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N Avdalovic

Publications and source records attributed to N Avdalovic.

At least 37 records · Page 2Linked to original sources
PubMed1996

Eliminating amino acid and peptide interference in high-performance anion-exchange pulsed amperometric detection glycoprotein monosaccharide analysis.

The monosaccharide content of a glycoprotein is often determined by acid hydrolysis at elevated temperature and subsequent high pH chromatography of the released, underivatized monosaccharides on pellicular anion-exchange resin (HPAE) using pulsed amperometric detection (PAD). We have found that for glycoproteins with low levels of glycosylation, monosaccharide quantitation can be compromised by amino acids fouling the working electrode surface. Specifically, lysine elutes on the CarboPac PA1 column just prior to galactosamine, whereas remaining amino acids and most peptides elute after the monosaccharides and do not interfere with monosaccharide quantification. A direct comparison of PAD vs Abs215 detection of lysine using the CarboPac PA1 column as the separator reveals that lysine does not cleanly come off the working electrode. The monosaccharide response inhibition caused by lysine could be corrected by the posthydrolysis addition of a rhamnose internal standard and the determination of "correction factors." We have developed a guard column with an altered selectivity for amino acids which, when used with a new separator, causes lysine to elute after the monosaccharides and also causes hydrophobic amino acids to elute further after the monosaccharides. Together the new separator and guard columns solve the lysine fouling problem, reduce sample-related baseline noise, and reduce the magnitude of correction factors.

Amino Acids↗
PubMed1996

Separation of human serum transferrin isoforms by high-performance pellicular anion-exchange chromatography.

Glycoproteins are microheterogeneous with respect to their attached oligosaccharides. When these oligosaccharides contain sialic acid, the oligosaccharide microheterogeneity will impart charge heterogeneity to the glycoprotein. We found that two commercial preparations of human serum transferrin (HST), a sialylated glycoprotein, have very different chromatographic profiles when the samples are separated by pellicular anion-exchange chromatography. Each anion-exchange profile contained multiple peaks, which suggested that both glycoproteins have charge heterogeneity. High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD) analysis of sialic acids and oligosaccharides released from the two preparations of HST revealed that the two preparations differed in sialic acid and sialylated oligosaccharide content. When oligosaccharides were released from the anion-exchange fractions of the two HST preparations, the HPAEC/PAD oligosaccharide profiles showed that protein retention was directly related to sialylated oligosaccharide content (i.e., the longer a fraction was retained, the greater its sialylated oligosaccharide content). Therefore, the anion-exchange profiles of the two HST preparations are related to their sialylated oligosaccharide content. We believe that pellicular anion-exchange chromatography can be used to quickly monitor gross changes in the sialylation of sialylated glycoproteins due to physiological state, or in the case of recombinant glycoproteins, culture conditions and/or purification.

Amidohydrolases↗
PubMed1994

Analysis of carbohydrates on IgG preparations.

Characterization of monoclonal antibodies (MAbs) produced for therapeutic or diagnostic purposes increasingly includes an assessment of their carbohydrate content. Using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD), we have analyzed the PNGase F released oligosaccharides of several IgG preparations including human polyclonal IgG, a humanized monoclonal IgG (MAb M115), and a murine monoclonal IgG (MAb MY9-6) derived respectively from serum, hybridoma cultures, and ascites fluid. The N-linked oligosaccharides released by PNGase F treatment of the above IgGs were found to consist mainly of neutral, fucosylated, biantennary species. Comparison of glycosylation of human polyclonal IgG, MAb M115, and MAb MY9-6 revealed differences in the levels of galactosylation and in the levels as well as the form of sialic acid present. HPAEC/PAD oligosaccharide profiling, combined with the use of enzymes (PNGase F, endoglycosidase F2, endoglycosidase H, neuraminidase, beta-galactosidase, and beta-N-acetylhexosaminidase), and monosaccharide analysis allowed making of tentative structural assignments. By performing monosaccharide analysis directly on PVDF electroblotted heavy and light chain bands separated by SDS-PAGE, it was verified that IgGs used in this study were glycosylated predominantly in their heavy chain.

Amidohydrolases↗
PubMed1993

Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We have developed an intermediate method toward the complete carbohydrate analysis of proteins, which should be universally applicable to all proteins and independent of sample matrix. Using only Coomassie Blue-stained proteins which have been electroblotted onto polyvinylidene fluoride membranes, we report a strategy for: (i) determining unequivocally whether a protein is glycosylated; (ii) obtaining a complete monosaccharide composition; (iii) oligosaccharide mapping which separates most forms according to size, charge and isomerity; and (iv) sequentially releasing and analyzing specific classes of oligosaccharides with endoglycosidases. The method was shown to be applicable to a variety of well characterized soluble glycoproteins and to the membrane-bound protein, the gastric H+, K(+)-ATPase. The monosaccharide composition of the H+,K(+)-ATPase revealed the absence of N-acetylneuraminic or N-glycolylneuraminic acids and a monosaccharide composition which indicated O-linked sugar chains. Oligomannosidic/hybrid and biantennary oligosaccharides were sequentially released and analyzed from one electroblotted band of recombinant tissue plasminogen activator using endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F2, respectively. Sialylated polylactosamine structures were identified and quantified by analyzing high performance liquid chromatography profiles of oligosaccharides first released by peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase and then treated with endo-beta-galactosidase, using a single, stained band of recombinant erythropoietin. This recombinant erythropoietin was found to contain eight times more tetrasialylated oligosaccharides than previously reported (Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076); 47% of released oligosaccharides were identified as polylactosamine structures.

Animals↗
PubMed1993

Neutralizing monoclonal antibodies that distinguish three antigenic sites on human cytomegalovirus glycoprotein H have conformationally distinct binding sites.

Seven neutralizing murine monoclonal antibodies specific for the glycoprotein H of human cytomegalovirus were produced and used to construct a topological map of two nonoverlapping antigenic sites that are bridged by a third antigenic site. Neutralization assays with 15 laboratory or clinical human cytomegalovirus strains indicated that the monoclonal antibodies recognize three antigenically variable and three conserved epitopes within the three antigenic sites. The variable-domain genes encoding monoclonal antibodies representing each of the three antigenic sites were cloned and sequenced, and molecular models of their binding sites were generated. Conformational differences in the antibody-binding sites suggested a structural basis for experimentally observed differences in gH epitope recognition.

Amino Acid Sequence↗
PubMed1991

Rapid restriction map constructions using a modified pWE15 cosmid vector and a robotic workstation.

This paper describes a number of techniques for rapid restriction mapping of cosmid clones. First, we have replaced the cloning site of cosmid vector pWE15 with a polylinker containing 15 infrequently cleaved restriction enzyme sites that are placed asymmetrically on each side of the BamHI cloning site. DNA cloned into this vector can be fully recovered by using several pairs of restriction enzymes. Second, we have designed a simple electrical circuit device that allows the performance of asymmetric voltage gradient field inversion gel electrophoresis (AFIGE) to improve the resolution of DNA molecules in the range of 20-50 kbp. AFIGE can be obtained by simply placing the device in between a commercially available switching unit and the gel box in a standard field inversion system. Finally, the restriction digestion procedure has been automated by using a Beckman Biomek 1000 robotic workstation. Using this automated system, 96 restriction reactions, including gel loading, can be performed in less than two hours. In summary, these methods represent at least a tenfold improvement in the speed and/or mapping data that can be obtained in a single gel.

Animals↗
PubMed1990

Development of an automated procedure for fluorescent DNA sequencing.

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.

Automation↗
PubMed1989

Characterization of primary transcripts and identification of transcription initiation sites on the heavy and light strands of mouse mitochondrial DNA.

Total RNA from Ehrlich ascites mitochondria pretreated with RNase-free DNase was capped in vitro with [alpha-32P]GTP and guanylyl transferase. The cappable RNAs representing the primary transcripts show a heterogeneous size distribution with four major species of 46, 63, 94, and 152 nucleotides and four minor species of 19, 24, 104, and 790 nucleotides in size. Hybridization with the D-loop DNA probes shows that the 19-nucleotide-long capped RNA is coded by the H-strand of mitochondrial DNA while the rest are coded by the L-strand. S1 nuclease mapping and primer extension analyses suggest the occurrence of a transcription initiation of H-strand at about 19 nucleotides upstream from the start of the tRNA(Phe) gene. All of the L-strand cappable RNAs have a common 5' end mapping to nucleotide 16,183 +/- 5 of the genome. The 3' ends of four major cappable RNA species line up to the conserved sequence boxes, putative start sites of DH-DNA; and in fact about 2% of these cappable species are found to exist as DNA-linked RNA under steady-state conditions. The 3' end of the 790-nucleotide cappable RNA lies close to the start of the tRNA(Pro) gene, suggesting that it may be the true precursor of L-strand transcript endonucleolytically processed at the 3' end. The level of L-strand-coded cappable RNAs varies markedly under different growth conditions. Treatment with cycloheximide results in a reduction while chloramphenicol caused over 3-fold induction, suggesting that these "primer" RNAs may have an additional regulatory function.

Animals↗
PubMed1989

Follicular exclusion of retroviruses in the bursa of Fabricius.

To gain insight into the regulation of retroviral infection at the cellular level, we analyzed the distribution of retroviral antigen and nucleic acid in the bursa of Fabricius of the parents and progeny of two highly inbred lines of chickens, one resistant and the other susceptible to infection. Line 15I5 chickens and line 7(2), which are C/C and C/A, respectively, and 15I5 x 7(2) F1 chickens were infected with either RAV-1 or RAV-49 avian leukosis virus (ALV). Most bursal follicles of F1 chickens infected with either virus contained a variable mixture of virus-positive and virus-negative cells and a few (1 to 20%) were void of detectable virus. However, in either parental line the respective virus was uniformly expressed among all follicles. The follicles which excluded virus in the F1 birds were indistinguishable from other infected follicles in the same bursa or in uninfected birds on the basis of histology or cellular antigen expression. It was concluded that virus susceptibility is most likely determined at the bursal stem cell level of differentiation, possibly by a process of allelic exclusion at the retroviral receptor locus.

Animals↗
PubMed1988

Transverse alternating field electrophoresis (TAFE).

The rapid development of electrophoretic technology during the past five years has given us a stream of improvements in the area of pulsed-field separation techniques. This progression has culminated in the conception of TAFE, a simple, high resolution technique providing straight lane geometry and separation of fragments up to 9 million base pairs in length. Pulsed-field techniques will unquestionably play a major role in the forthcoming analysis of the human genome by facilitating restriction mapping and cloning of large fragments. We have no doubt that TAFE will allow scientists to do this type of genetic analysis, faster and better than ever before.

Blotting, Southern↗
PubMed1985

Induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol 13-acetate in hamster fibroblasts. Relationship between levels of enzyme activity, immunoreactive protein, and RNA during the induction process.

Phorbol ester tumor promoters and growth factors rapidly stimulate ornithine decarboxylase activity in the transformed hamster fibroblast line HE68BP. We report here a close correspondence between the time courses and magnitudes of induction of ornithine decarboxylase activity and immunoreactive ornithine decarboxylase protein following treatment of HE68BP cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) and/or refeeding with fresh medium. Cycloheximide addition to induced cells caused a rapid fall in the levels of both ornithine decarboxylase activity and ornithine decarboxylase protein. Northern blot analysis of RNA isolated from HE68BP cells indicated that treatment with TPA and fresh medium increased the amount of two species of mRNA of lengths 2.4 and 2.1 kilobase. This increased accumulation of ornithine decarboxylase mRNA corresponded temporally to that observed at the protein level, with a 15-fold maximal induction 7 h after treatment followed by a rapid decline in hybridizable RNA. These data indicate that stimulation of ornithine decarboxylase activity by TPA or refeeding involves changes in levels of ornithine decarboxylase mRNA as well as changes in the rate of synthesis of ornithine decarboxylase protein.

Animals↗
PubMed1985

Bromodeoxyuridine treatment of rat liver cells in culture induces an increased metabolic conversion of exogenous ornithine.

In the presence of bromodeoxyuridine, rat liver cells show an increased incorporation of radiolabeled ornithine into cellular proteins. This increase was not due to activation of urea cycle enzymes but rather to an increase in metabolism of ornithine. Furthermore, treatment with bromodeoxyuridine changes profoundly and selectively the size of the free amino acid pool in these cells.

Amino Acids↗
PubMed1983

Use of gelatin/plasma coated flasks for isolating human peripheral blood monocytes.

A simple and efficient technique to purify human peripheral blood monocytes is described. This technique is based on the fact that monocytes have high affinity for fibronectin immobilized on a gelatin coated surface. Cell preparations obtained by the method described are more than 90% monocytes. The phenotype of these adherent cells was characterized with monoclonal antibodies.

Animals↗
PubMed1980

The effects of 5-bromodeoxyuridine on the growth and morphology of transformed rat liver cells.

The effects of bromodeoxyuridine (BrdUrd) on the growth, morphology, and tumorigenicity of the spontaneously transformed rat liver cell line R72/3 were studied. These cells grow either in suspension or in a monolayer and are tumorigenic. In monolayer cultures, cells treated with low concentrations (2.5 micrograms/ml) of BrdUrd were larger, more spread out, and more firmly attached to the substratum than were untreated controls. Treated cells failed to grow in suspension or on confluent monolayers of 3T3 cells and did not form colonies in soft agar. Scanning electron microscopy revealed extensive flattening of treated cells and a dramatic reduction in the number of microvilli on the cell surface. Transmission electron microscopy showed an increase in polyribosomes and rough endoplasmic reticulum, as well as an enlargement of endoplasmic reticulum cisternae and a complete absence of the bundles of intermediate size filaments that were conspicuous in untreated cells. The persistence of these changes required the continuous presence of BrdUrd in the medium. The effects of BrdUrd were readily reversed by withdrawal of BrdUrd and were not expressed in the presence of excess thymidine.

Animals↗
PubMed1979

Fibronectin alters the phenotypic properties of cultured chick embryo chondroblasts.

The state of chick embryo chondroblasts in culture was found to be sensitive to both fibronectin and another substance(s) (activity A) which could be extracted from chick embryo fibroblasts with 1 M urea or from conditioned medium. In the presence of either of these activities at concentrations of 25-150 micrograms/ml, chondroblasts, which normally grow as mixed cultures of floating and adherent cells, all immediately became attached to the tissue culture dish and spread. After several days, the morphology of these typically epithelioid cells became fibroblastic. This did not involve a selection process, since the effect was reversible. The synthetic program of these cells was also dramatically modified: the cultures no longer synthesized the chondroblast-unique type IV sulfated proteoglycan and began synthesizing alpha 2 collagen chains typical of fibroblastic or early limb bud cells. Fibronectin was resolved from activity A by gelatin affinity chromatography or gel filtration. Both activities were trypsin-sensitive. The two activities differed, however, on the basis of how the protein fractions in which they were found migrated in SDS-polyacrylamide gels, their specific activities and their effects on cell morphology and cell growth.

Animals↗