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Biomedical subjects

B Müller

Publications and source records attributed to B Müller.

At least 271 records · Page 15Linked to original sources
PubMed1993

Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product.

Four different DNA fragments each coding for poly(hydroxyalkanoic acid) depolymerase (phaZ1-phaZ4) were isolated in pUC plasmids from a genomic library of Pseudomonas lemoignei in Escherichia coli. All recombinant strains secreted a highly active poly(3-hydroxybutyric acid) depolymerase and produced large translucent halos on an opaque medium containing poly(3-hydroxybutyric acid) granules. One DNA region (phaZ1) was present in seven independently isolated clones. Three other cloned DNA fragments were different from phaZ1 and from each other (phaZ2-phaZ4). In phaZ1, an open-reading frame of 1245 bp was identified from the nucleotide sequence of a 5435-bp MboI fragment (57 mol G + C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymerase of P. lemoignei, poly(3-hydroxybutyric acid) depolymerase C. A leader-sequence peptidase-cleavage site was predicted from the deduced amino acid sequence between Ala37 and Leu38. The calculated relative molecular masses of the precursor and the putative mature protein were 43468 and 39581, respectively. The polypeptide contains a lipase consensus sequence (Gly-Xaa-Ser-Xaa-Gly) and an unusually high proportion of threonine residues (22 of 36 amino acids) near the C-terminus. The N-terminus of the deduced amino acid sequence of PhaZ1 differed from that of the purified poly(3-hydroxybutyric acid) depolymerases A, B and the poly(3-hydroxyvaleric acid) depolymerase of P. lemoignei. The phaZ1 gene product, poly(3-hydroxybutyric acid) depolymerase C, was partially purified from recombinant E. coli (pUC91::phaZ1). The purified protein was specific for poly(hydroxyalkanoic acid) consisting of monomers of four or five carbon atoms and for p-nithrophenylbutyrate as substrates. The polymer-hydrolyzing activity, but not the p-nitrophenylate esterase activity, was inhibited by complex media such as Luria-Bertani medium and by soluble E. coli proteins. The enzyme protein did not cross-react with antibodies raised against purified poly(3-hydroxyvaleric acid) depolymerase of P. lemoignei.

Amino Acid Sequence↗
PubMed1993

Effect of nitrogen dioxide inhalation on surfactant phosphatidylcholine synthesis in rat alveolar type II cells.

After exposure of rats to NO2 (10 ppm, 72 h) type II pneumocytes were isolated and compared to cells from control animals in order to determine whether nitrogen dioxide inhalation affects surfactant phospholipid synthesis. (1) Exposed cells contained more DNA, protein and phospholipid than type II cells from controls. (2) Choline kinase, CTP: cholinephosphate cytidylyltransferase, and cholinephosphotransferase showed higher specific activities in the exposed cells. (3) In correspondence with this finding, the incorporation rates of choline into intermediate metabolic products were also higher in the NO2-exposed cells. (4) The pool sizes of the intermediate metabolic products of the CDP-choline-pathway for the synthesis of phosphatidylcholine were also higher in the cells isolated from exposed animals. This suggests that acute nitrogen dioxide exposure leads to an enhanced phospholipid synthesis that may be responsible for the higher amount of phospholipid detectable in lung lavage.

Administration, Inhalation↗
PubMed1993

Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. I. Comparison of RuvAB- and RuvB-mediated reactions.

The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro. In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA. However, RuvA is required when the RuvB concentration is reduced 4-fold or more. Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides. To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB. The time courses of the two reactions were similar, and both required ATP and Mg2+. However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2). The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Müller, B., Tsaneva, I.R., and West, S. C. (1993) J. Biol. Chem. 268, 17185-17189) and can be overcome by addition of RuvA. These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.

Bacterial Proteins↗
PubMed1993

Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. II. Interaction of RuvB with DNA.

Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro. The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB. The presence of E. coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179-17184). To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I. The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction. RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA. In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner. At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.

Bacterial Proteins↗
PubMed1993

Kinetics of interaction of HIV reverse transcriptase with primer/template.

Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.

Base Sequence↗
PubMed1993

[Broad beans as a cause of acute hemolytic anemia].

