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U Marx

Publications and source records attributed to U Marx.

At least 19 recordsLinked to original sources
PubMed2000

Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach.

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver microsomes is measured by stopped-flow mixing of labeled microsomes with bovine serum albumin (BSA) solution. Extraction of analogs from the outer leaflet of microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BSA is different from that of the analogs inserted into membranes. From the signal kinetics, the transbilayer movement and the distribution of analogs in microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs. Half-times of the back-exchange for spin-labeled and fluorescent analogs were <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is much faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-labeled analogs was six- to eightfold slower than that of spin-labeled analogs.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding.

The variable-domain-attached oligosaccharide side chains of a human IgG produced by a human-human-mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) and crystallizing fragments. The oligosaccharides of the Fab fragment were released by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electrophoresis. The predominant components were disialyl- bi-antennary and tetra-sialyl tetra-antennary complex carbohydrates. Of note is the presence in this human IgG of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-acetylgalactosamine in the Fab fragment of this antibody, suggesting the presence of O-linked carbohydrates. A three-dimensional structure of the glycosylated variable (Fv) fragment was suggested using computer-assisted modelling. In addition, the influence of the Fv-associated oligosaccharides of the CBGA1 antibody on antigen binding was tested in several ELISA systems. Deglycosylation resulted in a decreased antigen-binding activity.

Amino Acid Sequence↗
PubMed1999

Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.

Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Membrane-based cell culture systems--an alternative to in vivo production of monoclonal antibodies.

A new generation of membrane-based cell culture devices especially designed for small scale production of monoclonal antibodies (mabs) has entered the market in the last few years. In contrast to conventional perfusion hollow fibre bioreactors, these devices contain two functionally different membranes--one ultrafiltration membrane for nutrient supply and one gas-permeable membrane for direct oxygenation of cells. The latest systems of this generation are static culture systems which are of moderate cost and either better than, or equal to, the ascites mice in terms of quality and quantity of produced monoclonal antibodies. We have investigated the advantages of the perfused Tecnomouse bioreactor and the static CELLine culture flasks in comparison to ascites production and conventional roller bottle cultures.

Animal Welfare↗
PubMed1998

European research and commercialisation activities in the field of tissue engineering and liver support in world wide competition.

Tissue engineering is seen as an interesting field of technology which could improve medical therapy and could also be considered as a commercial opportunity for the European biotechnological industry. Research in the state of the art of science using the MedLine and the Science Citation Index databases, in the patent situation and of the industry dealing with tissue engineering was done. A special method, based on the Science Citation Index Journal Citation Report 1993, for evaluating scientific work was defined. The main countries working in the field of tissue engineering were evaluated in regard to their scientific performance and their patents. The R&D of German industry was investigated as an exemplary European country. Out of all activities, different tissues were rated with respect to the attention received from research and industry and with regard to the frequency in which patents were applied for. USA, Germany and Japan rank first in most tissues, especially liver. After comparing German patents with the German scientific and industrial work, it seems that the potential in German patents and research is underestimated by German industry and inefficiently exploited.

Artificial Organs↗
PubMed1997

[6 minute walking test as stress test for patients treated by intermittent self-ventilation].

BACKGROUND: Until now there are no data about the exercise capacity of patients with chronic ventilatory pump failure before and during IPPV. PATIENTS AND METHODS: 55 patients with chronic ventilatory failure-20 patients before IPPV, 35 patients during mechanical ventilation with a mean of 16.5 months-were studied by the six-minute walking test. Eight patients were sampled in a follow-up study before and during IPPV with a mean of 2 months. RESULTS: The median walking distance was reduced to 273 in. The exercise capacity of the follow-up patients was not improved. The arterial pO2 during exercise fell markedly, arterial pCO2 increased only moderate. CONCLUSION: The exercise capacity of patients with chronic ventilatory failure is markedly reduced. Because of the significant oxygen desaturation on exercise we may frequently indicate ambulatory oxygen therapy.

Adult↗
PubMed1997

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach.

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.

Animals↗
PubMed1996

Generation and characterization of a human monoclonal IgM antibody that recognizes a conserved epitope shared by lipopolysaccharides of different gram-negative bacteria.

A hybridoma cell line secreting a human monoclonal antibody (humab) directed to an epitope in the lipid A region of lipopolysaccharides of Gram-negative bacteria was isolated. Peripheral blood lymphocytes (PBL) obtained from a healthy volunteer were immortalized by Epstein-Barr virus (EBV) transformation. Lymphoblastoid cell lines (LCL) secreting antibodies to the lipopolysaccharides of Gram-negative bacteria were determined by an enzyme-linked immunosorbent assay (ELISA) and subsequently fused with the human-mouse heteromyeloma cell line CB-F7 by polyethylenglycol (PEG)-mediated fusion. A hybridoma line producing a humab (LPD5H4), of the IgM/lambda isotype, which strongly reacted with the lipid A portion of Salmonella and E. coli spp. in ELISA, was established. The antibody was purified by hydrophobic interaction chromatography and gel filtration. Immunoblotting experiments showed a strong reactivity of the humab LPD5H4 with the lower molecular species of different rough and smooth lipopolysaccharide (LPS) types of the bacteria species Salmonella, E. coli, Klebsiella, and Neisseria meningitidis, whereas those of Pseudomonas spp. were negative. Binding of humab LPD5H4 to solid phase bound lipid A and different rough mutants of LPS could be inhibited by the corresponding antigens in solution. Competition assays with a murine monoclonal antibody to lipid A and with polymyxin B indicate that humab LPD5H4 recognizes its epitope in this extremely conserved part of the LPS molecule. In vitro tests demonstrated that the MAb is able to partially inhibit the LPS-induced release of TNF-alpha using isolated peripheral blood mononuclear cells (PBMC).

