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Biomedical subjects

M Schmidt

Publications and source records attributed to M Schmidt.

At least 505 records · Page 28Linked to original sources
PubMed1995

micF RNA is a substrate for RNase E.

Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli. While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined. In this study, micF RNA, an antisense RNA found in E. coli and related bacteria, was found to be a substrate for RNase E in vitro. Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures. These results help define a generalized RNase E cleavage/recognition pattern.

Base Sequence↗
PubMed1995

Independent efferent populations in the nucleus of the optic tract: an anatomical and physiological study in rat and cat.

The efferent projections of the nucleus of the optic tract (NOT) and dorsal terminal nucleus of the accessory optic system (DTN) to the contralateral NOT-DTN, ipsilateral inferior olive (IO), ipsilateral nucleus prepositus hypoglossi (NPH), and ipsilateral dorsal lateral geniculate nucleus (LGNd) were examined in pigmented rats and in cats by using anterograde and retrograde tract tracing, as well as extracellular recording and electrical stimulation. Anterograde tracing in the rat revealed a dense termination field of NOT-DTN efferents throughout the homologous contralateral territory. In both species three different cell populations, projecting to the contralateral NOT-DTN, ipsilateral IO, and ipsilateral LGNd, respectively, were distinguished by means of multiple retrograde tracing. No clear topographical segregation of the different NOT-DTN relay cell populations was observed. On the other hand, a large proportion (at least 60%) of NOT-DTN neurons projecting to the ipsilateral NPH were found to bifurcate upon the IO in the rat. Electrophysiologically, NOT-DTN neurons projecting to the IO were identified by their directionally selective responses. Such neurons were never activated by electrical stimulation of either the contralateral NOT-DTN or the ipsilateral LGNd. Neurons antidromically activated from the contralateral NOT-DTN could not be activated from the ipsilateral LGNd. Thus, in both cat and rat the NOT-DTN includes at least three independent relay cell populations. As a consequence, the NOT-DTN must serve functions additional to the generation of eye movements during optokinetic nystagnus, a function subserved by the directionally selective NOT-DTN cells.

Animals↗
PubMed1995

New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions.

A set of antibiotic-resistance and promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions was constructed. The cassettes contain the aacC1 gene of transposon Tn1696 conferring resistance to gentamicin in a large variety of Gram- and Gram+ bacteria. In addition to the antibiotic-resistance gene, a promoterless Escherichia coli lacZ gene was included in the cassettes, allowing the determination of the transcriptional activity at the insertion site. The cassettes can be excised from a plasmid mediating ampicillin resistance by many commonly used restriction enzymes. The new constructs have been successfully used for mutagenesis and studies of gene transcription in Rhizobium meliloti.

Acetyltransferases↗
PubMed1995

Rapid and persistent desensitization of m3 muscarinic acetylcholine receptor-stimulated phospholipase D. Concomitant sensitization of phospholipase C.

Activation of muscarinic acetylcholine receptors (mAChR) in human embryonic kidney (HEK) cells stably expressing the human m3 subtype leads to stimulation of both phospholipase C (PLC) and D (PLD). mAChR-stimulated PLD was turned off after 2 min of receptor activation with either the full (carbachol) or partial agonist (pilocarpine) and remained completely suppressed for at least 4 h. Partial recovery was observed 24 h after agonist removal. This rapid arrest of PLD response was not due to a loss of cell surface receptors and was also not caused by negative feedback due to concomitant activation of protein kinase C, tyrosine phosphorylation, increase in cytosolic calcium, or activation of Gi proteins. Furthermore, PLD stimulation by directly activated protein kinase C and GTP-binding proteins was unaltered in carbachol-pretreated cells. Finally, neither prevention of PLD stimulation during carbachol pretreatment by genistein nor inhibition of protein synthesis by cycloheximide, added before or after carbachol challenge, resulted in recovery of mAChR-stimulated PLD. The short term carbachol pretreatment nearly completely abolished agonist-induced binding of guanosine 5'-O-(3-thiotriphosphate) to membranes or permeabilized adherent cells. Full recovery of this response was achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by endogenously expressed purinergic receptors was also fully blunted after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to either receptor agonist partially, but not completely, reduced PLD stimulation by the other agonist. In contrast to desensitization of PLD stimulation, 2 min of carbachol treatment led to a sensitization, by up to 2-fold, of mAChR-stimulated inositol phosphate formation. This supersensitivity was also observed with pilocarpine, which acted as a full agonist on PLC. On the basis of these results, we conclude that the m3 mAChR stimulates PLD and PLC in HEK cells with distinct efficiencies and with very distinct durations of each response. The rapid and long lasting desensitization of the PLD response is apparently not due to a loss of cell surface receptors or PLD activation by GTP-binding proteins, but it may involve, at least initially, an uncoupling of receptors from GTP-binding proteins and most likely a loss of an as yet undefined essential transducing component.

