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Biomedical subjects

M Schmidt

Publications and source records attributed to M Schmidt.

At least 541 records · Page 30Linked to original sources
PubMed1995

In vivo assembly kinetics of fluorescently labeled Xenopus lamin A mutants.

The assembly kinetics of Xenopus wild type lamin A and 7 mutants were analyzed by the microinjection of renatured 5-iodoacetamidofluorescein (5-IAF)-labeled protein into mouse 3T3 cells. This experimental system has the advantage that the masking of the mutant phenotype by the formation of heterooligomeric complexes with newly synthesized lamins of the microinjected cell is negligibly small. The wild type protein and all mutants containing a nuclear localization signal were transported within 10 min into the nucleus. The wild type protein exhibited a strong lamina fluorescence 30 min after microinjection, whereas mutant molecules showed a delayed but complete, a delayed and incomplete, or no lamina assembly at all. The lamin A mutant lacking the carboxy-terminal cysteine of the CxxM-motif exhibited a delayed but complete assembly, whereas previously performed transfection experiments demonstrated no significant interaction of this mutant wih the lamina. According to present knowledge this phenotype in transfected cells can be explained by an overexpression of this mutant. We have identified an additional domain in the carboxy-terminal tail of lamin A that promotes its assembly into the lamina. In vitro this domain is required for the chromatin binding of lamin A. Lamin A molecules lacking the non-helical amino-terminal head domain showed no significant lamina staining, whereas point mutations in conserved regions of the alpha helix resulted in an incomplete assembly.

3T3 Cells↗
PubMed1994

Molecular characterization of human and bovine endothelin converting enzyme (ECE-1).

A membrane-bound protease activity that specifically converts Big endothelin-1 has been purified from bovine endothelial cells (FBHE). The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT-PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture and oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE-1.

Amino Acid Sequence↗
PubMed1994

Correlation between the stability of the GroEL-protein ligand complex and the release mechanism.

The protein-protein interactions during GroE-mediated protein refolding are of crucial importance for understanding how the assisted refolding of non-native proteins is achieved. Since GroEL seems to be a rather promiscuous polypeptide-binding protein it is not surprising that conditions for efficient dissociation from GroEL are promiscuous as well. To understand assisted protein refolding it is necessary to elucidate the underlying principles of the different partial steps of the functional cycle. Here we show a correlation between the overall stability of the complex between GroEL and ligand protein and the conditions for functional release from the chaperonin. As a model system, differently denatured species of an antibody Fab fragment were used. While weakly bound Fab fragments are functionally released in the absence of GroES, stably associated non-native forms of the same protein are dependent on the presence of the co-chaperonin for optimal GroE-mediated reactivation, suggesting that complex stability determines the release requirement. However, the observed overall stability of the complex between GroEL and substrate protein may be regarded as the net product of constant binding and rebinding of the ligand protein, once associated with GroEL, as shown by competition experiments.

Chaperonin 10↗
PubMed1994

The contribution of GABA-mediated inhibition to response properties of neurons in the nucleus of the optic tract in the rat.

The contribution of GABA-mediated inhibition to the generation of directional selectivity of neurons in the nucleus of the optic tract (NOT) and the dorsal terminal nucleus of the accessory optic system (DTN) was examined in anaesthetized rats by iontophoretic application of the GABAA receptor antagonist bicuculline methiodide. Spontaneous and visually evoked NOT-DTN cell activities were always increased by bicuculline application. The directional selectivity of NOT-DTN cells to slowly moving whole-field stimuli, expressed as the direction index, was reduced for most neurons. However, the difference between firing rates during stimulus movements in the preferred and in the non-preferred direction did not change systematically. On average, this difference was not significantly affected in the majority of the neurons, although bicuculline more strongly increased the activity during movement in the preferred or non-preferred direction in some of the neurons. These results indicate that directionally selective neurons in the rat NOT-DTN receive GABAergic inhibition which is most likely tonic and independent of the stimulus direction.

Animals↗
PubMed1994

Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors. Evidence for involvement of tyrosine phosphorylation.

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phopsholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins.

Acetylcholine↗
PubMed1994

Synthesis of new acyclic pyrimidine nucleoside analogs as potential antiviral drugs.

The synthesis of 6-[N-(4-hydroxybutyl)amino]pyrimidinone derivatives 18-23 and the acyclic phosphonate nucleoside analogs 29-30 is reported. Their cytotoxic and antiviral effects were investigated. 2,5-Diamino-6-[N-[2-(phosphonomethoxy)ethyl]amino]pyrimidin-4(3H)- one (30) showed strong antiviral effects, and 21 showed significant cytotoxic activity.

Amino Alcohols↗
PubMed1994

FMRFamide-like immunoreactivity in presumptive chemosensory afferents of the spiny lobster, Panulirus argus.

Stainings with an antibody against the neuropeptide FMRFamide in the CNS of the spiny lobster revealed strong immunoreactivity in a special class of sensory afferents. These afferents are extremely thin and numerous and innervate all sensory neuropils except the optical and olfactory lobes. In the target neuropils the terminals of the afferents branch in parallel and form very densely labeled net-like structures. Due to their size, number and distribution we conclude that the immunoreactive afferents represent a specialized chemosensory system not related to food detection. We propose that a FMRFamide-related peptide present in the afferent terminals serves as sensory transmitter.

