Glancing angle deposition of sculptured thin metal films at room temperature.

Metallic thin films consisting of separated nanostructures are fabricated by evaporative glancing angle deposition at room temperature. The columnar microstructure of the Ti and Cr columns is investigated by high resolution transmission electron microscopy and selective area electron diffraction. The morphology of the sculptured metallic films is studied by scanning electron microscopy. It is found that tilted Ti and Cr columns grow with a single crystalline morphology, while upright Cr columns are polycrystalline. Further, the influence of continuous substrate rotation on the shaping of Al, Ti, Cr and Mo nanostructures is studied with view to surface diffusion and the shadowing effect. It is observed that sculptured metallic thin films deposited without substrate rotation grow faster compared to those grown with continuous substrate rotation. A theoretical model is provided to describe this effect.

[Realisation of material costs in anaesthesia. Alternatives to the reimbursement via diagnosis-related groups]. / Sachkostenerfassung in der Anästhesie. Alternativen zur fallbezogenen "DRG-konformen" Vergütung.

BACKGROUND AND GOAL: For reimbursement via diagnosis-related groups (DRG), lump compensation-based payment of medical cases in German hospitals requires a case-related measuring and billing of resources that has to be consistent with DRG guidelines. Only through this, can the real costs be compared with the standard costs as calculated by the hospital reimbursment system (InEK) on a case-related basis and the DRG-specific break-even level be identified. METHODS: In the present paper the authors introduce and validate two newly created alternative methods for case-related allocation of material costs in the field of anaesthesia. Method 1 allows online documentation of material costs via pre-defined anaesthesia standards. This full cost method is suitable for hospitals that have implemented an electronic hospital information system in their daily clinical documentation routine. For other hospitals method 2 could be applicable as the case-related allocation of material costs is done retrospectively based on the data collected in an electronic anaesthesia protocol record system (andoc, medlinq). RESULTS: Method 1 makes it possible to allocate 90.3% of anaesthesia-related material costs to a specific case corresponding to a Pearsson coefficient of 0.77. After iterative improvement through optimisation of modules the documentation quality could be raised to >98% and a Pearsson coefficient of 0.96. Although the expense for implementation and maintenance is considerable, the necessary documentation work for the clinician is low. Method 2 demands no further clinical effort in documentation and implementation and 49.1% of all material costs can be assigned on a case-related basis. CONCLUSIONS: The online documentation of material costs via predefined anaesthesia standards accounts for nearly all material costs in anaesthesia and only a negligible documentation effort is necessary for the clinician. Nevertheless, a complex and time-consuming configuration of standards and a continuous iterative alignment of the modules with the actual processes are required. Due to its process-orientated character, method 1 can also be used for workflow optimisation in terms of standard operating procedures (SOPs). Allocation of material costs with data from the electronic anaesthesia record system is a method that can be easily implemented but only a partial case relation is rendered possible.

SLP65 deficiency results in perpetual V(D)J recombinase activity in pre-B-lymphoblastic leukemia and B-cell lymphoma cells.

Perpetual V(D)J recombinase activity involving multiple DNA double-strand break events in B-cell lineage leukemia and lymphoma cells may introduce secondary genetic aberrations leading towards malignant progression. Here, we investigated defective negative feedback signaling through the (pre-) B-cell receptor as a possible reason for deregulated V(D)J recombinase activity in B-cell malignancy. On studying 28 cases of pre-B-lymphoblastic leukemia and 27 B-cell lymphomas, expression of the (pre-) B-cell receptor-related linker molecule SLP65 (SH2 domain-containing lymphocyte protein of 65 kDa) was found to be defective in seven and five cases, respectively. SLP65 deficiency correlates with RAG1/2 expression and unremitting V(H) gene rearrangement activity. Reconstitution of SLP65 expression in SLP65-deficient leukemia and lymphoma cells results in downregulation of RAG1/2 expression and prevents both de novo V(H)-DJ(H) rearrangements and secondary V(H) replacement. We conclude that iterative V(H) gene rearrangement represents a frequent feature in B-lymphoid malignancy, which can be attributed to SLP65 deficiency in many cases.

Immunoglobulin class-switch recombination occurs in mantle cell lymphomas.