Animals↗
PubMed1995

Glycosylation analysis of a polyreactive human monoclonal IgG antibody derived from a human-mouse heterohybridoma.

Glycosylation of the human monoclonal IgG1 lambda antibody (mAb) CBGA1 was analysed by lectin blotting. The CBGA1 antibody binds to several antigens including donor self antigens, as detected by ELISA immunoblotting techniques and an erythrocyte binding assay. The mAb producing cell line was obtained by EBV transformation of peripheral blood lymphocytes of a healthy donor followed by fusion to the heteromyeloma cell line, CB-F7. The resulting heterohybridoma was cultivated in a hollow fibre bioreactor system. A bulk pool of 0.9 g antibody was produced. Fab and Fc fragments of the purified mAb were prepared and analysed. A noteworthy heterogeneity of CBGA1 and its fragments in SDS-PAGE and IEF was detected. We found glycosylation in the Fab fragment of CBGA1 in addition to the conserved glycosylation site in the Fc fragment at Asn 297. Fab glycosylation was detected in both the Fd region and the lambda-chain. The glycosylation pattern of the gamma-chain differs from that of the lambda-chain. Sequence analysis of the VH gene shows a potential N-glycosylation site located in framework III at position Asn 75.

Animals↗
PubMed1995

Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Animals↗
PubMed1994

Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody.

In vitro and in vivo experiments to explain the function of natural polyreactive antibodies, usually of the IgM isotype, require large amounts of purified antibodies. We have developed a two-step purification procedure using a human natural polyreactive monoclonal IgM antibody (CB03). This combines hydrophobic interaction chromatography on phenyl-Superose and gel filtration over Superose 12 and readily permits scaling-up to isolate mg to g amounts of antibody. Retention of the CB03 antibody during gel filtration by precipitation and interaction with the gel matrix was overcome by the addition of 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The yield of purified antibody was 34% and Fab fragments were obtained from the purified CB03 antibody by hot tryptic digestion (yield, 68% of theoretical amount). In an enzyme-linked immunosorbent assay, Fab and complete antibody had similar reaction patterns with different antigens.

Antibodies, Monoclonal↗
PubMed1994

Expression of gangliosides GM3 (NeuAc) and GM3 (NeuGc) in myelomas and hybridomas of mouse, rat, and human origin.

In this study gangliosides from various myelomas and hybridomas of mouse, rat, and human origin were characterized by thin-layer and high-performance liquid chromatography, immunological methods (overlay technique) and fast atom bombardment mass spectrometry. Exclusively GM3 substituted with C24:1- and C16:0-fatty acid, was found in all B cell-derived cell lines. C18 sphingosine was the single long chain base in each GM3 ceramide portion. The mouse myeloma (NS-1) and all hybridomas, obtained by fusion of mouse, rat, or human B lymphocytes with murine myelomas, showed high GM3 (NeuGc) content (> 75%) and low GM3 (NeuAc) expression. Absolute amounts of GM3 ranged from 0.2 up to 0.8 mg x 10(-9) cells. Normally, human cells do not express NeuGc, and an Epstein-Barr virus-transformed human B lymphocyte line analyzed in this study retained this sialylation status, expressing exclusively GM3 (NeuAc) (100%). The fusion of human B lymphocytes with mouse myelomas led to high GM3 (NeuGc) expression (average about 85%) in all mouse/human heterohybridomas examined. Our results indicate the chromosomal gene "transfer" and/or the activation of enzymes involved in NeuGc-biosynthesis due to the somatic cell fusion process, which might explain the mouse dominance in the manifestation of the NeuGc-phenotype in hybridomas of human origin.

Animals↗
PubMed1993

Suppression of anti-erythrocyte autoantibody-producing B cells by a physiological IgG-anti-F(ab')2 antibody and escape from suppression by tumour transformation; a model relevant for the pathogenesis of autoimmune haemolytic anaemia.

We showed previously that broadly reactive IgG anti-immunoglobulin autoantibodies produced by rats during the immune response suppress the B cell response. We report here on the effect of a similar human antibody on self-reactive human B cells. IgG anti-F(ab')2 was added to cultures of anti-erythrocyte autoantibody-producing B cells derived from healthy donors. A dose-dependent suppression of the antibody response was obtained (maximum at 1.3 ng IgG/10(6) cells). This effect was competitively inhibited by F(ab')2 gamma. Autoimmune haemolytic anaemia can be caused by chronic monoclonal B cell proliferation. To reproduce this condition in vitro we immortalized B cells with Epstein-Barr virus (EBV) and raised a B cell population with anti-erythrocyte autoantibody activity. These cells were electrically fused with CB-F7 tumour cells and an IgG1 cold-reactive anti-erythrocyte autoantibody-producing B cell line was established. Surprisingly, the tumour cells were not suppressed by IgG anti-F(ab')2. It is known that anti-immunoglobulins selectively suppress antigen-receptor (AgR)-occupied B cells by a Fc gamma-receptor (Fc gamma R)-mediated mechanism. To occupy their AgR, we preincubated the tumour cells with anti-AgR antibody. In spite of this, their susceptibility to suppression was not restored. As shown by rabbit IgG-sensitized ox erythrocyte (EA)-rosetting, this refractoriness was not due to a loss of Fc gamma R. Our experiments delineate a mechanism of peripheral B cell suppression to autoantigens, and show a way of escape from control relevant for the pathogenesis of autoimmune haemolytic anaemia.

Anemia, Hemolytic, Autoimmune↗