A previously healthy 17-year-old Greek boy suddenly developed jaundice of sclerae and skin. In addition, physical examination revealed a pale appearance. He also reported feeling tired and weak. The haemoglobin level was 9.6 g/dl, lactate dehydrogenase activity 335 U/l, bilirubin concentration 3.2 mg/dl (direct bilirubin 0.7 mg/dl, indirect bilirubin 2.5 mg/dl), haptoglobin concentration 48.8 mg/dl. As haemolytic anaemia was assumed, direct questioning elicited the fact that the patient had, for the first time in his life, eaten 300 g of broad beans (Vicia faba) on each of two days, namely 3 and 2 days before the appearance of jaundice. Absence of glucose-6-phosphate dehydrogenase activity in the red blood corpuscles confirmed the diagnosis of favism. On symptomatic treatment both the enzyme activities and the bilirubin level fell to normal within one week, and the haemoglobin level was 15.7 g/dl after 4 weeks.

Acute Disease↗
PubMed1993

Two-step binding mechanism of fibrinogen to alpha IIb beta 3 integrin reconstituted into planar lipid bilayers.

The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption to Bio-Beads. These vesicles were then used to generate planar lipid bilayers. The binding of fluorochrome labeled fibrinogen or the peptide ligand Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was monitored by total internal reflection fluorescence microscopy and a solid phase binding assay. Analysis of the kinetics revealed fast reversible formation of a fibrinogen/integrin precomplex (KD = 50 nM) followed by formation of a stable irreversible complex. This transition was monitored by measuring the fraction of precomplex which could be dissociated by addition of excess Gly-Arg-Gly-Asp-Ser (GRGDS). For the peptide, the KD was 1200 nM, and the rates of association and dissociation were faster than the time resolution of the method. Similar KD values were found by inhibition of fibrinogen binding to alpha IIb beta 3 in the immobilized receptor assay. Since the binding of fibrinogen was irreversible, KD values were dependent on the time period between fibrinogen incubation and peptide addition. These and results by other authors point to the biological importance of the biphasic binding process of fibrinogen to its receptor on platelets.

Amino Acid Sequence↗
PubMed1993

RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.

Amino Acid Sequence↗
PubMed1993

Impaired regulation of surfactant phospholipid metabolism in the isolated rat lung after nitrogen dioxide inhalation.

Various drugs have been shown to stimulate surfactant phospholipid metabolism. Particularly beta-adrenergic agonists play an important role under physiologic conditions. For the first time we have studied whether nitrogen dioxide (NO2) inhalation alters beta-adrenergic regulation of surfactant phospholipid metabolism in the model of the isolated lung. Rats were continuously exposed in vivo to a 5 ppm NO2-containing atmosphere for 48 hr. The lungs were isolated and perfused in presence of the beta-adrenergic agonist dopexamine and surfactant metabolism was studied in three lung compartments: (1) lung lavage, (2) lung tissue, and (3) lavagable free alveolar cells. We found that (1) in normal rat lungs dopexamine increased the incorporation of palmitate and choline from the perfusate into lung lavage phospholipids. In nitrogen dioxide exposed rat lungs beta-adrenergic stimulation did not cause an increase in precursor incorporation. No significant difference in unstimulated precursor incorporation was found for normal and NO2-exposed rat lungs. (2) Lung tissue from rats exposed to NO2 showed a decreased precursor incorporation into disaturated phosphatidylcholine due to an augmented cellular pool size. (3) Lavagable alveolar cells showed an increased palmitate uptake after nitrogen dioxide inhalation and by beta-adrenergic stimulation. From these data we conclude that nitrogen dioxide inhalation impairs the beta-adrenergic regulation of surfactant phospholipid metabolism. Moreover these data underline the importance of beta-adrenergic agonists in surfactant metabolism.

Administration, Inhalation↗
PubMed1993

Molecular evaluation of an Alu repeat including a polymorphic variable poly(dA) (AluVpA) in the vitamin D binding protein (DBP) gene.