Mantle cell lymphoma (MCL) is an IgM-expressing B cell lymphoma that originates from naive B cells and responds poorly to chemotherapy. We show here that several MCLs harbour isotype-switched subclones. Similar to the situation in normal B cells, in vitro stimulation of MCL cell lines with CD40 ligand (CD40L) and interleukin-4 induced expression of activation-induced cytidine deaminase (AID) and germline transcription at the immunoglobulin heavy chain gene locus. Additionally, the occurrence of switch-circle transcripts and mature IgG transcripts after stimulation indicated ongoing class-switch recombination in mantle cell lymphoma cell lines. Furthermore, stimulation of primary MCL cells in vitro induced expression of class-switched IgG mRNA in the tumour cells. Our data indicate that mantle cell lymphomas have retained the ability to undergo class-switch recombination if appropriate stimuli, such as the CD40 ligand, are provided.

Inhibition of striatal and retinal dopamine release via nociceptin/orphanin FQ receptors.

1. We determined the effects of nociceptin/orphanin FQ and the NOP receptor ligands acetyl-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)) and naloxone benzoylhydrazone on transmitter release in vitro. 2. The electrically evoked tritium overflow from guinea-pig and mouse striatal slices and guinea-pig retinal discs preincubated with [(3)H]-dopamine was inhibited by nociceptin/orphanin FQ (pEC(50) 7.9, 7.6 and 8.6; E(max) 30, 50 and 55%). Ac-RYYRIK-NH(2) 0.032 microM and naloxone benzoylhydrazone 5 microM antagonized the effect of nociceptin/orphanin FQ in striatal slices of the guinea-pig (apparent pA(2) 9.1 and 6.8) and the mouse (apparent pA(2) 9.2 and 7.5) and strongly attenuated the effect of nociceptin/orphanin FQ 0.1 microM in guinea-pig retinal discs. Ac-RYYRIK-NH(2) 0.032 microM did not affect the evoked overflow by itself whereas naloxone benzoylhydrazone 5 microM inhibited it in each tissue. 3. The electrically evoked tritium overflow from mouse brain cortex slices preincubated with [(3)H]-noradrenaline was inhibited by nociceptin/orphanin FQ (pEC(50) 7.9, E(max) 85%), Ac-RYYRIK-NH(2) (pEC(50) 8.3, E(max) 47%) but not affected by naloxone benzoylhydrazone 5 microM. Ac-RYYRIK-NH(2) and naloxone benzoylhydrazone showed apparent pA(2) values of 8.6 and 6.9. 4. In conclusion, the inhibitory effect of nociceptin/orphanin FQ on dopamine release in the striatum and retina and on noradrenaline release in the cerebral cortex is mediated via NOP receptors. Ac-RYYRIK-NH(2) behaves as an extremely potent NOP receptor antagonist in the striatum and retina and as a partial agonist in the cortex.

Core structures of polysialylated glycans present in neural cell adhesion molecule from newborn mouse brain.

Polysialylation of the neural cell adhesion molecule (N-CAM) is known to destabilize cell-cell adhesion and to promote plasticity in cell-cell interactions. To gain more insights into the molecular mechanisms regulating the selective expression of polysialic acid on distinct glycan chains, the underlying core structures of polysialylated N-CAM glycans from newborn mouse brain were examined. Starting from low picomolar amounts of oligosaccharides, a multistep approach was used that was based on various mass spectrometric techniques with minimized sample consumption. Evidence could be provided that polysialylated murine N-CAM glycans comprise diantennary, triantennary and tetraantennary core structures carrying, in part, type-1 N-acetyllactosamine antennae, sulfate groups linked to terminal galactose or subterminal N-acetylglucosamine residues and, as a characteristic feature, a sulfated glucuronic acid unit which was bound exclusively to C3 of terminal galactose in Manalpha3-linked type-2 antennae. Hence, our results reveal that part of the murine N-CAM carbohydrates are modified within a single oligosaccharide by polysialic acid plus a HSO3-GlcA-moiety, which is likely to represent a HNK1-epitope. As HNK1-carbohydrates are also known to modulate cell-cell interactions, the simultaneous presence of both carbohydrate epitopes may reflect a new mechanism involved in the fine-tuning of N-CAM functions.

Characterization of N-glycans from mouse brain neural cell adhesion molecule.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.

Influence of high pressure homogenisation equipment on nanodispersions characteristics.

