Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells

The native structures of the Asn-linked oligosaccharides and the O-glycans at Ser126 of human erythropoietin expressed from recombinant BHK cells have been elucidated. Enzymatically released N-glycans were studied by methylation analyses, fast-atom-bombardment mass spectrometry as well as one- and two-dimensional 1H-NMR spectrometry at 600 MHz. Many (82.7%) were found to be tetraantennary N-acetyllactosamine-type (22.8% with one, 3.6% with two and 0.4% with three N-acetyllactosamine repeats) being tetrasialylated (41%), trisialylated (29.6%) and disialylated (12.2%). A few (9.7%; 4.1% 2,4-branched, 5.6%, 2,6-branched) of the chains were triantennary (5.4% trisialyl, 4.3% disialyl) and 4.6% were of the disialyl diantennary type. Almost all of the innermost GlcNAc residues were alpha 1-6 fucosylated and NeuAc was exclusively alpha 2-3 linked to Gal beta 1-4GlcNAc-R; 60% of the protein was found to be O-glycosylated at Ser126; structures were monosialylated (70%) or disialylated (30%) forms of the Gal beta 1-3GalNAc core type. Glycosylation patterns at individual Asn-Xaa-Thr/Ser sites were determined by analytical high-pH anion-exchange chromatography with pulsed amperometric detection. Only tetraantennary chains with 0-3 N-acetyllactosamine repeats were detected at Asn38 and Asn83, while almost all of the di- and triantennary oligosaccharides were attached to Asn24. Batch analysis of different preparations of recombinant erythropoietin revealed the high reproducibility of the production procedure. Structures containing terminal GalNAc-GlcNAc were detected in small amounts in a few batches.     

Smoking cessation with alternative methods

Almost every former smoker has ceased smoking by himself. Everybody has collected his own experience with it and tried different methods to avoid withdrawal symptoms and the urge to start smoking again. During the last decades, many different methods for the therapy of smoking cessation have been applied. These techniques were often developed with the help of individual experiences. Many times, there is a lack of a scientific ground and verifiable investigations for these activities. The most important parameter for the smoking cessation is the clear and strong intention to quit. This intention may be supported by different methods. The mental requirements are discussed and the numerous methods and drugs available to quit smoking are described.     

Astrocytic neurotransmitter receptors in situ and in vivo

In the brain, astrocytes are associated intimately with neurons and surround synapses. Due to their close proximity to synaptic clefts, astrocytes are in a prime location for receiving synaptic information from released neurotransmitters. Cultured astrocytes express a wide range of neurotransmitter receptors, but do astrocytes in vivo also express neurotransmitter receptors and, if so, are the receptors activated by synaptically released neurotransmitters? In recent years, considerable efforts has gone into addressing these issues. The experimental results of this effort have been compiled and are presented in this review. Although there are many different receptors which have not been identified on astrocytes in situ, it is clear that astrocytes in situ express a number of different receptors. There is evidence of glutamatergic, GABAergic, adrenergic, purinergic, serotonergic, muscarinic, and peptidergic receptors on protoplasmic, fibrous, or specialized (Bergmann glia, pituicytes, Müller glia) astrocytes in situ and in vivo. These receptors are functionally coupled to changes in membrane potential or to intracellular signaling pathways such as activation of phospholipase C or adenylate cyclase. The expression of neurotransmitter receptors by astrocytes in situ exhibits regional and intraregional heterogeneity and changes during development and in response to injury. There is also evidence that receptors on astrocytes in situ can be activated by neurotransmitter(s) released from synaptic terminals. Given the evidence of extra-synaptic signaling and the expression of neurotransmitter receptors by astrocytes in situ, direct communication between neurons and astrocytes via neurotransmitters could be a widespread form of communication in the brain which may affect many different aspects of brain function, such as glutamate uptake and the modulation of extracellular space.     

Cell recognition, signal induction, and symmetrical gene activation at the dorsal-ventral boundary of the developing Drosophila wing

Appendage formation in insects and vertebrates depends upon signals from both the anterior-posterior and dorsal-ventral (DV) axes. In Drosophila, wing formation is organized symmetrically around the DV boundary of the growing wing imaginal disc and requires interactions between dorsal and ventral cells. Compartmentalization of the wing disc, dorsal cell behavior, and the expression of two dorsally expressed putative signaling molecules, fringe (fng) and Serrate (Ser), are regulated by the apterous selector gene. Here, we demonstrate that fng and Ser have distinct roles in a novel cell recognition and signal induction process. fng serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. Ser in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary.     

Thrombolytic and endovascular treatment of peripartum iliac vein thrombosis: a case report

This case report details the multidisciplinary treatment of peripartum left iliac vein thrombosis using percutaneous catheter-directed urokinase thrombolysis and balloon thromboplasty. Enhanced chances for long-term patency and the normalization of venous function make these minimally invasive procedures accepted options for the treatment of iliofemoral deep venous thrombosis in selected peripartum patients.     

Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.