Animals↗
PubMed1994

Symmetric complexes of GroE chaperonins as part of the functional cycle.

The particular structural arrangement of chaperonins probably contributes to their ability to assist in the folding of proteins. The interaction of the oligomeric bacterial chaperonin GroEL and its cochaperonin, GroES, in the presence of adenosine diphosphate (ADP) forms an asymmetric complex. However, in the presence of adenosine triphosphate (ATP) or its nonhydrolyzable analogs, symmetric complexes were found by electron microscopy and image analysis. The existence of symmetric chaperonin complexes is not predicted by current models of the functional cycle for GroE-mediated protein folding. Because complete folding of a nonnative substrate protein in the presence of GroEL and GroES only occurs in the presence of ATP, but not with ADP, the symmetric chaperonin complexes formed during the GroE cycle are proposed to be functionally significant.

Adenosine Diphosphate↗
PubMed1994

Macrophage colony-stimulating factor-induced bone marrow macrophages do not synthesize or release prostaglandin E2.

Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2-14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.

Animals↗
PubMed1994

On the role of groES in the chaperonin-assisted folding reaction. Three case studies.

The mechanism by which correctly folded proteins are recovered from stable complexes with groEL is not well understood. Certain target proteins require ATP and groES, while others seemingly dispense with the cochaperonin. Here, we examine the chaperonin-assisted folding of ribulose-1,5-bisphosphate carboxylase, malate dehydrogenase, and citrate synthase, three proteins that are believed to require both chaperonin components for successful reactivation. Surprisingly, in all cases, the need for groES depended on the folding environment. Under "non-permissive" conditions, where unassisted spontaneous folding could not occur, reactivation to the native state required the complete chaperonin system (e.g. groEL, groES, and MgATP). However, under "permissive" conditions where spontaneous folding could occur groES was no longer mandatory. Instead, upon the addition of ATP alone, all three target proteins could be released from groEL, in a form that was capable of reaching the native state. In the permissive setting, groES merely accelerated the rate of the ATP-dependent release process. The results suggest that the incompletely folded protein species that are released from groEL, in the absence of groES, are not necessarily committed to the native state. Similar to the unassisted folding reaction, they still partition between productive and unproductive folding pathways in an environment-dependent manner. It follows that the mechanistic contribution of the co-chaperonin, groES, and its physiological significance in cellular protein folding, could be entirely missed in a permissive in vitro environment.

Animals↗
PubMed1994

Inhibition of neuronal activity in the nucleus of the optic tract due to electrical stimulation of the medial terminal nucleus in the rat.

Morphologically, a GABAergic connection between the medial terminal nucleus of the accessory optic system and the nucleus of the optic tract, two primary visual nuclei involved in the optokinetic reflex, has been demonstrated. In this study it was investigated if the medial terminal nucleus forms an inhibitory input to movement direction selective units in the nucleus of the optic tract. Neurons in the nucleus of the optic tract were visually stimulated with moving large random square patterns in their preferred and non-preferred direction, and their activity was recorded extracellularly. Concomitantly, bipolar electrical stimulation was applied to the medial terminal nucleus and its effect was studied on the visual responses of units in the nucleus of the optic tract. Units in the nucleus of the optic tract were strongly inhibited during electrical stimulation of the medial terminal nucleus. The role of GABA in mediating this inhibition was investigated by applying bicuculline, a GABAA receptor antagonist, iontophoretically to the recorded units in the nucleus of the optic tract. However, although average spike rate levels of units in the nucleus of the optic tract increased with bicuculline, bicuculline did not reduce inhibition invoked by electrical stimulation of the medial terminal nucleus. A possible explanation for this observation is that this inhibition is GABAB receptor mediated.

Animals↗
PubMed1994

Induction of aromatase in stromal vascular cells from human breast adipose tissue depends on cortisol and growth factors.

The regulation of aromatase (estrogen synthase) activity of cultured stromal vascular cells from human breast adipose tissue by cortisol, db-cAMP and growth factors was studied in a serum-free culture system. While PDGF-BB alone inhibited the effect of db-cAMP on aromatase induction, it stimulated aromatase activity in the presence of cortisol with or without db-cAMP. In the presence of 1 microM insulin consistently higher aromatase activities were found as compared to 1 nM insulin. In contrast to PDGF-BB, bFGF led to an increase of aromatase activity only in the presence of both cortisol and db-cAMP.

Adipose Tissue↗
PubMed1994

LGN-projecting neurons of the cat's pretectum express glutamic acid decarboxylase mRNA.