We investigated an Alu element at the end of intron 8 of the human vitamin D-binding protein (hDBP, group-specific component, GC) gene that shows a polymorphic poly(A) tail due to a variable number of tandem repeats (AluVpA) forming the 3' end of this member of the most abundant class of short interspersed repeated DNA element (SINES). The Alu element sequence in intron 8 of the GC gene was identical in all three common GC alleles (GC*1F, GC*1S, and GC*2) and could be classified as an Alu-Sa or Alu class-II sequence. The polymerase chain reaction was used to amplify selectively a fragment of about 200 bp containing the identified (TAAA)n repeat from genomic DNA of 188 unrelated human subjects. The size of the amplified products was determined by polyacrylamide gel electrophoresis. Four alleles (named GC-18*6, GC-I8*8, GCI8*10, and GC-18*11) were found that differed in size by multiples of four nucleotides. The allele frequencies ranged from 0.0053 to 0.8511 and the observed heterozygosity was 26%. The stable inheritance of this polymorphic patterned poly(A) sequence was confirmed by a segregation study of a highly informative family with 19 members. Statistically significant linkage disequilibrium between the AluVpA and the GC iso-electric focusing (IEF) phenotypes was found in a sample of 188 unrelated individuals and delta values were calculated from the observed haplotype distribution.

Base Sequence↗
PubMed1993

Polymer materials in biosensors.

Fundamentals and application examples of polymeric materials in different types of biosensors and presented and discussed in view of their molecular structure and biosensor design and construction. The role of a series of polymers with respect to their typical application and their specific properties, like sensitivity and stability, is highlighted. Future trends of polymer materials for biosensors in the area of medical and environmental applications are outlined.

Biosensing Techniques↗
PubMed1993

[Biological standardization of Ginkgo extracts].

The determination of the inhibition of PAF (platelet-activating factor)-induced platelet aggregation has been proposed as a biological standardization method for commercially available Ginkgo biloba extracts by measuring the characteristic pharmacological effect of ginkgolides in vitro. The determination is specific for ginkgolides A, B, C, and J and is not influenced by other constituents present in Ginkgo biloba extracts. IC50 values of ginkgolide B can be used to standardize various Ginkgo extracts produced by special extraction methods with respect to equi-effective ginkgolide B contents. In order to compare values obtained by a chemical-analytical procedure with those obtained by the biological assay, the equi-effective total ginkgolide content of each Ginkgo extract had to be calculated. Accordingly, the concentrations of the individual ginkgolides in the various Ginkgo extracts were determined chromatographically by assaying ginkgolides as trime-thylsilyl derivatives. Their individual contributions towards the measured in vitro effects were derived from their respective IC50 values. The calculated equi-effective total ginkgolide contents of the Ginkgo extracts were in good agreement with those obtained by gas chromatography. The results demonstrate that, in addition to a chemical standardization, the biological standardization of Ginkgo extract preparations is also feasible.

Lactones↗
PubMed1993

Influence of medium components and metabolic inhibitors on citric acid production by Penicillium simplicissimum.

Penicillium simplicissimum excreted more than 100 mmol citric acid l-1 [2.9 mmol (g dry wt)-1; 9 d] if an industrial filter dust (> 50% ZnO) providing a high extracellular buffering capacity was present in the medium. A similar specific [2 mmol (g dry wt)-1], but lower absolute (26 mmol l-1), citric acid excretion occurred in the absence of an extracellular buffer and if amino acids or urea were used as nitrogen source. P. simplicissimum excreted no citric acid under conditions where Aspergillus niger produces citric acid (deficiency of trace elements, low pH and reduced biomass formation). Citric acid excretion by P. simplicissimum always paralleled biomass formation and occurred in a pH range between 4 and 7. This indicated that different imbalances of metabolism were responsible for citric acid excretion in A. niger and P. simplicissimum. However, provided a high extracellular buffering capacity was present, the response of the Penicillium system to different carbon and nitrogen sources was similar to the Aspergillus system. In contrast, the metals iron and copper had virtually no effect on citric acid excretion compared with A. niger. Estimation of intracellular citric acid, as well as the effects of the uncoupler 2,4-dinitrophenol, and the H(+)-ATPase inhibitor sodium orthovanadate, led to the conclusion that the buffer-stimulated citric acid efflux was dependent on metabolic energy and an energized plasma membrane, respectively. Despite similarities to the Aspergillus system, a different mechanism for buffer-stimulated citric acid excretion by P. simplicissimum seems probable.

Antimetabolites↗
PubMed1993

Bronchoalveolar lavage. Quantitation of intraalveolar fluid?

A precise calculation of the amount of intraalveolar fluid is the basis of a quantitative analysis of intraalveolar compounds. Different approaches have been made to cover this important problem. Here, we report a comparative study with five markers: 99mTc-DTPA, 51Cr-EDTA, inulin, urea, and methylene blue in animal experiments as well as in human experiments. The marker substances were added to the lavage fluid, and the "dilution" of the markers, i.e., the alveolar fluid, was calculated. The results showed that in animals with healthy lungs the tracer methods are able to calculate amounts of intraalveolar fluid that are comparable to morphologic findings. In animals as well as in humans, methylene blue and inulin were shown to be useless in determining alveolar fluid volume compared with the tracer methods. In humans, the calculations with the urea method and with Tc-DTPA were in the same magnitude, but there was no individual correlation. We conclude that, at present, the methods to quantitate alveolar fluid volume lack precision and add nothing to a deeper understanding of alveolar biology.

Animals↗
PubMed1993

Horizontal cells in the cone-dominated tree shrew retina: morphology, photoreceptor contacts, and topographical distribution.

While most mammalian retinas are rod dominated, in the tree shrew retina 95% of the photoreceptors are cones. We studied three shrew horizontal cells to look for features associated with this unusual photoreceptor arrangement. The morphology of horizontal cells was revealed by intracellular injections of Lucifer yellow, and their photoreceptor contacts were assessed by light and electron microscopy. Horizontal cell topography was studied in material stained with a neurofilament antibody and with toluidine blue. The tree shrew has two types of horizontal cell that are basically the same as A- and B-type horizontal cells of other mammals. All the photoreceptor contacts of the larger, axonless, A-type cell and the dendritic contacts of the smaller, axon-bearing, B-type cell are with cones. Both types contact nearly all the cones in their dendritic field and both types synapse with both red and blue cones. There is no anatomical evidence for chromatic selectivity. The sparsely branched B-type horizontal cell axon probably contacts rods as in other mammals. The unusual features of the A-type cell are the profuse dendritic terminal arborizations and the large dendritic field size. These features may be related to the abundance of cones but do not justify the conclusion for a special type of horizontal cell as has previously been supposed. Both types of horizontal cell have a central-peripheral density gradient; at any location B-type cells are up to three times as numerous as A-type cells. There are detailed features of the distributions that differ from those of other mammalian horizontal cells. The density maximum of B-type cells is in inferior retina and roughly coincides with that of the cones; the A-type maximum is located more superiorly. Neither maximum is colocalized with the ganglion cell peak in the central area. The mosaic of B-type cells is much more regular than that of A-type cells.

Animals↗
PubMed1993

[The content of hemoglobin in the blood and the abundant and trace elements in blood plasma and erythrocytes of sheep, fallow deer, dwarf goats and reindeer].

Of the 4 species, the blood of fallow deer had the highest concentration of Hb (10.9 +/- 0.7) and that of pygmy goats (6.5 +/- 1.4 mmol/l) the lowest. The highest concentration of protein in the plasma was found in the reindeer (81.2 +/- 2.1) and the lowest in the pygmy goats (59.1 +/- 4.3 g/l). High values of ascorbic acid were recorded in the reindeer (16.4 +/- 2.4) and in the Finnish sheep (16.3 +/- 2.5 micrograms/ml). There existed some differences in the concentrations of Na, K, Ca, Mg, Pa, Fe, Cu and Zn in the plasma of the sheep of 7 different breeds under similar nutritional conditions. The erythrocytes of the sheep of 3 breeds and that of fallow deer, of pygmy goats and of reindeer uniformly had a low Na- and a high K-concentration. The Ca-concentration of the erythrocytes of the sheep of most breeds and that of the fallow deer and pygmy goats was very low, that of Mg and Zn was higher than in the plasma.

Animals↗