PubMed Health⌕ Search

Biomedical subjects

D Schober

Publications and source records attributed to D Schober.

9 recordsLinked to original sources
PubMed2001

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.

Amino Acid Sequence↗
PubMed2000

Inhibition of clathrin-dependent endocytosis has multiple effects on human rhinovirus serotype 2 cell entry.

Minor group human rhinoviruses (exemplified by human rhinovirus serotype 2 (HRV2)) use members of the low density lipoprotein receptor family for cell entry; all these receptors possess clathrin-coated pit localization signals. Viral infection should thus be inhibited under conditions of impaired clathrin-mediated endocytosis. However, Madshus et al. reported an increase in the cytopathic effect of HRV2 infection in HEp-2 cells upon suppression of clathrin-dependent endocytosis by hypotonic shock and potassium depletion (Madshus, I. H., Sandvig, K., Olsnes, S., and van Deurs, B. (1987) J. Cell. Physiol. 131, 14-22.) To resolve this apparent contradiction, we investigated the binding, internalization, conformational changes, and productive uncoating of HRV2 in HeLa cells subjected to hypotonic shock and potassium depletion. This treatment led to an increase in HRV2 binding, with internalization being barely affected. The generation of C-antigenic particles requiring pH </=5.6 was strongly reduced due to an elevation of the pH in endosomal compartments. However, K(+) depletion only slightly affected de novo viral protein synthesis, suggesting that productivity of viral RNA in the cytoplasm is enhanced and thus compensates for the reduction in C-antigenic particles. The distinct steps in the entry pathway of HRV2 are thus differently influenced by potassium depletion. Viral internalization under conditions of inhibited clathrin-dependent endocytosis without the need to disturb the ionic milieu was confirmed in HeLa cells overexpressing the nonfunctional dynamin-1 mutant K44A. Unexpectedly, overexpression of dynamin-1 K44A resulted in elevated endosomal pH compared with overexpression of wild-type dynamin.

Cell Membrane↗
PubMed1998

Uptake of poliovirus into the endosomal system of HeLa cells.

To understand the topology and mechanism of poliovirus uncoating, the question of whether intact virions can be endocytosed by the host cell was studied by a combination of various techniques. In order to prevent alteration of the virus to subviral particles, Hela cells were infected at 26 degrees C. At this temperature the majority of cell-associated virions remained at the plasma membrane, whereas a smaller amount accumulated in vesicles having the same mobility (upon free-flow electrophoresis) and migration behaviour on Nycodenz density gradients as early and late endosomes. Co-localization of native poliovirions with endosomal markers was verified by peroxidase-induced diaminobenzidine density-shift of endosomal vesicles. Internalization of poliovirions into endosomes makes it likely, but does not prove that viral RNA can be released into the cytoplasm from the vesicular compartment.

Endocytosis↗
PubMed1998

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection.

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.

Anti-Bacterial Agents↗
PubMed1998

Major and minor receptor group human rhinoviruses penetrate from endosomes by different mechanisms.

Intercellular adhesion molecule 1 and the low-density lipoprotein receptor are used for cell entry by major and minor receptor group human rhinoviruses (HRVs), respectively. Whereas minor-group viruses, exemplified by HRV2, transfer their genomic RNA to the cytoplasm through a pore in the endosomal membrane (E. Prchla, C. Plank, E. Wagner, D. Blaas, and R. Fuchs, J. Cell Biol. 131:111-123, 1995), the mechanism of in vivo uncoating of major-group HRVs has not been elucidated so far. Using free-flow electrophoresis, we performed a comparative analysis of cell entry by HRV2 and the major group rhinovirus HRV14. Here we demonstrate that this technique allows the separation of free viral particles from those associated with early endosomes, late endosomes, and plasma membranes. Upon free-flow electrophoretic separation of microsomes, HRV14 was recovered from endosomes under conditions which prevent uncoating, whereas the proportion of free viral particles increased with time under conditions which promote uncoating. The remaining virus eluted within numerous fractions corresponding to membraneous material, with no clear endosomal peaks being discernible. This suggests that uncoating of HRV14 results in lysis of the endosomal membrane and release of subviral 135S and 80S particles into the cytoplasm.

Endosomes↗
PubMed1997

Use of free-flow electrophoresis for the analysis of cellular uptake of picornaviruses.

Free-flow electrophoresis is a powerful tool to separate subcellular vesicles such as early and late endosomes from plasma membranes. Using this technique, the intracellular distribution of poliovirus type 2 Sabin (PV2) and its derived subviral particles was analyzed upon infection of HeLa cells. Comparison of various infection conditions showed that maximally 30% of total cell associated PV2 was found in endosomal compartments with the remainder being associated with plasma membrane fractions; 2% of viral label was recovered from the cytoplasm in form of free virions. Sucrose gradient centrifugation analysis of the viral material recovered from the respective fractions revealed that intracellular virus was exclusively in its native conformation. This is in sharp contrast to human rhinovirus serotype 2 (HRV2), which is rapidly modified to RNA-free subviral particles upon accumulation in endosomes. The data suggest that productive poliovirus uncoating can occur at the plasma membrane whereas internalized virus is most probably aborted.

Cold Temperature↗
PubMed1997

LY303870, a centrally active neurokinin-1 antagonist with a long duration of action.

The selective neurokinin (NK)-1 antagonist LY303870 has high affinity and specificity for human and guinea pig brain NK-1 receptors labeled with 125I-substance P. It has approximately 15- to 30-fold lower affinity for rat and mouse brain NK-1 receptors, consistent with previously reported species differences in the affinities of nonpeptide antagonists for NK-1 receptors. In vivo, LY303870 blocked the characteristic, caudally directed, biting and scratching response elicited by intrathecal administration of the selective NK-1 agonist Ac-[Arg6,Sar9,Met(O2)11]substance P6-11 in conscious mice. The potentiation of the tail-flick response elicited by intrathecal administration of the NK-1 agonist [Sar9,Met(O2)11]substance P in rats was also selectively blocked by LY303870. When tested in a model of persistent nociceptive activation induced by tissue injury (the formalin test), LY303870 blocked licking behavior in the late phase of the formalin test, in a dose-dependent manner. After oral administration of 10 mg/kg, the blockade of the late-phase licking behavior was evident for at least 24 hr. Ex vivo binding studies in guinea pigs showed that orally administered LY303870 potently inhibited binding to central and peripheral NK-1 receptors labeled with 125I-substance P. This inhibition was long-lasting, consistent with other in vivo activities. LY306155, the opposite enantiomer of LY303870, was less active in all of the functional assays. In rodents, LY303870 did not exhibit any neurological, motor, cardiovascular, gastrointestinal or autonomic side effects at doses of < or = 50 mg/kg p.o. Thus, LY303870 is a potent, centrally active, NK-1 antagonist in vivo, with long-lasting oral activity.

Animals↗
PubMed1989

[Physiologic occupational study of changing technologies in refuse collection].

The strain on drivers and on driver's mates was investigated while emptying 1.1 m(3) containers by means of new dust compressing vehicles, and while removing dustbins of 2201 capacity. A classification was made on the basis of heart rates sampled during the whole shift by portable memories. Extrapolating for the whole shift, the authors measured heart rates of 106.0 min(-1) (drivers) from the 2201 technology. The complementary values from the 1.1 m(3) technology were 107.7 min(-1) (driver's mates) and 105.2 min(-1) (drivers). The men's physical endurance limit would be exceeded when they handled dustbins without break. The investigated tasks are classified to be heavy in both technologies.

Adult↗