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Biomedical subjects

J Brunner

Publications and source records attributed to J Brunner.

At least 127 records · Page 7Linked to original sources
PubMed1983

Direct accurate gas flow measurement in the patient: compensation for unavoidable error.

It is shown that the conditions for accurate flow measurement are not met if the resistant flow meter (e.g., Fleisch pneumotachograph or screen pneumotachograph) is attached directly at the mouth or endotracheal tube and the breath flows directly through it, firstly because its gas composition, temperature, and humidity change radically even within the course of one respiratory cycle, secondly because the expiratory peak flow of the patient being ventilated rapidly tends to become too high, and thirdly because the entire system is sensitive to turbulence. Methods are proposed to compensate continuously for the influence of the changing gas concentrations and to reduce expiratory peak flow without increasing resistance. With the resulting reduction in the error from 20% to about 2%, tidal volume can be more reliably determined, and the higher quality of primary data allows a more differentiated and more complex evaluation (N2-washout compartment analysis, VDS measurement, analysis of complicated patterns of spontaneous breathing or mechanical ventilation such as IMV, etc.).

Humans↗
PubMed1982

Isolation of the hydrophobic membrane binding domain of rat renal gamma-glutamyl transpeptidase selectively labeled with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine.

The membrane-permeable photoactivatable reagent 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine was used to selectively label the hydrophobic domain of the amphipathic form of gamma-glutamyl transpeptidase reconstituted into phosphatidylcholine vesicles. The reagent labels only a limited segment of the large subunit of the heterodimeric transpeptidase. Treatment of labeled and reconstituted enzyme with papain causes the release of the unlabeled catalytic domain and the cleavage of the membrane binding domain into two discrete 125I-labeled peptides. The hydrophobic peptides which remain associated with the vesicles were isolated by chromatography on Sephadex LH-60. They exhibit apparent molecular weights of 8700 and 3400. Amino acid analysis indicates that they contain 68 and 58% hydrophobic residues, respectively. The procedures developed in this study should make possible the large scale isolation of the unlabeled membrane binding domain of gamma-glutamyl transpeptidase.

Amino Acids↗
PubMed1982

Hydrophobic labeling, isolation, and partial characterization of the NH2-terminal membranous segment of sucrase-isomaltase complex.

A photogenerated carbene, 3-trifluoromethyl-3-(m-[125I]iodophenyl)carbene (Brunner, J., and Semenza G. (1981) Biochemistry, 20, 7174-7182), was used to label the hydrophobic core of small intestinal brush-order membrane vesicles. Reaction of the carbene with sucrase-isomaltase complex was restricted to a polypeptide segment which is essential for binding the enzyme complex to the native membrane or to liposomes. The same labeling selectivity was obtained when purified sucrase-isomaltase complex was labeled either in Triton X-100 solution or when it was incorporated in egg-lecithin liposomes. During cleavage of sucrase-isomaltase with papain, the radiolabel remained covalently associated with the anchor peptide. It was thus possible to detect easily the polypeptide in the course of subsequent separation and purification operations. The molecular weight of the peptide was determined by gel filtration on Sephadex LH-60 in ethanol-formic acid (Takagaki, Y., Gerber, G. E., Nihei, K., and Khorana, H. G. (1980) J. Biol. Chem. 255, 1536-1541). The figure thereby obtained, 6500, is somewhat lower than that obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (approximately 8000). Circular dichroism of the peptide indicates a secondary structure of high alpha-helical content. A possible structure of the membranous segment is discussed.

Animals↗
PubMed1981

Selective labeling of the hydrophobic core of membranes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a carbene-generating reagent.

The synthesis of a new photoactivatable probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]-TID), with a high specific radioactivity (10 Ci mmol-1) is described. It was tested as a probe for the hydrophobic core of membranes. TID partitions strongly in favor of the lipid phase of membranes, and the photogenerated carbene labels intrinsic membrane proteins in a highly selective manner. This conclusion was reached from the distribution of radioactivity among the proteins of [125I]TID-labeled human erythrocyte membranes. By far the most heavily labeled protein is band 3 [nomenclature of Fairbanks, G., Steck, T. L., & Wallach, F. G. H. (1971) Biochemistry 10, 2606-2617] while the labeling of glycophorin is approximately 5 times less than that of band 3. There is little or no labeling of known extrinsic proteins.

Affinity Labels↗
PubMed1980

Ion and sugar permeabilities of lecithin bilayers: comparison of curved and planar bilayers.

Na+ and sugar permeabilities of egg lecithin bilayers were measured using curved bilayers and planar bilayers as represented by single-bilayer vesicles and black lipid films, respectively. The Na+ permeability coefficient measured with single-bilayer vesicles at 25 degrees C is (2.1 +/- 0.6) x 10(-13) cm sec-1. Because of technical difficulties it has been impossible to measure ionic permeabilities of values lower than about 10(-10) cm sec-1 in planar (black) lipid bilayers using tracer methods. The D-glucose and D-fructose permeabilities were measured with both curved and planar bilayers. The permeability coefficients measured with vesicles at 25 degrees C are (0.3 +/- 0.2) x 10(-10) cm sec-1 for glucose and (4 +/- 1) x 10(-10) cm sec-1 for D-fructose; these are in reasonable agreement with the corresponding values obtained for planar (black) lipid bilayers which are (1.1 +/- 0.3) x 10(-10) cm sec-1 for D-fructose, respectively.