Carbachol↗
PubMed1995

cDNA analysis of the mite allergen Lep d 1 identifies two different isoallergens and variants.

For the first time the complete cDNA encoding two isoallergens of the non-pyroglyphid dust mite Lepidoglyphus destructor, Lep d 1, allergen has been sequenced. In addition, one of the isoallergens was found to have two variants. Oligonucleotides were designed from known amino acid sequences. The cDNA sequences were obtained by hybridizing these primers to mRNA and enhancement by the RT-PCR technique. To obtain the different complete encoding cDNA sequences and eliminate heteroduplex artifacts, we performed PCR + 1 reactions. Comparison of the amino acid sequence of the allergen shows leader sequences of 16 amino acids for both isoforms.

Amino Acid Sequence↗
PubMed1995

Functional characterization of the higher plant chloroplast chaperonins.

The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10) have been purified and their structural/functional properties examined. In all plants surveyed, both proteins were constitutively expressed, and only modest increases in their levels were detected upon heat shock. Like GroEL and GroES of Escherichia coli, the chloroplast chaperonins can physically interact with each other. The asymmetric complexes that form in the presence of ADP are "bullet-shaped" particles that likely consist of 1 mol each of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional molecular chaperone. Under "nonpermissive" conditions, where spontaneous folding was not observed, it was able to assist in the refolding of two different target proteins. In both cases, successful partitioning to the native state also required ATP hydrolysis and chaperonin 10. Surprisingly, however, the "double-domain" ch-cpn10, comprised of unique 21-kDa subunits, was not an obligatory co-chaperonin. Both GroES and a mammalian mitochondrial homolog were equally compatible with the ch-cpn60. Finally, the assisted-folding reaction mediated by the chloroplast chaperonins does not require K+ ions. Thus, the K(+)-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.

Adenosine Diphosphate↗
PubMed1995

Replicating foamy virus-based vectors directing high level expression of foreign genes.

Replication-competent retroviral vectors (pFOV-1 to -3 and -7) were constructed on the basis of an infectious human foamy virus molecular clone which has deletions in the U3 region of the long terminal repeat and in the 3' region of the genome, previously identified to be nonessential for virus replication in vitro. The CAT and luciferase indicator genes were expressed as C-terminal fusion proteins to 215 amino acids of the viral Bet protein in the pFOV-1 vector. Introduction of the foot-and-mouth disease 2A protease sequence between the truncated bet coding sequence and the cloning site for the insertion of foreign genes in the pFOV-7 vector resulted in self-cleaving of the recombinant fusion protein. Alternatively, an internal ribosomal binding site was introduced, allowing expression of authentic foreign protein (pFOV-2 and -3 vectors). DNA fragments derived from the mouse hepatitis virus surface gene up to the length of 1.3 kb were inserted into pFOV-1. The vector constructs gave rise to viruses which were fully infectious in diploid human fibroblasts and recombinant viruses stably expressed high levels of foreign protein indicating that the pFOV vectors may be useful tools to study the effects of proteins of interest at least in tissue culture cells.

Amino Acid Sequence↗
PubMed1995

Biotechnological and gene therapeutic strategies in cancer treatment.

New anti-cancer agents are being developed which incorporate cancer-cell-specific recognition functions and are thus able to distinguish between normal and tumor cells. Recognition is dependent on the enhanced expression of antigenic determinants on the surface of tumor cells. The ErbB-2 receptor (ErbB-2R) is overproduced in a high percentage of adenocarcinomas arising in the breast, ovary, lung and stomach, when compared to normal cells. The tumor-enriched expression and extracellular accessibility make this receptor a suitable target for directed tumor therapy. A gene expressing the single-chain antibody molecule (scFv), specific for the extracellular domain of the ErbB-2R, was constructed by joining cDNAs encoding the light- and heavy-chain variable domains of the monoclonal antibody (mAb) FRP5. This scFv-encoding gene has been used as a targeting domain for two effectors: (i) A recombinant immunotoxin-encoding gene was constructed by adding sequences encoding a modified Pseudomonas aeroginosa exotoxin A (ETA) to the scFv-encoding DNA. (ii) Cytotoxic T-lymphocytes (CTL) with specificity for ErbB-2R-producing tumor cells were generated by retroviral transfer of a chimeric gene which encodes the scFv(FRP5), a hinge region and the zeta-chain of the T-cell receptor (TCR) complex. The bacterially produced recombinant immunotoxin scFv(FRP5)-ETA binds specifically to the ErbB-2R and displays both in vitro and in vivo cytotoxic effects selective for tumor cells producing high levels of the ErbB-2R. Target cells expressing the ErbB-2R gene were lysed in vitro with high specificity by the scFv::hinge::zeta-expressing T-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Physiological characterization of pretectal neurons projecting to the lateral posterior-pulvinar complex in the cat.

