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Biomedical subjects

M Schmidt

Publications and source records attributed to M Schmidt.

At least 451 records · Page 25Linked to original sources
PubMed1996

Analysis of mammalian 20S proteasome biogenesis: the maturation of beta-subunits is an ordered two-step mechanism involving autocatalysis.

Maturation of eukaryotic 20S proteasomes involves the processing of beta-subunits by limited proteolysis. To study the processing mechanism we analysed different point mutations of the beta-subunit LMP2 in transfected human T2 cells. Here we show that the presence of the intact Gly-1Thr1 consensus motif and Lys33 are essential for correct processing. Mutation of Thr1, the active site residue in mature subunits, or of Lys33, results in complete inhibition of processing at the consensus site. In addition, proprotein processing in vitro of wild-type LMP2, incorporated in immature 16S precursor complexes, can be blocked by a proteasome-specific inhibitor. While the processing of inhibitor-treated wild-type proprotein was completely prevented, the site-directed mutagenesis of LMP2 results in processing intermediates carrying an extension of 8-10 residues preceding Thr1, suggesting an additional cleavage event within the prosequence. Furthermore, exchange of mammalian prosequences interferes with processing efficiency and suggests subunit specificity. Based on our data we propose a model for self-activation of proteasomal beta-subunits in which residue Thr1 serves as nucleophile and Lys33 as proton donor/acceptor. We provide evidence that subunit processing of mammalian beta-subunits proceeds via a novel ordered two-step mechanism involving autocatalysis.

Amino Acid Sequence↗
PubMed1996

Quantification of Bcr-Abl transcripts in chronic myelogenous leukemia (CML) using standardized, internally controlled, competitive differential PCR (CD-PCR).

The quantification of Bcr-Abl transcript numbers in chronic myelogenous leukemia (CML) patients described here uses simultaneous competitive PCR amplification of the target gene (Bcr-Abl) and a reference gene (porphobilinogen deaminase; Pbgd) together with a single composite competitor molecule for both targets based on heterologous sequences. Using this technique, Bcr-Abl transcript numbers could be reproducibly determined even in clinical samples known to harbour poor quality RNA.

DNA Primers↗
PubMed1996

Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.

The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.

Animals↗
PubMed1996

Restoration of Clostridium difficile toxin-B-inhibited phospholipase D by phosphatidylinositol 4,5-bisphosphate.

Receptor signalling to phospholipase D (PLD) in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor apparently involves Rho proteins. Since phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been recognized as an essential cofactor for PLD activity and since activated Rho proteins have been reported to stimulate the synthesis of PtdIns(4,5)P2, we studied whether in HEK cells PLD activity is regulated by PtdIns(4,5)P2 and, in particular, whether PtdIns(4,5)P2 can restore PLD activity inhibited by Clostridium difficile toxin B, which inactivates Rho proteins. Addition of MgATP to permeabilized HEK cells increased basal PLD activity and potentiated PLD stimulation by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), concomitant with a large increase in PtdIns(4,5)P2. On the other hand, neomycin, which binds to PtdIns(4,5)P2, inhibited basal and GTP[S]-stimulated PLD activities. Addition of PtdIns(4,5)P2 increased PLD activity in HEK cell membranes by 2-3-fold, whereas various other phospholipids were ineffective. Prior treatment of HEK cells with toxin B reduced the level of PtdIns(4,5)P2, measured either in intact cells or in membrane preparations, by about 40%. In membranes of toxin-B-treated cells, basal and GTP[S]-stimulated PLD activities were reduced, when measured with exogenous phosphatidylcholine as enzyme substrate. Inclusion of PtdIns(4,5)P2 with phosphatidylcholine in the substrate vesicles or addition of PtdIns(4,5)P2 fully restored basal and GTP[S]-stimulated PLD activities in membranes of toxin-B-treated cells. In conclusion, the data indicate that PtdIns(4,5)P2 is an essential cofactor for PLD activity in HEK cells and that inhibition of PLD activity by the Rho-inactivating toxin B is apparently caused by depletion of the PLD cofactor, PtdIns(4,5)P2.

Bacterial Proteins↗
PubMed1996

Dendritic morphology of projection neurons in the cat pretectum.

The distribution and dendritic morphology of neurons in the cat pretectal nuclear complex were analyzed with respect to their projection to the ipsilateral dorsal lateral geniculate nucleus (LGNd) and the ipsilateral inferior olive (IO). Single and double retrograde tracing techniques were combined with intracellular injections of either horseradish peroxidase into electrophysiologically identified pretectal neurons or Lucifer Yellow into retrogradely labeled somata. Pretectal cells afferent to the LGNd were located in the nucleus of the optic tract (NOT), adjacent dorsal terminal nucleus of the accessory optic system (DTN), and posterior pretectal nucleus (NPP). Cells projecting to the IO were also distributed throughout the NOT-DTN and dorsal part of the NPP. Separate tracer injections (fluorogold and horseradish peroxidase [HRP] or granular blue) into the LGNd and the IO showed considerable overlap of labeled neurons in the NOT and dorsal NPP. Double-labeled neurons, however, were not observed after double tracer injections into LGNd and IO. Partial topographical segregation of the two populations was observed along the dorsoventral axis because LGNd-projecting neurons exhibited maximum density ventral to that of IO neurons. Pretectal cells to the LGNd had cell body diameters between 16 and 48 microns. Somatic shapes varied between fusiform and multipolar with considerable overlap between these two morphological appearances. Neurons projecting to the IO exhibited similar cell body sizes and their morphology also varied from fusiform to multipolar. Quantitative analysis of dendritic field size and orientation, number and order of dendritic arborizations, and symmetry of the dendritic tree revealed no statistically significant difference between the two neuronal populations. Hence, neurons of the two populations cannot be unequivocally identified just from the dendritic morphology. By contrast, dendritic morphology was correlated with the topographical location of either cell type within the pretectal nuclei rather than projection. Thus, the morphological appearance of neurons located dorsally predominantly was fusiform while neurons located ventrally mostly were multipolar.