There have been conflicting reports on the chemical nature of the projection of the pretectal nuclei [nucleus of the optic tract and dorsal terminal nucleus of the accessory optic tract (NOT-DTN complex) and posterior pretectal nucleus] to the lateral geniculate nucleus and inferior olive. There is evidence that the pretecto-geniculate pathway is inhibitory. However, most attempts to verify the GABAergic nature of the projection neurons have failed. In order to answer this question, we employed a combination of retrograde transport and in situ hybridization. Rhodamine-labelled latex microspheres were injected into the electrophysiologically identified lateral geniculate nucleus. In addition, fluorescein-labelled latex microspheres were injected into the inferior olive. Retrograde axonal transport labelled large pretectal neurons. We then applied riboprobes specific for glutamic acid decarboxylase mRNA. We were able to demonstrate glutamic acid decarboxylase mRNA expression in up to 70% of lateral geniculate nucleus-projecting NOT-DTN and posterior pretectal nucleus neurons but in none of the pretecto-olivary projection neurons. The results suggest that the pretecto-geniculate projection is GABAergic in nature, which would confirm previous electrophysiological and morphological observations. The pretecto-olivary projection is not GABAergic.

Animals↗
PubMed1994

A randomized, multicenter study comparing the efficacy and tolerability of tropisetron, a new 5-HT3 receptor antagonist, with a metoclopramide-containing antiemetic cocktail in the prevention of cisplatin-induced emesis.

BACKGROUND: Chemotherapy-induced emesis is one of the most disturbing side effects in cancer therapy. Thus, antiemetic treatment is a mandatory adjunct in emetogenic chemotherapy. METHODS: Tropisetron (Navoban, Sandoz Pharma Ltd., Basel, Switzerland), a new 5-HT3 receptor antagonist, was compared in a randomized multicenter trial with a high-dose metoclopramide-dexamethasone cocktail for the prevention of nausea and emesis during cisplatin-containing chemotherapy. Two hundred fifty-nine chemotherapy-naive patients were included and followed during two consecutive courses. The main cancer types were gynecologic tumors, followed by lung cancer, head and neck cancer, and bladder cancer. The cisplatin dose usually was in the range of 50-89 mg/m2. The efficacy and quality of life assessments and the safety recordings were done during the first 6 days of both courses of chemotherapy. RESULTS: Acute vomiting was prevented in 63-64% of patients by both antiemetic regimens. The total rate of control of vomiting increased from 63% on day 1 to 93% on day 6 in the group receiving tropisetron. Acute nausea was prevented in 40% of the patients with tropisetron monotherapy and in 61% of patients receiving the antiemetic cocktail. With regard to delayed nausea, there were no significant differences between the two antiemetic regimens. Mild headache and constipation were more frequently associated with tropisetron, and extra-pyramidal side effects and sedation were associated with the antiemetic cocktail. CONCLUSIONS: Tropisetron was easier to administer and better tolerated than the cocktail, and it seems to be a highly efficacious and safe new antiemetic drug.

Adult↗
PubMed1994

Glycosylation of two recombinant human uterine tissue plasminogen activator variants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain.

Recombinant human uterine tissue plasminogen activator (tPA) glycosylation mutants carrying an additional N-glycosylation site in the epidermal-growth-factor-like domain due to the replacement of either Tyr67 by Asn (YN-tPA) or Gly60 by Ser (GS-tPA) were expressed in mouse epithelial cells (C127) in the presence of [6-3H]glucosamine. Glycopeptides comprising individual glycosylation sites were isolated and oligosaccharides attached were liberated by treatment with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols obtained after reduction were either directly characterized by high-pH anion-exchange chromatography (high-mannose and hybrid-type glycans) or preparatively subfractionated after enzymic desialylation and separation from sulphated asialooligosaccharides (complex-type sugar chains). Individual (sub)fractions of glucans were studied by methylation analysis, liquid secondary-ion mass spectrometry and, in part, by exoglycosidase digestion, whereas corresponding deglycosylated peptides were identified by amino acid analysis and N-terminal amino acid sequencing. The results revealed that Asn117 of YN-tPA carried exclusively high-mannose-type glycans with five to nine mannose residues similar to wild-type tPA expressed in this cell line [Pfeiffer, G., Schmidt, M., Strube, K.-H. & Geyer, R. (1989) Eur. J. Biochem. 186, 273-286]. In contrast, Asn117 of GS-tPA carried only small amounts (about 25%) of high-mannose and hybrid-type species and predominantly complex-type sugar chains (about 75%) which were partially incomplete and mostly devoid of fucose. Newly introduced N-glycosylation sites at Asn67 (YN-tPA) or Asn58 (GS-tPA) as well as those at Asn184 and Asn448 were solely substituted by complex-type glycans. Each carbohydrate attachment site displayed a peculiar oligosaccharide pattern with regard to branching and substitution by Gal alpha 3-residues, sulphate groups, intersecting GlcNAc and lactosamine repeats. Our study clearly demonstrates that creation of a new glycosylation site at Asn58 influenced the oligosaccharide processing and, hence, the glycosylation pattern at Asn117, whereas introduction of a new site at Asn67 did not. The relative amounts of complex-type glycans at Asn117 of GS-tPA correlated with the degree of carbohydrate substitution of Asn58. Therefore, it can be concluded that the presence of a sugar chain at the position and not the Gly to Ser mutation itself is responsible for the observed alteration of GS-tPA glycosylation.

Amino Acid Sequence↗