Pretectal neurons projecting to the lateral posterior-pulvinar complex (LP-P) in cats were electrophysiologically identified by their antidromic activation from the LP-P. Their responses to various visual stimuli and to electrical stimulation of the optic chiasm and the lateral geniculate nucleus (LGN) were characterized. In retrograde double-labelling experiments, the pretectal projections to the LP-P and to the LGN were tested for possible overlap. Forty-five neurons were antidromically activated from the LP-P; 55% of them could also be activated orthodromically from the optic chiasm. Most antidromically activated neurons responded to rapid movements of large textured visual stimuli as well as to 'on' or 'off' visual stimulation with short bursts. When stimulated with a slowly moving, large, structured visual stimulus, most cells showed a slight but significant activity increase. None of the neurons projecting to the LP-P was also activated antidromically from the LGN. Thus, the two populations are separate. This is supported by the results from the retrograde double-labelling experiments. None of the LP-P projecting neurons showed any directional selectivity to slow movements of large visual stimuli, a property by which pretectal neurons projecting to the inferior olive are characterized. Thus, neurons projecting to the LP-P do not bifurcate to the inferior olive. Response properties of neurons projecting to the LP-P were very similar to those of the 'jerk' neurons described by Schweigart and Hoffmann (Exp. Brain Res., 91, 273-283, 1992); therefore we believe them to be identical.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Analysis of the primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves by protein and cDNA sequencing indicates a eukaryotic origin of its gene.

The primary structure of the chloroplast isozyme of triosephosphate isomerase from rye leaves was identified by protein and cDNA sequencing and compared to the deduced amino acid sequence of a cDNA for the cytosolic isozyme. The mature cytosolic and chloroplast isozyme proteins share 64% amino acid sequence identity. The cDNA for the chloroplast isozyme codes for a precursor protein consisting of an N-terminal transit peptide of Mr 4351 and a mature subunit of Mr 27,282. Southern blot analysis indicates that the two rye isozymes are encoded by two independent single genes. Amino acid residues or sequence regions of basic functional relevance in known triosephosphate isomerases are strictly conserved in the chloroplast isozyme. The chloroplast isozyme contains 6 cysteine residues, instead of 4 in the cytosolic isozyme. A cysteine at position 143 of the chloroplast isozyme appears to be modified. Phylogenetic trees constructed on the basis of sequence comparisons for triosephosphate isomerases from different species of all major taxonomic groups indicate that the chloroplast isozyme is much more closely related to eukaryotic cytosolic enzymes than to eubacterial enzymes. The results indicate that the nuclear gene for the chloroplast isozyme originated with that for the cytosolic isozyme through duplication of an ancestral eukaryotic gene, rather than through gene transfer from a prokaryotic endosymbiont.

Amino Acid Sequence↗
PubMed1995

Secondary structures of Escherichia coli antisense micF RNA, the 5'-end of the target ompF mRNA, and the RNA/RNA duplex.

The Escherichia coli micF RNA is a prototype for a class of antisense RNAs encoded by genes at different loci from those that code for their target RNAs. RNAs in this class exhibit only partial complementarity to their targets. micF RNA binds to and regulates the stability of ompF mRNA in response to a variety of environmental stimuli. The secondary structures of micF RNA, ompF-213 mRNA (a segment containing the 213 nucleotides at the 5'-terminus of the target message), and the micF RNA/ompF-213 mRNA duplex were analyzed in vitro by partial digestion with structure-specific ribonucleases and chemical modification. Both micF RNA and ompF mRNA have single-stranded 5'-ends and contain stable stem-loop structures. Strong phylogenetic support for the proposed secondary structure for E. coli micF RNA is provided by a comparison of structural models derived from micF sequences from related bacteria. The micF RNA/ompF-213 mRNA duplex interaction appears to involve only a short segment of micF RNA. Unfolding of only one stem-loop of micF RNA and a minor stem-loop of ompF-213 mRNA appears to be necessary to form the duplex. The probing data suggest that the Shine-Dalgarno sequence and AUG start codon of ompF mRNA, found in single-stranded regions in the free message, are base-paired to micF RNA in the RNA/RNA duplex.

Bacterial Outer Membrane Proteins↗
PubMed1995

[Second malignancy in patients with Hodgkin disease in full remission. Interim results].

Among 345 patients with Hodgkin's disease (HD) in full remission and followed for a mean of 7.2 years (11 months-26 years) since the initial diagnosis, 33 were found to have a second malignancy. The cumulative incidence after 10 years was 13.5%, after 15 years 21%. Non-Hodgkin lymphoma was the most frequent (n = 7), followed by bronchial carcinoma (n = 5) and acute myeloid leukaemia (n = 3). There was a definitely increased risk in elder patients (P < 0.001) and in those who had had a splenectomy. There was no demonstrable effect of type of therapy, primary stage and frequency of recurrence. It is concluded that patients with Hodgkin's disease should have life-long oncological follow-up. This preliminary study is being extended into a multicentre investigation of a larger number of patients.

Adult↗
PubMed1995

EGF receptor and p185erbB-2-specific single-chain antibody toxins differ in their cell-killing activity on tumor cells expressing both receptor proteins.

Many human tumors over-express erbB-2 and EGF receptors. The membrane localization of these receptor tyrosine kinases make them appropriate targets for directed tumor therapy. We have used recombinant DNA technology to produce single-chain antibody exotoxin A (scFv-ETA) fusion proteins which specifically bind the erbB-2 and EGF receptors. The scFv portion is composed of the heavy- and light-chain variable domains of monoclonal antibodies which recognize the extracellular portion of each receptor. We have previously described the anti-tumor activity of the bacterially produced scFv(FRP5)-ETA directed to the erbB-2 receptor. In this paper we describe the characteristics of scFv(225)-ETA, a protein which binds the EGF receptor. The bacterially produced recombinant protein binds to the receptor with high affinity and inhibits the in vitro growth of the EGF receptor over-expressing tumor cell lines A431 and MDA-MB468. Combination treatment with scFv-(FRP5)-ETA and scFv(225)-ETA led to an additive inhibitory effect on the in vitro growth of A431 cells. SKBR3 cells expressing low levels of EGF receptor but high levels of p185erbB-2 were not affected by scFv(225)-ETA treatment but were sensitive to scFv(FRP5)-ETA. Stimulation of SKBR3 cells and HCII RI#11 mouse mammary epithelial cells expressing the human erbB-2 with EGF led to an increase in scFv(FRP5)-ETA activity, showing that the EGF-induced activation of erbB-2 can potentiate the action of the erbB-2-directed toxin. Treatment of athymic nude mice with scFv(FRP5)-ETA and the combination of both scFv-ETA proteins led to the transient arrest of growth of established A431 tumors. scFv(225)-ETA treatment alone was the most effective, leading to tumor shrinkage during the course of treatment, whereas treatment with the parental monoclonal antibody 225 led to retarded tumor growth.

ADP Ribose Transferases↗
PubMed1995

Clinical and histological features retain their prognostic impact under interferon therapy of CML: a pilot study.

In 55 patients with Ph1+ CML under interferon (IFN) monotherapy, an immunohistochemical and morphometric study on pretreatment bone marrow biopsies was performed to evaluate the prognostic impact of clinical as well as histological disease features. For identification of megakaryocytes we used the PAS stain and CD61 to calculate the subfraction of precursors (pro- and megakaryoblasts). Demonstration of macrophages and their different subsets was carried out by PG-M1 (CD68) and the GSA-1 lectin. The erythroid precursors were stained by Ret40f (anti-glycophorin C). Density of argyrophilic (reticulin plus collagen) fibers was determined by applying Gomori's silver impregnation method. Clinical variables like state of hematological response to IFN administration, age, spleen and liver size, myeloblasts plus promyelocytes, basophils as well as basophils and eosinophils exerted a predictive capacity by univariate statistical analysis. However, when entering these factors into previously published risk models, i.e., the so-called Sokal score and its modifications, to assess subgroups with different survival patterns or relative risk groups, a clear-cut discrimination was not feasible. Bone marrow features of prognostic value consisted of megakaryocytes and their precursors, fibers, and pro- and erythroblasts. Only when including histological variables into a formerly reported Cox model, could a significant separation of patients into the different categories or relative risk groups be computated. In conclusion, the present data emphasize the prognostic impact of histological parameters to be considered in all clinical trials on CML.

Adult↗
PubMed1995

Analysis of receptor-G protein interactions in permeabilized cells.

Receptor-induced binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP [gamma S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus alpha-toxin, binding of GTP[gamma S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP[gamma S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP[gamma S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[gamma S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP[gamma S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP[gamma S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1995

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type.

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

Calcium↗