Herbert Stadler

Simple and rapid measurement of acetylcholine and choline by HPLC and enzymatic-electrochemical detection.

A simple, rapid and sensitive method for the detection of acetylcholine and choline in tissue extracts is reported. Acetylcholine and choline are first separated by HPLC then react in a mini-column with acetylcholinesterase and choline oxidase immobilized on Sepharose. The resulting H(2)O(2) produced by choline oxidase is then detected electrochemically. The assay is more sensitive than existing methods. We believe that the principle involved in this method namely the combination of immobilized enzymes and the high sensitivity of electrochemical detection may be applied to other substances that can be converted by immobilized enzymes into an electrochemically detectable compound.

The primary structure of the 16 S rRNA binding protein S 8 from Escherichia coli ribosomes

Protein S 8 from the 30 S ribosomal subunit of Escherichia coli was sequenced for the following reasons: a) It binds specifically to the 16 S RNA [l-3] and a specific complex containing S 8 and 40 nucleotides of known sequence has been isolated by controlled ribonuclease digestion [4]. This small ribonucleoprotein particle, which can be dissociated and reassociated, is a promising model for investigating the chemical basis of protein-RNA interaction [5]. b) Two temperature sensitive mutants with an altered S 8 protein have been described (6). The identification of the type and location of the amino acid substitutions may also facilitate the understanding of the S 8 function in the ribosome. c) Some general properties concerning the shape and secondary structure of the protein have been determined from hydrodynamic and low angle X-ray diffraction studies (see Discussion).

Structure and function of cholinergic synaptic vesivles.

The function of a nerve terminal is to release neurotransmitter to transfer a signal to a receptor cell. The nerve terminal is a specialized region of the neuron, it is characterized by the presence of numerous membrane-bound organelles, the synaptic vesicles. The vesicles store and release the neurotransmitter and thus play a central role in synaptic transmission. The nerve terminals are separated from the cell body by their long and thin axon and the neuron therefore represents an extremely polarized cell. In the axon and in the nerve terminal there is no protein synthesis, and in order to maintain synaptic function the nerve terminal has to be supplied continuously with synaptic vesicle membranes and other material from the cell body. In addition to the nerve terminal, the cell body has to supply the dendrites. As a consequence the problem of membrane sorting is especially intriguing and challenging in the neuron (Fig. 1).

Identification of a Synaptic Vesicle Antigen (Mr 86,000) Conserved Between Torpedo and Rat

Abstract: Antisera were raised in guinea pigs to synaptic vesicles purified from the electric organ of Torpedo marmorata. In cholinergic nerve terminals from Torpedo the major antigens identified had Mr 300,000‐150,000, 86,000, and 18,000. The Mr 86,000 antigen was conserved between Torpedo and rat, where it is neuron‐specific and concentrated in nerve terminals. When rat brain synaptosomes are subfractionated the antigen is associated with synaptic vesicles. The antigen is not found in the cytoskeleton and in the vesicle‐free cytosol. Immunohistochemical localization of the antigen in rat shows it to be associated with synapses in diaphragm, cerebellum, hippocampus, and cerebral cortex. The staining pattern of the antigen indicates that the antigen is not cholinergicspecific. The function of the Mr 86,000 antigen remains to be identified.

Calmodulin Binding Proteins of the Cholinergic Electromotor Synapse: Synaptosomes, Synaptic Vesicles, Receptor−Enriched Membranes, and Cytoskeleton

Abstract: Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs—Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane‐associated. The Triton X‐100‐insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin‐coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.