Animals↗
PubMed1996

Heat-labile uracil-DNA glycosylase: purification and characterization.

A uracil-DNA glycosylase (UNG) from a psychrophilic marine bacterium (BMTU 3346) has been purified to apparent homogeneity. The enzyme has a molecular weight of 23400 Da. It is stable in complex buffers (containing glycerol/BSA), whereas it is heat-labile in dilute buffers (free of stabilizers) with a half-life of 2 min at 40 degrees C. Due to the thermolability, uracil-DNA glycosylase is suitable for application in the carryover prevention technique showing less residual activity and/or a slower reactivation rate than the usually applied UNG from Escherichia coli.

Bacteria↗
PubMed1996

Identification of a 200 kDa polypeptide as type 3 phosphatidylinositol 4-kinase from bovine brain by partial protein and cDNA sequencing.

Two phosphatidylinositol 4-kinase isozymes, type 3 and type 2, have been separated on hydroxylapatite after solubilizing bovine brain microsomes with Triton X-114. Employing a newly developed renaturation procedure following SDS-PAGE, we demonstrate that a 200 kDa polypeptide carries the enzyme activity of this type 3 isoform. Chromatography on hydroxylapatite, Heparin-Sepharose, Superdex 200 and finally SDS-PAGE results in an approximately 30,000-fold purification. Tryptic peptides generated from the 200 kDa polypeptide after SDS-PAGE have been sequenced and the obtained data have been used for constructing and synthesizing degenerated oligonucleotides. Polymerase chain reaction as well as screening of cDNA libraries allowed several clones to be isolated from which a 4.7 kb contiguous sequence can be built up. The open reading frame covers 4.4 kb with a 0.3 kb untranslated 3' end which yields a deduced amino acid sequence of 1,467 amino acids. The C-terminal part of ca. 300 amino acids represents the catalytic domain. Sequence alignment of this domain with the mammalian counterpart, the human type 2 phosphatidylinositol 4-kinase, the yeast kinases STT4 and PIK1, as well as with the catalytic domains of bovine, human, mouse and yeast phosphatidylinositol 3-kinases reveals a high degree of identity: 26 of these approximately 300 amino acids are invariable in all of these eight catalytic domains. Five motifs indicate nuclear localization and DNA binding properties of the enzyme. Two leucine zipper motifs (amino acids 358-386, 862-882) are detectable. Furthermore, a helix loop helix motif (amino acids 716-729) as well as two nuclear localization signals (amino acids 838-854, 345-349) indicate the presence of the type 3 isoform in the nucleus.

1-Phosphatidylinositol 4-Kinase↗
PubMed1996

Effects of acute hypoxia and hyperoxia on ventilation in spontaneously hypertensive and normotensive rat.

The aim of this study was to investigate whether there are differences between particular characteristics of the ventilatory responses to acute hypoxia and hyperoxia in primary hypertensive and normotensive states which might indicate significant differences in arterial chemoreceptor reflex function. Pneumotachographic monitoring of ventilation was carried out in anesthetized, spontaneously breathing, normotensive randomly bred Wistar rats (NWR), Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Under air breathing conditions, minute ventilation in SHR (50 +/- 2 ml/min per 100 g) was not significantly different from that of WKY (54 +/- 3 ml/min per 100 g) but NWR had a significantly lower minute ventilation (39 +/- 1 ml/min per 100 g) than both SHR and WKY. Our data indicate that there is no elevation of the ventilatory drive under air breathing conditions which can be unequivocally associated with primary hypertension in adult animals. During acute hypoxia, minute ventilation increased by a similar magnitude in SHR and NWR (by 97 and 77%, respectively, above baseline values), whereas in WKY the increase was only 58%. When exposed to acute hyperoxia, minute ventilation was inhibited by a similar degree in all animals investigated. We conclude that there is no characteristic pattern of peripheral chemoreceptor-mediated ventilatory responses in close association with primary hypertension.

Animals↗
PubMed1996

A bivalent single-chain antibody-toxin specific for ErbB-2 and the EGF receptor.

ErbB-2 and EGF receptors are often co-expressed in human tumors and have been shown to synergize in the transformation of cells in experimental model systems. Transactivation of ErbB-2 can occur via ligand-induced heterodimerization with EGF receptor or other members of the ErbB family of receptor tyrosine kinases. We have previously described the potent anti-tumoral activity of the monospecific single-chain antibody-toxins scFv(FRP5)-ETA and scFv(225)-ETA binding to, respectively, ErbB-2 and the EGF receptor. Here we report the construction and functional characterization of a novel bivalent, bispecific single-chain antibody-toxin, scFv2(FRP5/225)-ETA. The fusion protein consists of 2 scFv domains specific for ErbB-2 and the EGF receptor linked to a modified Pseudomonas exotoxin A. ScFv2(FRP5/225)-ETA displayed in vitro cell killing activity on tumor cells overexpressing either ErbB-2 or the EGF receptor similar to that of the monospecific toxins. It was more potent in vitro and in vivo in inhibiting the growth of tumor cells expressing both receptors. Treatment of A431 cells with scFv2(FRP5/225)-ETA led to an increase in EGF receptor and ErbB-2 phosphotyrosine content, most likely via the induction of receptor heterodimers. This may explain the enhanced toxicity of the bispecific antibody-toxin.

ADP Ribose Transferases↗
PubMed1996

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins.

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.

ADP Ribose Transferases↗