Time-resolved Raman scattering with incoherent light.
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Journal Article↗
Biomedical subjects
M Müller
Publications and source records attributed to M Müller.
Significance and redox state of SH groups in pyruvate carrier isolated from bovine heart mitochondria.
The role and properties of -SH groups of purified pyruvate (monocarboxylate) carrier were investigated. After isolation, this protein has all -SH groups in the oxidized state. Upon reduction, the carrier can be labelled with eosin-5-maleimide. The shift in apparent Mr after the labelling points to the presence of at least two cysteine residues. Pyruvate uptake in the reconstituted system is inhibited by both permeable (eosin-5-maleimide at 1 mM concentration) and impermeable (mersalyl, p-chloromercuribenzoate) -SH group reagents. Phenylarsine oxide inhibits pyruvate transport only slightly (20%), but the inhibition is enhanced after preincubation with the substrate.
Animals↗
Nucleotide sequence of porcine insulin-like growth factor. 1:5' untranslated region, exons 1 and 2 and mRNA.
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Amino Acid Sequence↗
[Glutaraldehyde residues in heart valve prostheses].
Bioprosthetic heart valves prepared from glutaraldehyde-pretreated bovine pericardium are used to replace diseased human cardiac valves. Mineralisation in the course of time and toxic effects are possibly caused by glutaraldehyde residues. Different washing methods carried out before transplantation are compared with regard to the different ability of glutaraldehyde extraction.
Animals↗
Protein--DNA contacts in the structure of a homeodomain--DNA complex determined by nuclear magnetic resonance spectroscopy in solution.
The 1:1 complex of the mutant Antp(C39----S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three-dimensional structure were based on the use of the fully 15N-labelled protein, two-dimensional homonuclear proton NOESY with 15N(omega 2) half-filter, and heteronuclear three-dimensional NMR experiments. Based on nearly complete sequence-specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H-1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix-turn-helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix-turn-helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39----S) homeodomain no defined spatial structure could be found for the apparently flexible N-terminal segment comprising residues 0-6.
Animals↗
The interaction with DNA of wild-type and mutant fushi tarazu homeodomains.
The in vitro DNA binding properties of wild-type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein-DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.
Amino Acid Sequence↗
Determinants of membrane-targeting and transmembrane translocation during bacterial protein export.
We have separately analyzed membrane-targeting and membrane translocation of an exported bacterial protein. The precursor of the outer membrane protein LamB of Escherichia coli was synthesized in vitro and translocated into inverted plasma membrane vesicles under co- and post-translational conditions. The translation/translocation products of LamB were subsequently resolved into soluble and membrane-associated material. Dissipation of the H(+)-motive force, depletion of ATP and treatment of membranes with N-ethylmaleimide each inhibited processing and translocation of preLamB without preventing its binding to the membranes. Hence, all three conditions block transmembrane passage rather than membrane-targeting. The latter was abolished by pretreatment of salt-extracted membrane vesicles with trypsin. It was also drastically reduced when preLamB was synthesized in cell extracts derived from either a secA amber or a secB null mutant. Membrane-targeting of preLamB therefore requires soluble SecA and SecB as well as a protease-sensitive membrane receptor. The finding that SecA is involved in targeting whereas ATP is required for the transmembrane passage suggests that SecA, which harbors an ATPase activity [Lill et al. (1989), EMBO J., 8, 961-966], might have a dual function in bacterial protein export.
Adenosine Triphosphate↗
Transgenic offspring by transcaryotic implantation of transgenic ovaries into normal mice.
Female transgenic mice may be unable to reproduce successfully if the product encoded by the transgene results in pathological changes or affects the fertility of the mouse. To approach this problem, we have produced chimaeras by transferring the ovaries of transgenic mice into normal mice of the same strain. Such chimaeras will be an ideal tool for investigating the interactions between transgenic ovaries and normal mice or vice versa. Here we show that, using this method, we were able to get large numbers of transgenic offspring even from founder transgenic female mice that were themselves infertile as a result of the overexpression of growth hormone genes. Although none of the ovary recipients were given immunosuppressant treatment, 60% of the recipients had biologically active ovaries over a mean period of about 100 days.