Lymphocyte enrichment using CD81-targeted immunoaffinity matrix

In mass cytometry, the isolation of pure lymphocytes is very important to obtain reproducible results and to shorten the time spent on data acquisition. To prepare highly purified cell suspensions of peripheral blood lymphocytes for further analysis on mass cytometer, we used the new CD81+ immune affinity chromatography cell isolation approach. Using 21 metal conjugated antibodies in a single tube we were able to identify all basic cell subsets and compare their relative abundance in final products obtained by density gradient (Ficoll‐Paque) and immune affinity chromatography (CD81+ T‐catch™) isolation approach. We show that T‐catch isolation approach results in purer final product than Ficoll‐Paque (P values 0.0156), with fewer platelets bound to target cells. As a result acquisition time of 105 nucleated cells was 3.5 shorter. We then applied unsupervised high dimensional analysis viSNE algorithm to compare the two isolation protocols, which allowed us to evaluate the contribution of unsupervised analysis over supervised manual gating. ViSNE algorithm effectively characterized almost all supervised cell subsets. Moreover, viSNE uncovered previously overseen cell subsets and showed inaccuracies in Maxpar™ Human peripheral blood phenotyping panel kit recommended gating strategy. These findings emphasize the use of unsupervised analysis tools in parallel with conventional gating strategy to mine the complete information from a set of samples. They also stress the importance of the impurity removal to sensitively detect rare cell populations in unsupervised analysis.

Structure and Function of Synaptic Vesicles:New Aspects

Synaptic vesicles play a central role in neurotransmission as the transmitter storing and releasing organelles of the nerve terminal.In recent years progress has been made in functional identification of vesicle specific components.Most of this work has been done on synaptic vesicles isolated from electric organs of Torpedo marmorata (Stadler et al.,1985), a purely cholinergic model system.More recently a few proteins specific to brain synaptic vesicles have been isolated and characterized as well.Integration of the findings leads to a first model of the vesicle structure including aspects of uptake and storage of the solutes within these organelles. The major core protein of cholinergic vesicles, a heparan - sulfate proteoglycan (Stadler and Dowe,1982),is a secretory protein.This protein can be labelled in vivo with S-sulfate and this labelling technique enabled us to study the heterogeneity of these vesicles and their life cycle in the nerve terminal in more detail than previously.These findings provide new aspects towards understanding quantal release and synapse formation in this system.Furthermore we present evidence that mammalian brain synaptic vesicles may contain a proteoglycan-like component suggesting that it is a secretory protein as well.

Eine vollständig reversible, nicht-magnetische Zellisolation

A new automated bench top instrument quantitatively selects cells of interest in high yields and purity from whole blood or other blood preparations using the traceless affinity cell selection technology (TACS). This enables a fully reversible capture and release of target cells. TACS uses immune affinity chromatography based on CD-specific Fab-fragments which delivers label-free, non-activated target cells in a standardized manner of reproducible quality.

The Proton Pump of Synaptic Vesicles

Synaptic vesicles are the secretory organelles of the nerve terminal that store and release neurotransmitters. In the case of the acetylcholine-containing synaptic vesicles from electric organs, the bestcharacterized vesicle preparation at present, it was found that high concentrations of acetylcholine are stored in a fluid phase’ at an acidic pH around 5.5.2 These findings suggested that a proton ATPase might be present in the vesicle membrane, creating an electrochemical potential necessary for accumulation of transmitter. In agreement with these findings, experiments using the technique of [“C]methylamine uptake on isolated highly purified vesicles of Torpedo electric organ showed that ATP-dependent acidification of the vesicle interior occurred, indicating the presence of an ATP-driven proton pump. Further experiments showed in addition that the vesicle membrane contains a ouabainand oligomycin-insensitive Mg-ATPase that might be part of the proton pump.” Similar experiments were carried out using a synaptic vesicle preparation from guinea pig brain freed from membrane contaminants by chromatography on Sephacryl S 1OOO. As in the case with Torpedo electric organ vesicles, these vesicles showed ATP-dependent acidification of the interior and association with a ouabainand oligomycin-insensitive Mg-ATPase, indicating as well the presence of an ATP-driven proton pump. Furthermore, morphological examination of the preparation using a quick freeze, deep-etch, rotary-shadowing technique showed that protrusions were present on the surface of the vesicle, suggesting that they might be part of the vesicular proton pump analogous to proton pumps in other systems.’ Altogether these findings provide evidence that synaptic vesicles are equipped with one or more copies of ATP-driven proton pumps that create an electrochemical gradient over the vesicle membrane necessary for uptake and storage of transmitter. Isolation and detailed characterization of the composition of the proton pump have not been carried out yet.