week). The study examined in two ways the frequency of the 3 R's in their recalled dreams. First, in answer to direct questions as to how frequently they dreamt about each activity, roughly 90% of the respondents reported that they never or hardly ever dreamt about each of four activities: reading, writing, typing, and calculating. In answers to other questions, this group reported spending a mean of six hours per day engaged in these activities. Second, responses as to the relative prominence of six activities (walking, writing, talking with friends, reading, sexual activity, typing) in dreaming versus waking produced two clear groupings of activities. Walking, talking with friends, and sexual activity were each rated almost as prominent in dreaming as in waking whereas the second group consisting of writing, reading, and typing were rated as far more prominent in waking than in dreaming. The two activity groups differed at p < .0001. Thus, the 3 R's appear to occur very infrequently in dreams. These findings are placed in a theoretical frame which suggests that dreaming (compared to waking) deals very little with serial activities characterized by input—rapid-processing—output in which the neural nets function in a feed-forward mode. Rather, dreaming may be characterized by relatively broad or loose connection making in which the nets function more in an autoassociative mode.     

Architecture of the Alzheimer’s AβP Ion Channel Pore

We have proposed that the cytotoxic action of Alzheimers amyloid beta protein might be initiated by the interaction with the neuronal cell membrane, and subsequent formation of toxic ion channels. Consequently, AP toxicity can be explained on the basis of harmful ion fluxes across AP channels. The conformation of AP in membranes is not known. However, several models suggests that a transmembrane annular polymeric structure is responsible for the ion channel properties of the membrane-bound AP. To identify that portion of the AP molecule making up the conducting pore we have hypothesized that the region of the AP sequence in the vicinity of the hypothetical pore might interact with complementary regions in the adjacent AP subunits. We have further hypothesized that an interaction by a peptide segment would block AP conductance. To test this hypothesis we synthesized peptides that encompass the histidine dyad (H-H) previously hypothesized to line the pore. We report here that peptides designed to most closely match the proposed pore are, in fact, the most effective at blocking ion currents through the membrane-incorporated AP channel. As previously shown for Zn²⁺ blockade, peptide blockade is also asymmetric. The results also provide additional evidence for the asymmetric insertion of the AP molecules into lipid membranes, and give support to the concept that rings of histidines line the entry to one side of the AP pore.     

Utilization and degradation of lindane by soil microorganisms

Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane -1,2,3,4,5,6-hexachlorocyclohexane - -PCCH -2,3,4,5,6-pentachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - PCB pentachlorobenzene - 1,2,3,4-TCB 1,2,3,4-tetrachlorobenzene - 1,2,3,5-TCB 1,2,3,5-tetrachlorobenzene - 1,2,4,5-TCB 1,2,4,5-tetrachlorobenzene - 1,2,3-tCB 1,2,3-trichlorobenzene - 1,2,4-tCB 1,2,4-trichlorobenzene - 1,3,5-tCB 1,3,5-trichlorobenzene - 1,2-DCB 1,2-dichlorobenzene - 1,3-DCB 1,3-dichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine