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M Müller

Publications and source records attributed to M Müller.

At least 433 records · Page 24Linked to original sources
PubMed1999

The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism.

Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.

3' Untranslated Regions↗
PubMed1999

Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins.

Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA). Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane. In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins. Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane. Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes. In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium. We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm. Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.

Bacterial Outer Membrane Proteins↗
PubMed1999

Interaction of the P-glycoprotein multidrug transporter (MDR1) with high affinity peptide chemosensitizers in isolated membranes, reconstituted systems, and intact cells.

P-glycoprotein-mediated multidrug resistance can be reversed by the action of a group of compounds known as chemosensitizers. The interactions with P-glycoprotein of two novel hydrophobic peptide chemosensitizers (reversins 121 and 205) have been studied in model systems in vitro, and in a variety of MDR1-expressing intact tumor cells. The reversins bound to purified P-glycoprotein with high affinity (77-154 nM), as assessed by a quenching assay using fluorescently labeled purified protein. The peptides modulated P-glycoprotein ATPase activity in Sf9 insect cell membranes expressing human MDR1, plasma membrane vesicles from multidrug-resistant cells, and reconstituted proteoliposomes. Both peptides induced a large stimulation of ATPase activity; however, higher concentrations, especially of reversin 205, led to inhibition. This pattern was different from that of simple linear peptides, and resembled that of chemosensitizers such as verapamil. In both membrane vesicles and reconstituted proteoliposomes, 1-2 microM reversins were more effective than cyclosporin A at blocking colchicine transport. Reversin 121 and reversin 205 restored the uptake of [3H]daunorubicin and rhodamine 123 in MDR1-expressing cells to the level observed in the drug-sensitive parent cell lines, and also effectively inhibited the extrusion of calcein acetoxymethyl ester from intact cells. In cytotoxicity assays, reversin 121 and reversin 205 eliminated the resistance of MDR1-expressing tumor cells against MDR1-substrate anticancer drugs, and they had no toxic effects in MDR1-negative control cells. We suggest that peptides of the reversin type interact with the MDR1 protein with high affinity and specificity, and thus they may be good candidates for the development of MDR1-modulating agents to sensitize drug resistance in cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

AMPA-type glutamate receptor subunits are expressed in the avian cochlear hair cells and ganglion cells.

The cellular localization of AMPA-type glutamate receptor subunits was examined in the pigeon inner ear using subunit specific polyclonal antibodies (GluR1-4). In the auditory ganglion cell bodies immunoreactivity for the subunits GluR2/3 and GluR4, but not for GluR1 was detected. The hair cells showed diffuse immunoreactivity for GluR4. Additionally, immunostaining for the subunits GluR2/3 and GluR4 was present below the hair cells. These results indicate that the AMPA type glutamate receptors play a role in neurotransmission at the hair cell afferent synapse in the avian auditory system.

Animals↗
PubMed1999

Immunocytochemistry of GABA in the central complex of the locust Schistocerca gregaria: identification of immunoreactive neurons and colocalization with neuropeptides.

The central complex is a highly organized neuropil structure in the insect brain and plays a role in motor control and visual orientation. We describe the distribution of gamma-aminobutyric acid (GABA) immunostaining in the central complex of the locust Schistocerca gregaria in an effort to analyze inhibitory neural circuits within this brain area. Antisera against GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase resulted in identical patterns of immunostaining. Cell counts revealed about 100 bilateral pairs of GABA-immunoreactive neurons with arborizations in the central complex. Five types of immunostained neurons could be identified through reconstruction of the staining pattern, comparison with individually stained neurons, and double labeling experiments with Neurobiotin-injected neurons. All of these GABA-immunostained neurons are tangential neurons that connect the lateral accessory lobes to distinct layers of the central body. Three types of immunostained neurons (TL2, TL3, TL4) invade the lower division of the central body, and two additional types of neurons (TU1, TU2) have ramifications in layers I and II of the upper division of the central body. Double-labeling experiments with peptide antisera suggest that peptides related to Phe-Met-Arg-Phe-NH2/bovine pancreatic polypeptide and Dip-allatostatin might act as cotransmitters with GABA in TL4 neurons of the lower division and (Dip-allatostatin only) in TU2 neurons of the upper division of the central body. The high conservation in the pattern of GABA immunostaining in all insect species investigated so far suggests that GABA plays an essential role in the basic neural circuitry of the central complex in insects.

Animals↗
PubMed1999

Photometric determination of human serum bromide levels--a convenient biomonitoring parameter for methyl bromide exposure.

Methyl bromide is one of the most important pesticides for the control of insects, fungi and nematodes. Serum bromide has been proposed as a biomonitor for occupational exposure to methyl bromide. Therefore, a novel, sensitive photometric method was developed for the determination of serum bromide at concentrations relevant for such exposure. Further possible applications are monitoring of intoxication victims and halothane narcosis. Using the method we have established a mean serum bromide level of 4.13 +/- S.D. 1.05 mg/l (n/64) in a group of healthy female and male volunteers not knowingly exposed to bromide or bromine containing organics. Serum of a subject accidently exposed to methyl bromide revealed a bromide level of 11.5 mg/l serum, while two individuals exposed to methyl iodide had no elevated levels. A group of 30 agricultural workers showed a mean serum bromide level of 15.33 +/- S.D. 1.90 mg/l at the end of the methyl bromide application season.

Bromides↗
PubMed1999

Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection.

Expression of human papillomavirus type 16 (HPV 16) fusion proteins LI deltaCE7(1-55) and LI deltaCE7(1-60) (carboxy-terminal deletion of LI replaced by 55 or 60 amino-terminal amino acids of E7) leads to formation of chimeric papillomavirus-like particles (CVLPs). After "infection" of cells by CVLPs, the chimeric proteins can be detected in the cytosol and the endoplasmic reticulum (ER), suggesting that they are intracellularly processed via the MHC class I pathway and, therefore, able to activate cytotoxic T lymphocytes (CTLs). To investigate the cytotoxic immune response against HPV 16 LI deltaCE7(1-60) and LI deltaCE7(1-55) CVLPs, we immunized C57Bl/6 mice with various CVLP doses without adjuvant. Two weeks after immunization, spleen cells were prepared and stimulated in vitro using HPV 16 E7-expressing transfectants of the tumor cell line RMA. In 51Cr-release cytotoxicity assays, spleen cells of mice vaccinated with LI deltaCE7(1-60) CVLPs specifically lysed the RMA-E7 transfectants as well as RMA cells loaded with the peptide E7(49-57), which represents an H2-Db-restricted CTL epitope. This demonstrates that CVLPs induce an E7-specific CTL response in mice in the absence of an adjuvant. Furthermore, immunization with CVLPs prevented outgrowth of E7-expressing tumor cells even if inoculation of cells was performed 2 weeks before vaccination. We conclude from our data that CVLPs show promise for therapy of HPV-associated lesions.

Animals↗
PubMed1999

Influence of MUC18/MCAM/CD146 expression on human melanoma growth and metastasis in SCID mice.

The cell surface glycoprotein MUC18MCAM/CD146 was originally defined as a marker of melanoma progression and has been suspected to be directly linked to the metastatic process of this malignancy. In order to address this question, 2 MCAM negative human melanoma cell lines, SK-2 and XP44RO(Mel), were transfected with MCAM-encoding cDNA. Surface MCAM expression on SK-2 and XP44RO(Mel) transfectants was similar to that observed in naturally occurring MCAM positive human melanoma cells and transfectants demonstrated MCAM-dependent increase in homotypic adhesion in vitro. The growth behavior of 7 MCAM transfectants and their respective vector controls was evaluated in SCID mice. Tumor size at 4-5 weeks after s.c. implantation was highly variable, but did not correlate with MCAM expression. Despite massive primary tumor formation at the injection site, no spontaneous metastasis was observed with any of the investigated MCAM transfectants. The influence of MCAM expression on lung metastases formation in an experimental metastasis assay was system dependent, converting only XP44RO(Mel) transfectants into metastatic cells, although increased homotypic adhesion, leading to formation of tumor cell clusters, was observed with transfectants of both cell lines in vitro. Our findings indicate that MCAM expression of human melanoma cells has an influence on later stages of the metastatic process only, namely, extravasation and establishment of new foci of growth, but is per se not sufficient for this process.

Animals↗
PubMed1999

Cloning and sequencing of an acetyl-CoA synthetase (ADP-forming) gene from the amitochondriate protist, Giardia lamblia.

A Giardia lamblia gene, Glacs, was cloned, sequenced and expressed in Escheria Coli. This gene codes for a 726 residue long acetyl-CoA synthetase (ADP-forming). This enzyme is responsible for the formation of acetate, a metabolic endproduct of G. lamblia. It is known from only two Type I amitochondriate eukaryotes, G. lamblia and Entamoeba histolytica and from the archaebacterium, Pyrococcus furiosus. With Glacs as query, homologous unidentified open reading frames were detected in the complete genomes of only a few archaebacteria and eubacteria. These form a new protein family present in all three domains of life, which probably plays a central role in the acyl-CoA metabolism but is of restricted taxonomic distribution.

Animals↗
PubMed1999

Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase.

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

Amino Acid Sequence↗
PubMed1999

Author errata.

Explore the source record for details and available documents.

Retraction Notice↗
PubMed1999

Piperidine-renin inhibitors compounds with improved physicochemical properties.

Piperidine renin inhibitors with heterocyclic core modifications or hydrophilic attachments show improved physical properties (lower lipophilicity, improved solubility). Tetrahydroquinoline derivative rac-30 with a molecular weight of 517 and a log D(pH 7.4) of 1.9 displays potent and long lasting blood pressure lowering effects after oral administration to sodium depleted conscious marmosets.

Animals↗
PubMed1999

Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.

Bacterial periplasmic nickel-containing hydrogenases are composed of a small subunit containing a twin-arginine signal sequence and a large subunit devoid of an export signal. To understand how the large subunit is translocated into the periplasm, we cloned the hyb operon encoding the hydrogenase 2 of Escherichia coli, constructed a deletion mutant, and studied the mechanism of translocation of hydrogenase 2. The small subunit (HybO) or the large subunit (HybC) accumulated in the cytoplasm as a precursor when either of them was expressed in the absence of the other subunit. Therefore, contrary to most classical secretory proteins, the signal sequence of the small subunit itself is not sufficient for membrane targeting and translocation if the large subunit is missing. On the other hand, the small subunit was required not only for membrane targeting of the large subunit, but also for the acquisition of nickel by the large subunit. Most interestingly, the signal sequence of the small subunit determines whether the large subunit follows the Sec or the twin-arginine translocation pathway. Taken together, these results provide for the first time compelling evidence for a naturally occurring hitchhiker co-translocation mechanism in bacteria.

Biological Transport↗
PubMed1999

Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response.

The use of chimeric virus-like particles represents a new strategy for delivering tumor antigens to the immune system for the initiation of antitumor immune responses. Immunization of DBA/2 mice with the P1A peptide derived from the P815 tumor-associated antigen P1A induced specific T-cell tolerance, resulting in progression of a regressor P815 cell line in all animals. However, immunization with a human papillomavirus type 16 L1 virus-like particle containing the P1A peptide in the absence of adjuvant induced a protective immune response in mice against a lethal tumor challenge with a progressor P815 tumor cell line. Additionally, we demonstrated that these chimeric virus-like particles could be used therapeutically to suppress the growth of established tumors, resulting in a significant survival advantage for chimeric virus-like particle-treated mice compared with untreated control mice. Chimeric virus-like particles can thus be used as a universal delivery vehicle for both tolerizing and antigenic peptides to induce a strong protective and therapeutic antigen-specific antitumor immune response.

Animals↗
PubMed1999

Ionic mechanism of 4-aminopyridine action on leech neuropile glial cells.

Extracellular 4-aminopyridine (4-AP), tetraethylammonium chloride (TEA) and quinine depolarized the neuropile glial cell membrane and decreased its input resistance. As 4-AP induced the most pronounced effects, we focused on the action of 4-AP and clarified the ionic mechanisms involved. 4-AP did not only block glial K+ channels, but also induced Na+ and Ca2+ influx via other than voltage-gated channels. The reversal potential of the 4-AP-induced current was -5 mV. Application of 5 mM Ni2+ or 0.1 mM d-tubocurarine reduced the 4-AP-induced depolarization and the associated decrease in input resistance. We therefore suggest that 4-AP mediates neuronal acetylcholine release, apparently by a presynaptic mechanism. Activation of glial nicotinic acetylcholine receptors contributes to the depolarization, the decrease in input resistance, and the 4-AP-induced inward current. Furthermore, the 4-AP-induced depolarization activates additional voltage-sensitive K+ and Cl- channels and 4-AP-induced Ca2+ influx could activate Ca2+-sensitive K+ and Cl- channels. Together these effects compensate and even exceed the 4-AP-mediated reduction in K+ conductance. Therefore, the 4-AP-induced depolarization was paralleled by a decreasing input resistance.

4-Aminopyridine↗
PubMed1999

Species-specific alternative splicing of transgenic RNA in the mammary glands of pigs, rabbits, and mice.

Gene-farming techniques provide an effective tool for the production of recombinant proteins in livestock. Transgenes consisting of genomic DNA sequences are as a rule more efficiently expressed than those in which the product of interest is encoded by a cDNA. However, the processing of pre-mRNA from genomic constructs may yield unexpected messenger RNAs and subsequently protein variants. We describe the appearance of different alternative mRNA splice patterns of a gene construct in which a mutant human growth hormone (hGH-N) gene is transcriptionally controlled by 2.5 kb of mouse whey acidic protein (WAP2) regulatory sequences in the mammary gland of different livestock species. Compared to the transcription products in transgenic mice harboring the same gene construct and to cell transfection experiments, expression analysis in transgenic pigs and rabbits revealed different mRNA splice patterns with regard to the proportion of the processed transcripts. Apart from already-known physiological mRNA splice products, previously undescribed processed hGH transcripts were observed in these species. Sequence analysis of the transgenes suggests that the species-specific hGH mRNA patterns may be caused by species- and tissue-specific differences in trans-acting splice factors.

Alternative Splicing↗
PubMed1999

Localization of tissue factor in actin-filament-rich membrane areas of epithelial cells.

Tissue factor (TF), the cellular receptor and cofactor for clotting factor VII/VIIa (FVII/VIIa), is known mainly as the initiator of the coagulation protease cascade. Recently, it was shown that inactivation of the murine TF gene (TF-/-) results in embryonic lethality which is most likely due to some failure of vascular integrity. On the other hand, gene disruption in mice of coagulation proteins like FVII, prothrombin, and fibrinogen results in phenotypes of embryonic development that contrast with that of TF-/-, suggesting a role for TF beyond fibrin formation in embryogenesis. In addition, there is a growing body of evidence that cellular TF may be involved in nonhemostatic functions. To determine the microtopography of membrane TF with regard to the cytoskeleton organization, we examined the expression patterns of TF and cytoskeletal proteins in various cell lines by means of double immunofluorescence and electron microscopy (EM). In spreading cells, a granular membrane TF expression of the cell cortex and a pronounced granular TF staining of microspikes, lamellipodes, and ruffled membrane areas were observed. Especially, actin and alpha-actinin were in close proximity to TF in these regions. Colocalization of TF and nonmuscle filamin (ABP-280) at the leading edge of spreading cells indicated an association of TF with the actin filament system, too. Using scanning EM we found gold-labeled TF at long processes and actin-filament-containing microspikes of neighboring cells in both branching and contact sites. By the means of immunogold EM we observed that TF is localized at the cell surface in a spotty pattern, at the base and at the top of budding processes. The observed staining pattern points to a connection of TF with elements of the cytoskeleton in these highly dynamic membrane regions, a fact which is underlined by the recently described molecular interaction of TF's cytoplasmic domain with ABP-280. In cells undergoing cytokinesis, we detected also strong TF expression in dynamic membrane areas and protrusions of the midbodies, indicating an accumulation of TF in actin-rich membrane areas with high contractile activity. In addition, we were able to demonstrate that immobilized ligands for TF, both catalytically active and inactive FVIIa or anti-TF mAbs, accelerated adhesion and spreading of TF-expressing cancer cells. Thus, our findings support the contention that ligation of cellular TF may be involved in morphogenic processes such as adhesion and spreading by an association to cytoskeletal structures. On the other hand, incubation of these cells with proteolytically active FVIIa but not with covalently inactivated FVIIa (DEGR-FVIIa) or anti-TF mAbs in solution resulted in increased motility of these cells, indicating that not only ligation of TF but also the proteolytic activity of TF-FVIIa complex is involved in cell migration.

Actin Cytoskeleton↗