Animals↗
Quantitative comparison of frequency representation in the auditory brainstem nuclei of the gerbil, Pachyuromys duprasi.
Frequency representation in the auditory brainstem nuclei of the fattailed gerbil (Pachyuromys duprasi) was studied by focal iontophoretic HRP injections into the cochlear nucleus at physiologically characterized positions. Tuning curves of cochlear nucleus neurons were determined by extracellular recording of multi-unit spike activity prior to HRP application. Neurons with characteristic frequencies of 0.55 to 17.5 kHz were recorded. Anterograde labeling of synaptic terminal fields demonstrated a tonotopic arrangement of frequencies within the cochlear nucleus subdivisions and the lateral and medial superior olivary nuclei. In all nuclei investigated terminal fields were arranged in isofrequency planes. In the subdivisions of the cochlear nucleus and in the lateral superior olive the frequency range from 0.55 to 17.5 kHz was represented. In the medial superior olive on the other hand, frequency representation was restricted to frequencies below 3.5 kHz. Quantitative evaluation of tonotopy showed that the frequency representation in the anteroventral and posteroventral cochlear nucleus and lateral superior olive were very similar. The amount of tissue-volume processing one octave varied within the hearing range. The frequency range of highest auditory sensitivity (between 1 and 5 kHz) occupied up to 25% nuclear tissue/octave, while at the upper and lower limits of the hearing range, frequencies were represented by a ratio of 10-15% nuclear tissue/octave. The frequency representation pattern in these brainstem nuclei reflected exactly the frequency place map of the cochlea.
Animals↗
Synapsin I expression in the rat retina during postnatal development.
The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.
Animals↗
Carrier-mediated transport in the hepatic distribution and elimination of drugs, with special reference to the category of organic cations.
Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.
Animals↗
Flow cytometric analysis of tissue factor (TF) expression on stimulated monocytes--comparison to procoagulant activity of mononuclear blood cells.
Whereas tissue factor (TF), a 47 kDa transmembrane glycoprotein, is constitutively present in certain tissues such as epithelial tissue, brain, and placenta, it is normally not expressed by cells within the vasculature. However, inflammatory mediators including bacterial lipopolysaccharide (LPS) can stimulate the expression of cell surface procoagulant activity (PCA) on monocytes. In our present study the kinetics (over 24 h) of molecular TF expression on LPS-stimulated monocytes analyzed by flow cytometry corresponds closely to functional PCA of human mononuclear blood cells (MBC). Both PCA and TF expression on monocytes were rapid events reaching their maximum after about 6 h of stimulation. At this time approximately 70-80% of monocytes had also achieved maximum anti-TF MAb receptor density. For certain analytical applications, monitoring of molecular TF expression on monocytes by flow cytometry using anti-TF MAb is favorable because there is no influence by PCA inhibitors.
Antibodies, Monoclonal↗
Regulation of stimulus-induced calcium transport pathways in human T (Jurkat) lymphoblasts.
In human T (Jurkat) lymphoblasts we have studied the calcium signals induced by monoclonal antibodies reacting with the T-cell antigen receptor complex (TCR and CD3). Jurkat cells were preloaded with the fluorescent calcium indicator Indo-1 and the stimulus-induced rise in cytoplasmic free calcium concn was followed in the absence or in the presence of external calcium. The technique allowed the separate investigation of the intracellular calcium release and the external calcium influx processes. The changes in the membrane potential of Jurkat cells were followed simultaneously by using fluorescent indicators. We found that the activation of protein kinase C by phorbol ester (PMA) or by the permeable diacyl glycerol, DiC8, rapidly eliminated the calcium signal, independently of the presence or absence of external calcium, while these treatments did not appreciably change the membrane potential. In contrast, cell membrane depolarization achieved by various treatments selectively blocked the stimulus-induced calcium influx, while did not affect stimulus-induced calcium release from internal stores. The magnitude of the stimulus-induced calcium influx was found to be largely independent of the external calcium concns between about 2-2500 microM. It is demonstrated that the inhibitory effect of membrane depolarization on calcium influx is not simply due to the reduction of the inward calcium gradient under these conditions. These observations indicate a significant down-regulation of the stimulus-induced calcium signal by protein kinase C activation and a selective inhibition of the receptor-operated calcium channels by membrane depolarization.
4-Chloromercuribenzenesulfonate↗
The structure of the homeodomain and its functional implications.
The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.
Amino Acid Sequence↗
Effect of sulfonamides and 4-MET on adhesion to tooth substrates.
Adhesion of a composite to tooth substrates was studied. The bonding liner was a mixture: camphorquinone, 0.5%; one of four sulfonamides (A, B, C, and D), 0.5%; 4-methacryloxyethyl trimellitate (4-MET), 0, 2, and 5%; in triethyleneglycol dimethacrylate (TEGDMA). The best tensile bond strength to bovine dentin treated with 0.3 M EDTA 2 Na-0.2 M EDTA FeNa (EDTA 3-2) was 7.5 MPa. The bond strength to bovine dentin decreased with the addition of 4-MET. The bond strength to bovine enamel treated with 65% phosphoric acid (H3PO4) was 12.0 MPa. The tensile bond strength to bovine enamel treated with EDTA 3-2 was lower than that to enamel treated with 10% citric acid-3% ferric chloride (10-3) and with H3PO4. The polymerization of these liners was characterized by differential scanning calorimetry (DSC). The photocurable bonding liner studied was adequate for clinical studies.
Acid Etching, Dental↗
DNA binding properties of the purified Antennapedia homeodomain.
The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.
Alkylation↗
Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis.
We have determined the primary structure of the [2Fe-2S]ferredoxin of the anaerobic protist Trichomonas vaginalis. This protein, situated in the hydrogenosome, is composed of 93 amino acids. A comparison of T. vaginalis ferredoxin with greater than 80 other ferredoxins shows the closest similarity to [2Fe-2S]putidaredoxin of the aerobic bacterium Pseudomonas putida and a lesser one to mitochondrial [2Fe-2S]ferredoxins of vertebrates. This similarity is reflected in the overall primary structure and in the spacing of cysteine residues coordinating the iron-sulfur center. The primary structure, but not the environment of the iron-sulfur center, also shows similarity with [2Fe-2S]ferredoxins of photosynthetic organisms and halobacteria. We have cloned and analyzed the T. vaginalis ferredoxin gene. The gene is present in a single copy and devoid of introns. It gives rise to a transcript with unusually short 5' and 3' untranslated regions of 16 and 18 nucleotides, respectively. DNA sequence analysis of the gene predicts an additional 8 amino acids at the amino terminus which are absent from the purified protein. This amino-terminal region of the protein is characterized by properties typical of mitochondrial presequences.
Amino Acid Sequence↗
Development of a cell-free system to study the membrane assembly of photosynthetic proteins of Rhodobacter capsulatus.
A cell-free translation system from the facultatively photoheterotrophic bacterium Rhodobacter capsulatus is described. Synthesis of two proteins of the bacterium's photosynthetic apparatus (light-harvesting complex B870 alpha and beta) was performed by SP6 polymerase transcription of the subcloned genes, isolation of the mRNA and translation in vitro using a cell-free extract of R. capsulatus cells. The integration of these proteins in vitro into added intracytoplasmic membrane vesicles (ICM) is demonstrated. Without addition of ICM approximately 70% of the synthesized B870 proteins were soluble. If, however, ICM were present during synthesis, the majority of the soluble protein was found to associate with the membranes. The membrane-associated polypeptides could be solubilized only by detergent treatment but could not be extracted by treatment at alkaline pH (Na2CO3), suggesting that the proteins had been firmly inserted into the lipid bilayer. Moreover, the B870 alpha and beta proteins that integrated in vitro into ICM were also found to associate with pigment ligands and to assemble into a native reaction center/B870 complex. The native conformation of this complex isolated from ICM by Triton fractionation was demonstrated by microspectral analysis of the bound pigments.
Bacterial Proteins↗