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Biomedical subjects

F Müller

Publications and source records attributed to F Müller.

At least 397 records · Page 22Linked to original sources
PubMed1985

Nuclear magnetic resonance studies of the old yellow enzyme. 1. 15N NMR of the enzyme recombined with 15N-labeled flavin mononucleotides.

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with specifically 15N-labeled flavin mononucleotide and investigated by 15N NMR spectroscopy in the oxidized and reduced state. The results indicate that in the oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition, hydrogen bonds exist between the N(1) and N(3) atoms of FMN and the apoprotein. The resonance position of N(10) indicates that this atom is somewhat sp3-hybridized, i.e. lifted out of the molecular plane of the isoalloxazine ring system. In the reduced state the N(1) atom is negatively charged and the N(3) atom forms a hydrogen bond with the apoprotein. The N(10) atom in protein-bound FMN exhibits about the same hybridization state as in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp3-hybridized. This interpretation can also be derived from the 15N(5)-1H coupling constant. Among the flavoproteins thus far studied by NMR techniques, old yellow enzyme is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lifted out of the molecular plane. The isoelectric focussing properties of old yellow enzyme and a new easy method for the preparation of the apoprotein are also reported.

Apoenzymes↗
PubMed1985

Nuclear magnetic resonance studies of the old yellow enzyme. 2. 13C NMR of the enzyme recombined with 13C-labeled flavin mononucleotides.

The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with selectively 13C-enriched flavin mononucleotides and investigated by 13C NMR spectroscopy. The 13C NMR results confirm the results obtained by 15N NMR spectroscopy and yield additional information about the coenzyme-apoenzyme interaction. A strong deshielding of the C(2) and C(4) atoms of enzyme-bound FMN both in the oxidized and reduced state is observed, which is supposed to be induced by hydrogen-bond formation between the protein and the two carbonyl groups at C(2) and C(4) of the isoalloxazine ring system. The chemical shifts of all 13C resonances of the flavin in the two-electron-reduced state indicate that the N(5) atom is sp3-hybridized. From 31P NMR measurements it is concluded that the FMN phosphate group is not accessible to bulk solvent. The unusual 31P chemical shift of FMN in old yellow enzyme seems to indicate a different binding mode of the FMN phosphate group in this enzyme as compared to the flavodoxins. The 13C and 15N NMR data on the old-yellow-enzyme--phenolate complexes show that the atoms of the phenolate are more deshielded whereas the atoms of the enzyme-bound isoalloxazine ring are more shielded upon complexation. A non-linear correlation exists between the chemical shifts of the N(5) and the N(10) atoms and the pKa value of the phenolate derivative bound to the protein. Since the chemical shifts of N(5), N(10) and C(4a) are influenced most on complexation it is suggested that the phenolate is bound near the pyrazine ring of the isoalloxazine system. 15N NMR studies on the complex between FMN and 2-aminobenzoic acid indicate that the structure of this complex differs from that of the old-yellow-enzyme--phenolate complexes.

Apoenzymes↗
PubMed1985

Carbon-13 and nitrogen-15 nuclear-magnetic-resonance investigation on Desulfovibrio vulgaris flavodoxin.

Desulfovibrio vulgaris apoflavodoxin has been reconstituted with 15N and 13C-enriched riboflavin 5'-phosphate. For the first time all carbon atoms of the isoalloxazine ring of the protein-bound prosthetic group have been investigated. The reconstituted protein was studied in the oxidized and in the two-electron-reduced state. The results are interpreted in terms of specific interactions between the apoprotein and the prosthetic group, and the chemical structure of protein-bound FMN. In the oxidized state weak hydrogen bonds exist between the apoprotein and the N(5), N(3) and O(4 alpha) atoms of FMN. The N(1) and O(2 alpha) atoms of FMN form strong hydrogen bonds. The isoalloxazine ring of FMN is strongly polarized and the N(10) atom shows an increased sp2 hybridisation compared to that of free FMN in aqueous solution. The N(3)-H group is not accessible to bulk solvent, as deduced from the coupling constant of the N(3)-H group. In the reduced state the hydrogen bond pattern is similar to that in the oxidized state and in addition a strong hydrogen bond is observed between the N(5)-H group of FMN and the apoprotein. The reduced prosthetic group possesses a coplanar structure and is ionized. The N(3)-H and N(5)-H groups are not accessible to solvent water. Two-electron reduction of the protein leads to a large electron density increase in the benzene subnucleus of bound FMN compared to that in free FMN. The results are discussed in relation to the published crystallographic data on the protein.

