Search PubMed⌕ Search

Biomedical subjects

R Jahn

Publications and source records attributed to R Jahn.

At least 109 records · Page 6Linked to original sources
PubMed1994

Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain.

The synaptic vesicle protein synaptotagmin has been implicated in the docking and subsequent calcium-regulated exocytosis of synaptic vesicles. We demonstrate that synaptotagmin is a major constituent of synaptic vesicle membranes, comprising 7-8% of the total vesicle protein. A proteolytic fragment of synaptotagmin, containing two repeats homologous to the C2-domain of protein kinase C, bound to a variety of natural membranes in a calcium-dependent manner (EC50 approximately 30 microM calcium). Binding was insensitive to proteolysis of the acceptor membranes suggesting an interaction with the lipid constituents. This interaction was confirmed using a recombinant fusion protein, containing both C2-like domains of synaptotagmin, that bound to artificial liposomes in a calcium-dependent manner. Phospholipid binding properties were preserved in a 114-amino acid domain corresponding to the first C2-like repeat of the protein and represents the shortest functional cassette yet reported. Furthermore, deletion of a highly conserved 9-amino acid motif, within this region, was sufficient to abolish the calcium-dependent phospholipid binding properties of this domain. This mutation may provide a means to selectively disrupt individual C2-domains in order to assess their relative contributions to function.

Amino Acid Sequence↗
PubMed1994

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.

Amino Acid Sequence↗
PubMed1994

Regional, cellular, and ultrastructural distribution of N-methyl-D-aspartate receptor subunit 1 in monkey hippocampus.

The regional, cellular, and subcellular distributions of N-methyl-D-aspartate (NMDA) receptor subunit 1, NMDAR-1, were investigated in monkey hippocampus by using a monoclonal antibody directed against a fusion protein corresponding to aa 660-811 of NMDAR-1. The data indicate that many neurons in each subfield of the hippocampus contain NMDAR-1 protein, although the intensity and distribution of immunoreactivity varied across regions, strata, and cellular compartments. In stratum lucidum of CA3, mossy fiber axons were immunoreactive for NMDAR-1, which may correspond to previously hypothesized presynaptic receptors. NMDAR-1-labeled postsynaptic profiles were present in stratum radiatum of CA3 but were largely absent from stratum lucidum. Such intraneuronal segregation of glutamate receptor subunits or classes may be spatially correlated with afferent systems that exhibit laminar segregation and terminate in different portions of the postsynaptic dendritic tree. For example, in CA3 pyramidal cells, NMDA receptors are postsynaptic in distal apical dendrites (stratum radiatum) where NMDA-dependent long-term potentiation in rats is mediated by associational/commissural afferents, and are absent from proximal apical dendrites (stratum lucidum), where NMDA-independent long-term potentiation is mediated by the mossy fiber input.

Animals↗
PubMed1994

[Thermal side effects after use of the pulsed IR laser on meniscus and bone tissue].

Thermal effects on meniscus and bone tissue after application of 314 boreholes using five different infrared (IR) lasers: Nd:YAG, Tm:YAG, Ho:YAG, Er:YAG, Cr,Er:YSGG (application energy 200 mJ, 400 mJ, 600 mJ, 800 mJ, 1000 mJ; repetition rate 2 Hz, 5 Hz; medium air, water rinse) were analyzed. The experimental set-up comprised for the beam guiding a focussing lens (f = 100 mm) or a flexible fiber (Ho:YAG). Damaged tissue was investigated macroscopically, histologically, and by scanning electron microscopy. Application in air caused carbonisation in all cases. Application in water showed thermal brown discoloring using Tm:YAG, Ho:YAG laser on meniscus tissue. The Nd:YAG did not ablate. The Er:YAG laser showed macroscopically precise boreholes without any discoloring of the adjacent tissue as well in meniscus as in bone. Cr:ErYSGG laser results were comparable with the results using an Er:YAG laser although ablation on bone tissue created higher thermal effects. For the aim of developing minimal invasive operating techniques the Er:YAG laser showed best results.

Animals↗
PubMed1994

Synaptotagmin I is present mainly in autonomic and sensory neurons of the rat peripheral nervous system.

The distribution of synaptotagmin I in the peripheral nervous system of the rat was investigated by immunofluorescence and confocal laser scanning microscopy. After crushing of the sciatic nerve, synaptotagmin I-like immunoreactivity accumulated proximally as well as distally to the crushes in thin and medium-sized axons. Double labelling studies revealed that synaptotagmin I co-localized with tyrosine hydroxylase, a marker of sympathetic adrenergic neurons, and with substance P, a marker for sensory neurons. No synaptotagmin I-like immunoreactivity was found in large axons, while accumulations of the synaptic vesicle proteins synaptophysin and synapsin I were found in all types of axons. Furthermore, no synaptotagmin I-like immunoreactivity was detected in motor endplates. In contrast, the protein was found in muscle spindles of young rats and in perivascular terminals, where it co-localized with synaptophysin and synapsin I. Lumbar sympathectomy resulted in a marked reduction of the amount and intensity of synaptotagmin I-like immunoreactivity in sciatic nerve. High magnification revealed that synaptotagmin I-like immunoreactivity was mainly distributed in a fine granular pattern, but large, brightly fluorescent granules which were not labelled by anti-synaptophysin or anti-synapsin I were occasionally observed. We conclude that synaptotagmin I is mainly expressed in adrenergic and sensory neurons and is absent from, or below detection levels, in motoneurons.