Animals↗
PubMed1980

Analysis of membranes photolabeled with lipid analogues. Reaction of phospholipids containing a disulfide group and a nitrene or carbene precursor with lipids and with gramicidin A.

Analogues of fatty acids have been synthesized which contain an S-S bridge in the aliphatic chain and at the omega-carbon the photoactivatable p-(3-trifluoromethyldiazirino)phenyl or p-azidophenyl group as a carbene and nitrene precursor, respectively. These acids were used to acylate 1-palmitoyl phosphatidylcholine by the procedure described by Gupta et al. (Gupta, C.M., Radhakrishnan, R., and Khorana, H.G. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4315-4319). Multilamellar liposomes prepared from various radioactively labeled lipids and 2 to 10 mol% of one of the photosensitive phospholipid analogues were photolyzed and the extent of cross-linking was determined. The carbene-generating probe labeled dipalmitoyl phosphatidylcholine in yields of 15 to 20%, while labeling with the nitrene probe was about 10 times less efficient. In contrast these probes were found to react with the unsaturated fatty acid of 1-palmitoyl-2-oleoyl phosphatidylcholine with almost equal efficiency. The nitrene was 2 to 3 times more efficient than the carbene in cross-linking to gramicidin A. Amino acid analysis and Edman degradation of the labeled peptide indicated that both probes predominantly attacked tryptophan residues. No geometrical correlation was found between the expected penetration of the reactive group of the probe within the bilayer and the labeled sites of the peptide. This result is discussed in terms of chemical selectivities of the photoactivated intermediates. After photocross-linking, the disulfide bridge can be cleaved to generate a free sulfhydryl group which subsequently can be utilized for preparative or analytical purposes. Photolabeled lipids and peptides can be separated easily from the bulk of the unlabeled material. These phospholipid analogues are substrates for the phospholipid exchange protein isolated from calf liver. Transfer from sonicated dispersions of the analogues into an acceptor membrane was demonstrated. Individual monolayers can thus be labeled separately with these reagents.

Azirines↗
PubMed1980

3-Trifluoromethyl-3-phenyldiazirine. A new carbene generating group for photolabeling reagents.

The synthesis of 3-trifluoromethyl-3-phenyldiazirine (TPD) is reported in an overall yield of 60% based on 2,2,2-trifluoroacetophenone as starting material. TPD is rapidly photolyzed on irradiation near 350 nm to yield 35% of the diazoisomer and 65% of the corresponding carbene. No internal rearrangement of the latter compound by fluorine migration was detected. Photolysis in methanol yielded the product of a formal OH insertion in greater than 95% yield. Photolysis in cyclohexane gave at least a 50% yield of the CH insertion product at a diazirine concentration of 15 mM. The products were identified by gas chromatographic mass spectra and by 19F NMR spectra. In the dark the diazirine is stable in 1 M acid or base and at temperatures as high as 75 degrees C for at least 30 min. The diazoisomer is much less photolabile and is stable in 0.1 M acetic acid for at least 12 h. The synthesis of a derivative of TPD containing the 2-hydroxyethyl[O-tosylate] group on the para position of the phenyl ring is described. This compound permits the easy attachment of the TPD function to other molecules. It is suggested that the ease of synthesis, dark stability, rapid photolysis, and reactivity of the carbene may make this group useful in the preparation of various photochemical probes.

Affinity Labels↗
PubMed1980

[Histochemical study of peroxidase and tyrosinase activities in the cuticle of Protophormia terraenovae].

Quantitative histochemical measurements of activities of enzymes were performed by temporal defined stages in development of Protophormia terraenovae. The effect of the specific insecticide of moult Duphar pH 60-38 activity of enzyme was evaluated. Activities of peroxidase and tyrosinase showed parallel oscillations, at which proof of both enzymes was restricted to the cuticle. The insects, treated with insecticide, showed a moulting, dependent on concentration. A transfer of maxima of activities is described.

Animals↗
PubMed1976

Single bilayer vesicles prepared without sonication. Physico-chemical properties.

Single shelled lecithin vesicles of uniform size (diameter = 300 A) are prepared without sonication by solubilizing unsonicated lecithin dispersions with sodium cholate and removing the detergent from the mixed lecithin - cholate micelles by gel filtration on Sephadex G-50. A homogeneous population of pure lecithin single-bilayer vesicles free of multilamellar structures is obtained. The vesicle diameter is somewhat larger than the average diameter of sonicated vesicles. The curvature of the bilayer seems to be sufficiently large to allow for similar packing densities (areas/molecule) on the outer and inner layer of the bilayer. The morphology and some physico-chemical properties of these vesicles are described and compared with those of sonicated vesicles.

Cholic Acids↗