Binding Sites↗
PubMed1985

Formation of pentachlorophenol as the major product of microsomal oxidation of hexachlorobenzene.

On incubation of [14C]-hexachlorobenzene with microsomes from livers of rats induced with hexachlorobenzene, the major product (80-90%) was pentachlorophenol. The only other detectable metabolite, tetrachlorohydroquinone (4-15%), was presumably formed from pentachlorophenol. A considerable amount of radioactivity (5-10% of the amount of extracted metabolites) was covalently bound to protein. Microsomes derived from male hexachlorobenzene--induced rats gave by far the highest conversion (approx. 1% of substrate). Microsomes from female hexachlorobenzene--induced rats were 3 times less efficient. Microsomes from untreated and 3-methyl-cholanthrene--treated animals gave less than 5% of the amount of pentachlorophenol formed by microsomes from hexachlorobenzene--induced male rats, while phenobarbital and aroclor 1254-induction resulted in formation of 51% and 34% respectively.

Animals↗
PubMed1985

The first appearance of the neural tube and optic primordium in the human embryo at stage 10.

Thirteen embryos of stage 10 (22 days) were studied in detail and graphic reconstructions of most of them were prepared. The characteristic feature of this stage is 4-12 pairs of somites. Constantly present are the prechordal and notochordal plates (the notochord sensu stricto is not yet apparent), the neurenteric canal or at least its site, the thyroid primordium, probably the mesencephalic and rhombencephalic neural crest and the adenohypophysial primordium. During this stage, the following features appear: terminal notch, optic sulcus, initial formation of neural tube, oropharyngeal membrane, pulmonary primordium, cardiac loop, aortic arches 1-3, intersegmental arteries, and laryngotracheal groove. The primitive streak is still an important feature. Graphic reconstructions have permitted the detection of the telencephalic portion of the forebrain, for the first time at such an early stage. It is proposed that the remainder of the forebrain comprises two subdivisions: D1, which becomes largely the optic primordium during stage 10, and D2, which is the future thalamic region. The optic sulcus is found in D1 but does not extent into D2, as has been claimed in the literature. An indication of invagination of the otic disc appears towards the end of the stage. As compared with the previous stage, the prosencephalon has increased in length, the mesencephalon has remained the same, the rhombencephalon has decreased, and the spinal part of the neural plate has increased fivefold in length. The site of the initial closure of the neural groove is rhombencephalic, upper cervical, or both. The neural plate extends caudally beyond the site of the neurenteric canal. Cytoplasmic inclusions believed to indicate locations of great activity were always detected in the forebrain (especially in the optic primordium), and also in the rhombencephalon, spinal part, and mesencephalon.

Brain↗
PubMed1985

Monoclonal antibodies to immunodominant surface-exposed protein antigens of Treponema pallidum.

Specific murine monoclonal antibodies directed against immunodominant surface-exposed protein antigens of Treponema pallidum with molecular weights of 15,500, 33,000, 44,000, and 46,000 were isolated. Of seventeen monoclonal antibodies characterized by Western blotting, three were directed against 15,500, three against 33,000, nine against 44,000, and two against 46,000 molecular weight protein antigens of Treponema pallidum. Three of the monoclonal antibodies were reactive in the haemagglutination assay, 11 in the in vitro immobilization assay, and 13 in the immunofluorescence assay. It is suggested that the different monoclonal antibodies could be useful in isolating immunodominant protein antigens of Treponema pallidum and in obtaining information on their biological relevance for use in the diagnosis of syphilis.

Animals↗
PubMed1985

Identification of glycosylated protein antigens of Treponema pallidum and Treponema phagedenis.

Comparison of autoradiographies of intrinsically [35S] methionine and [14C] glucosamine labeled Treponema pallidum (Nichols strain) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed four glycosylated proteins with molecular weights 30,500, 33,000, 35,000, and 59,000. T. phagedenis (biotype Reiter) was comparatively investigated and showed only two glycosylated proteins with molecular weights 33,000 and 34,000. The at the first time in treponemes identified glycosylated proteins could be precipitated with homologous human antibodies and characterized as antigens. By comparison with 125I surface labeling of T. pallidum and T. phagedenis it is suggested that the glycosylated protein antigens are localized on the surface of these treponemes.

Antigens, Bacterial↗
PubMed1985

Digestion and absorption rates of lactose, glucose, galactose, and fructose in three infants with congenital glucose-galactose malabsorption: perfusion studies.

The digestion rates of lactose and the absorption rates of glucose, galactose, and fructose were studied by continuous perfusion of the jejunum in three patients aged 7 weeks to 9 months with congenital glucose-galactose malabsorption (infusion rate:1.0 ml min-1; concentration of each sugar: 200 mM; perfusion distance: 30 cm). The mean absorption rates of glucose and galactose were 26.5 and 43.8 mumol min-1 30 cm-1, respectively, and were significantly reduced (p less than 0.001) to 13 and 22%, respectively, of intake. On the other hand, the absorption of fructose was 133.3 mumol min-1 30 cm-1, i.e., as high as in the controls. The hydrolysis rate of lactose was also normal (134.0 mumol min-1 30 cm-1). However, the absorption rates of glucose and galactose released from the disaccharide were as low as the perfusion of free monosaccharides. In patients with glucose-galactose malabsorption the glucose absorption rate is as low as that of galactose. No additional glucose transport system seems to exist. A hydrolase related transport system is also of no importance in compensating for the primary defect of monosaccharide absorption.

Biological Transport, Active↗
PubMed1985

Exfoliative cytology after transurethral resection of superficial bladder tumours.

Fifty-one patients with predominantly superficial bladder tumours underwent prospective cytological examination at predetermined intervals following surgery in order to evaluate the prognostic reliability of bladder irrigation cytology. The results suggest that positive cytology 3 days after radical transurethral resection is a reliable indicator of incomplete resection or additional occult tumour foci. The implications for post-operative management and follow-up are discussed.

Follow-Up Studies↗
PubMed1985

Improved method for isolating synaptosomes from 11 regions of one rat brain: electron microscopic and biochemical characterization and use in the study of drug effects on nerve terminal gamma-aminobutyric acid in vivo.

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.

3-Mercaptopropionic Acid↗
PubMed1985

Influence of gliadin on fetal chick intestine in tissue culture.

The purpose of this study was to develop a model system for detecting biological effects of gliadin which may be related to coeliac disease. The technique applied was tissue culture of chicken duodenum at several stages of fetal development. A normally occurring increase in disaccharidase activities in cultured tissue explants was diminished by the presence of peptic-tryptic digested gliadin or of a tryptic fragment of alpha-gliadin (alpha-GT 18,000). The effect of peptic-tryptic digested gliadin was only demonstrable in the early phase of fetal development (days 10-14), and disappeared at day 16. The release of enzymes into the culture medium was decreased by gliadin at the 12th day of fetal development. The results suggest that gliadin inhibits the differentiation processes of the fetal intestine.

Age Factors↗
PubMed1985

[Immunology of neurosyphilis: intrathecal synthesis of Treponema pallidum-specific IgG and IgM antibodies].

Due to uncharacteristic symptoms of central nervous system (CNS) alterations during Treponema pallidum (TP) infection, the exact diagnosis of neurosyphilis has become more and more difficult. Particularly, there is a lack of sufficiently specific and sensitive assays for the demonstration of intrathecal synthesis of TP-specific immunoglobulins. TP-specific IgG or IgM class antibodies were estimated quantitatively in serum and cerebrospinal fluid (CSF) of 302 syphilitic patients by a modified enzyme immunoassay (ELISA), and antibody concentrations were quantitated as ELISA units per mg total IgG or IgM. In 197 of 237 patients with neurosyphilis an intrathecal synthesis of TP-specific IgG or IgM could be demonstrated by a 3- to 450-fold higher antibody concentration in the CSF than in the corresponding serum. In contrast, 65 patients without CNS involvement on the infection did not show any difference in the antibody concentration of CSF and serum. Thus, the diagnosis of neurosyphilis respectively the exclusion of CNS participation on the TP infection on an immunological base has become possible.

Adult↗
PubMed1985

The origin of the ectodermal ring in staged human embryos of the first 5 weeks.

Seven embryos of stages 10-16 (3-5 weeks) were studied for their external form by means of graphic reconstructions. This is the first systematic report on the general surface anatomy of the early human embryo. The brain has been described and illustrated in a previous publication, and the present article is concerned particularly with an important although neglected feature: the ectodermal ring, described by Schmitt [Morph. Arb., Jena 8: 236-303, 1898] and named by Blechschmidt [Funktionsentwicklung. I. Mechanische Gegenwirkungen. Musterschmidt, Göttingen 1948]. Late in stage 10, three longitudinal bands can be seen on the surface: somitic, intermediate, and lateral plate. The intermediate band, which partly overlies nephrogenic tissue, is the site of the future ectodermal ring, which begins as an inverted U at stage 11 and is completed caudally in stage 12. The ectodermal ring, which may well be an important example of epithelial-mesenchymal interaction, comprises six parts: (a) rostral part, containing the situs neuroporicus, and olfactory and lens discs; (b) pharyngeal part, the covering of the pharyngeal arches; (c) occipital and cervicothoracic parts, related at first to the four occipital somites and later to the cervicothoracic junction; (d) membral part, represented by a preliminary ectodermal thickening, followed within 2 days by the apical ectodermal ridge; (e) intermembral part, related at first to the underlying coelom, and mesonephric duct and ridge; (f) caudal part, containing the cloacal membrane and a temporary 'caudal ectodermal ridge'. It is stressed that the incorrectly named 'Milchstreifen' is merely the intermembral part, in which the mammary crest ('Milchlinie' or 'Milchleiste') appears 1 week later.

Ectoderm↗
PubMed1985

Chicken fetal intestine in tissue culture.

A simple and reproducible method for cultivation of fetal chicken small intestine is presented. The culture was performed in a defined medium without serum. Duodena were excised from embryos at the 14th day of fetal development and cut in small segments that were maintained in culture until day 16. It could be shown that the morphology of cultured intestine resembles that of noncultured gut of corresponding age as judged by light microscopy. The increase in activity of sucrase and maltase in cultured explants is comparable with that of intestine in ovo, whereas that of alkaline phosphatase is lower than under in vivo conditions. Hormones (thyroxine, dexamethasone) influence the enzymic pattern of fetal intestine in a known manner. Therefore, the method permits maintenance of fetal intestine in tissue culture for 2 days, a period sufficient for investigation of maturation processes of intestinal mucosa.

Alkaline Phosphatase↗
PubMed1984

31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase.

In addition to the phosphate residues contained in the acid-dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active-center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis has identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH-dependent chemical shift of the phosphorus residue in the molybdenum cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show phosphorus resonances at about -3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at +8.8 ppm and at +13.5 ppm are assigned to those of the pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T.L., Edmondson, D.E., and Husain, M. (1981) Biochemistry 20, 617] and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about -3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50-60% functional enzyme, but increases dramatically in intensity in the desulpho enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line-broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the approximately equal to -3-ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) 'inhibited' form of the enzyme with ethylene glycol results in extensive line-broadening of the resonances at -3 ppm and +1 ppm, but has no observable affect on the FAD resonances. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the activesite molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.

Animals↗