A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Werner A, Duvar S, Müthing J, et al. Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers. BIOTECHNOLOGY AND BIOENGINEERING. 2000;68(1):59-70.Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 mu g/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 mu g/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary meta-belie functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system. (C) 2000 John Wiley & Sons, Inc

Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes

Müthing J, Spanbroek R, PeterKatalinic J, et al. Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes. GLYCOBIOLOGY. 1996;6(2):147-156.The isolation and structural characterization of fucosylated neolacto-series gangliosides with linear poly-N-acetyllactosaminyl chains from normal human granulocytes is described. Gangliosides were purified by consecutive use of anion exchange HPLC on Fractogel TMAE-650(S), adsorption and reversed phase HPLC on Nucleosil 50-7 and Nucleosil 7C(18) columns, respectively, TLC immunostaining with carbohydrate specific monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) of the permethylated derivatives and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates were used for structure elucidations, One ganglioside was identified as sialyl Lewis(x) antigen with nLcOse(6)Cer core, Neu5-Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-3Gal beta 1-4Glc beta 1-1Cer. Furthermore, monosialylated ceramide: deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse(8)Cer) and four (nLcOse(10)Cer) linear lactosaminyl repeats were identified, carrying one to three fucoses. The ceramide portions were found to contain C-18 sphingosine and predominantly C-16:0 fatty acids, All monosialogangliosides were homogenous concerning their terminal alpha 2-3Neu5Ac-sialylation, but different in their fucosylation status, Beside VI(3)Neu5Ac, V(3)Fuc-nLcOse(6)Cer, in two of the fucosylated polylactosaminyl ganglioside fractions the sialyl Lewis(x) epitope was found, whereas five species expressed the terminal VIM-2 motif. The role of protein linked sialyl Lewis(x) epitope of human granulocytes as a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1; E-selectin) and platelet activation-dependent granule external membrane protein (PADGEM; P-selectin) is well documented, However, the involvement of endothelial cells E- and/or P-selectin mediated cell-cell adhesion via lipid bound sialyl Lewis(x) and/or VIM-2 epitopes on human granulocytes has to be proved in further investigations

Delegation heilkundlicher Maßnahmen an Notfallsanitäterinnen und Notfallsanitäter durch die Ärztlichen Leiter Rettungsdienst in Bayern

Background After a recent change in German paramedic education, paramedics are qualified to independently conduct medical treatment according to standard operating procedures (SOP) in the otherwise physician-based German Emergency Medical Services (EMS). These SOP are delegated by the local medical director of EMS. This article summarizes the SOP system in the state of Bavaria, Germany. Methods Within an iterative group process, eligible standard emergencies were identified and corresponding SOP were designed. The authors focused on the principles of clarity, delineation of critical emergencies, compliance with guidelines and current evidence, patient safety, restriction to the general competence level of paramedics, statewide uniformity, and legal security. Results Five SOP for "injured person," "isolated limb injury," "isolated burn injury," "hypoglycemia," and "suspected sepsis" were designed. Subsequently, these algorithms were agreed upon by all medical directors in the state of Bavaria. The SOP system is accompanied by a systematic quality management program. Discussion The Bavarian SOP system stands out from that of other federal states with regard to its clear differentiation from critical emergency scenarios with EMS physician involvement and its statewide uniformity. Quality assurance efforts focus on the compliance of paramedics with the SOP and on the SOP safety and effectiveness