Animals↗
PubMed1994

Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.

rab3A, a low molecular weight GTP-binding protein of synaptic vesicles with a putative function in synaptic vesicle docking, interacts in a GTP-dependent manner with rabphilin-3A, a peripheral membrane protein that binds Ca2+ and phospholipids. We now show that rabphilin-3A is an evolutionarily conserved synaptic vesicle protein that is attached to synaptic vesicle membranes via its N terminus and exhibits a heterogeneous distribution among synapses. In rab3A-deficient mice, rabphilin-3A is decreased in synapses belonging to neurons that primarily express rab3A and accumulates in the perikarya of these neurons. In contrast, neurons expressing significant levels of rab3C still contain normal levels of rabphilin-3A in a synaptic pattern, and rabphilin-3A binds rab3C in vitro. These results suggest that analogous to the membrane recruitment of raf by ras, rab3A and rab3C may function in recruiting rabphilin-3A to the synaptic vesicle membrane in a GTP-dependent manner.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins.

Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release. In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins. Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins: synaptobrevin (BoNT/D, BoNT/F); SNAP-25 (BoNT/A, BoNT/E), or HPC-1/syntaxin (BoNT/C1). All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F. BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes.

Animals↗
PubMed1994

Clostridial neurotoxins: new tools for dissecting exocytosis.

Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis. Within the past five years the protein sequences of all eight neurotoxins have been determined, their mode of action as metalloproteases has been established, and their intraneuronal targets have been identified. The toxins act by selectively proteolysing the synaptic vesicle protein synaptobrevin (VAMP) or the presynaptic membrane proteins syntaxin (HPC-1) and SNAP-25. These three proteins form the core of a complex that mediates fusion of carrier vesicles to target membranes. Tetanus and botulinal neurotoxins could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.

Journal Article↗
PubMed1994

Rab proteins in regulated exocytosis.

Regulated exocytosis is responsible for neuronal communication, hormone secretion, food digestion, control of glucose uptake and many other basic processes. Despite the structural and functional diversity of the cells undergoing regulated exocytosis, all regulated exocytosis involves specialized vesicles that are stored in the cytoplasm and fuse with the plasma membrane in response to a trigger event. Recent evidence suggests that a subset of small GTP-binding proteins, Rab3 and its relatives, participate in the control of regulated exocytosis.

Animals↗
PubMed1994

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis.

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

Animals↗
PubMed1994

Localization of Rab5 to synaptic vesicles identifies endosomal intermediate in synaptic vesicle recycling pathway.

After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.

Amino Acid Sequence↗
PubMed1994

[Injury of the popliteal artery as a complication of knee dislocation].

Dislocation of the knee is rarely seen. Only 15 patients with a knee dislocation were treated in the surgical department of the university hospital in the last 18 years. One patient had bilateral dislocation of the knee. Injury to the popliteal artery is severe and often endangers the extremity. In seven patients with knee dislocation arterial ischemic was detected. Five patients were treated for arterial damage (31.25%).

Adult↗
PubMed1993

An anion binding site that regulates the glutamate transporter of synaptic vesicles.

Glutamate, the major excitatory neurotransmitter of the mammalian central nervous system, is stored in synaptic vesicles and released by exocytosis upon depolarization of the presynaptic nerve terminal. Synaptic vesicles possess an active glutamate-specific transporter that is driven by an electrochemical proton gradient across the vesicle membrane and requires chloride for maximal activity. In this study, we have characterized the role of chloride in vesicular glutamate transport using 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a potent inhibitor of anion translocators. DIDS inhibited glutamate uptake with an IC50 of 0.7 microM or less. In contrast, all energy gradient parameters (membrane potential, pH gradient, and ATPase activity) required at least 5-fold higher concentration of DIDS for inhibition. Furthermore, high concentrations of chloride but not of glutamate or other anions prevented DIDS inhibition of glutamate uptake. In contrast to uptake, glutamate efflux from glutamate-loaded vesicles was independent of chloride over a wide concentration range. However, efflux was still susceptible to DIDS inhibition. DIDS inhibition was prevented by excess chloride. We conclude that the vesicular glutamate transporter possesses a DIDS-sensitive chloride binding site on the cytoplasmic side, distinct from the substrate binding site, which regulates transport activity.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Expression of endogenous NMDAR1 transcripts without receptor protein suggests post-transcriptional control in PC12 cells.

Expression of RNA for the NMDAR1 subunit of the N-methyl-D-aspartate receptor was detected by Northern hybridization in both nerve growth factor-differentiated and undifferentiated rat pheochromocytoma (PC12) cells. The NMDA receptor type 1 (NMDAR1) message in PC12 cells was similar in size to that expressed in hippocampal neurons. PC12 cell cDNAs that were amplified by polymerase chain reaction with primers flanking the coding region of NMDAR1 corresponded to the NMDAR1 splice variant NMDA receptor type 1 isoform C (NMDAR1C). Using calcium imaging or patch-clamp recording, no functional NMDA-gated ion channels were found in PC12 cells. A monoclonal antibody against NMDAR1 was developed in order to investigate whether or not NMDAR1 protein was present in PC12 cells. Only trace amounts of NMDAR1 protein were found in native PC12 cells. However, expression of NMDAR1 protein was detected in PC12 cells that were transfected with an expression vector containing an NMDAR1C clone under control of a cytomegalovirus promoter. These findings suggest that the expression of NMDAR1 protein in PC12 cells may be controlled by post-transcriptional mechanisms. The PC12 cell line may serve as a model system for the study of the transcriptional, post-transcriptional, and translational regulation of NMDAR1. Furthermore, the presence of NMDAR1 RNA in a particular cell type may not necessarily indicate expression of NMDAR1 protein.

Alternative Splicing↗