In this study a comparison of the influence of the homogenising equipment supplied by different manufacturers on the quality of the lipid nanodispersions is given. An Avestin EmulsiFlex-B3 (B3) and APV Micron Lab 40 (LAB 40) were used for high pressure homogenisation. Particle size and particle size distribution were chosen as quality parameters. The influence of different process parameters was evaluated. The two homogenisers were compared in their quality of nanoparticles-production by hot and cold homogenisation technique and in processing nanoemulsions. Working with the B3 appeared as useful for preformulation studies and processing of expensive or rare drugs and excipients. This first scaling up within laboratory scale is evaluated and the problems and remarkable aspects working with the B3 are pointed out.

Host-cell-specific glycosylation of HIV-2 envelope glycoprotein.

Neutral complex-type N-glycans of the envelope glycoprotein 120 of HIV-2, propagated in different host cells, display cell-type specific variations. In order to identify typical structural elements, glycans were analysed by gel filtration, by enzymic sequencing and, in part, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The characteristic substituents of di- tri- and tetraantennary carbohydrate units thus observed include N-acetyllactosamine repeats, bisecting N-acetylglucosamine and fucose linked to the chitobiose core as well as to N-acetyllactosamine antennae. Each glycoprotein preparation displayed a characteristic set of glycoforms.

Elongation of the N-glycans of fowl plague virus hemagglutinin expressed in Spodoptera frugiperda (Sf9) cells by coexpression of human beta 1,2-N-acetylglucosaminyltransferase I.

Spodoptera frugiperda (Sf9)-cells differ markedly in their protein glycosylation capacities from vertebrate cells in that they are not able to generate complex type oligosaccharide side chains. In order to improve the oligosaccharide processing properties of these cells we have used baculovirus vectors for expression of human (beta 1,2-N-acetylglucosaminyltransferase I (hGNT-I), the enzyme catalysing the crucial step in the pathway leading to complex type N-glycans in vertebrate cells. One vector (Bac/GNT) was designed to express unmodified GNT-I protein, the second vector (Bac/tagGNT) to express GNT-I protein with a tag epitope fused to its N-terminus. In Sf9-cells infected with Bac/tagGNT-virus a protein of about 50 kDa representing hGNT-I was detected with an antiserum directed against the tag epitope. HGNT-I activity was increased at least threefold in lysates of infected cells when N-acetylglucosamine (GlcNAc)-free ovalbumine was used as substrate. To monitor hGNT-I activity in intact Sf9-cells, the glycosylation of coexpressed fowl plague virus hemagglutinin (HA) was investigated employing a galactosylation assay and chromatographic analysis of isolated HA N-glycans. Coexpression of hGNT-I resulted in an at least fourfold increase of HA carrying terminal GlcNAc-residues. The only structure detectable in this fraction was GlcNAcMan3GlcNAc2. These results show that hGNT-I is functionally active in Sf9-cells and that the N-glycans of proteins expressed in the baculovirus/insect cell system are elongated by coexpression of glycosyltransferases of vertebrate origin. Complete complex type oligosaccharide side chains were not observed when hGNT-I was overexpressed, thus supporting the concept that Sf9-cells do not contain glycosyltransferases acting after hGNT-I.

Oligosaccharide profiles of HIV-2 external envelope glycoprotein: dependence on host cells and virus isolates.

The glycosylation pattern of the external envelope glycoprotein of human immunodeficiency virus type 2 (HIV-2) was studied in dependence on host cells and virus isolates. Strains HIV-2ALT, HIV-2ROD and HIV-2D194, differing in their biological properties and in the amino acid sequences of their env genes, were propagated in MOLT4, HUT78 and U937 cells, in human peripheral blood lymphocytes and monocytes/macrophages in the presence of [6-3H]glucosamine. Radiolabelled viral glycoproteins were isolated from the cell-free supernatants and digested with trypsin. Glycans were sequentially liberated by endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F, and fractionated according to charge and size. Comparison of the oligosaccharide profiles revealed that the envelope glycoproteins of different virus isolates, propagated in the same host cells, yielded very similar glycan patterns, whereas cultivation of an isolate in different host cells resulted in markedly divergent oligosaccharide maps. Variations concerned the proportion of high-mannose-, hybrid- and complex-type substituents, as well as the state of charge and structural parameters of the complex-type species. As a characteristic feature, complex-type glycans of macrophage-derived viral glycoprotein were almost exclusively substituted by lactosamine repeats. Hence, glycosylation of the HIV-2 external envelope glycoprotein seems to be primarily governed by host cell-specific factors rather than by the amino acid sequence of the corresponding polypeptide backbone.