Biomedical subjects
Publications and source records attributed to K Decker.
Radiographs of the lumbar spine in sitting and standing positions demonstrate sliding movements of the vertebral bodies to an unexpected extent, as a sign of the instability of the lumbar spine. These observations suggest some ideas for the construction of chairs.
1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.
The conditions for the quantitative determination of UDP-Gal:glucosylceramide galactosyltransferase and of UDP-Gal:GM2 galactosyltransferase in Golgi-enriched preparations of rat liver were optimized. Triton X-100 was the detergent routinely used as octyl glucoside acted as a galactose acceptor forming octyl lactoside. Manganese ions were required for full activity, but Co2+ and Mg2+ could substitute to some extent. The nucleotide pyrophosphatase activity of the Golgi preparations which interfered with the GL2-synthase assay was inhibited by addition of 20 mM IMP; the latter is without appreciable effect on the rate of GL2 synthesis. Apparent Km values for UDP-Gal were 130 microM and 140 microM with Gl2-synthase and Gm1-synthase, respectively. That for glucosylceramide was 80 microM with GL2-synthase; for GM2 it was 10 microM with GM1-synthase. Competition experiments with variable concentrations of the lipid acceptors showed that the two synthase activities are independent catalytic entities. The specific activity of GM1-synthase exceeds that of GL2-synthase by a factor of ca. 25 under the optimized conditions used here.
Rat liver lactate dehydrogenase (LDH) has been synthesized in hepatocytes and in a cell-free translation system. The subunit synthesized in both systems displayed the same electrophoretic mobility upon sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. Sequence analysis of the subunits translated in vitro and synthesized in vivo indicated N-acetyl-alanyl-alanine for both N termini. Thus, the newly synthesized subunit does not exhibit an amino-terminal extension. The mRNA for the lactate dehydrogenase subunit was exclusively found in free polysomes. A size of 2120 +/- 240 nucleotides was estimated for the mRNA. Only about 50% of these nucleotides are needed to code for the polypeptide chain of the enzyme.
Very small quantities of FAD were able to reactivate apo-D-amino acid oxidase. In the presence of D-alanine, luminol, horseradish peroxidase, and an excess of the apoenzyme, a quantitative luminometric determination of FAD was possible. The maximal photon emission measured in a bicarbonate buffer, pH 9.2, at 37 degrees C was proportional to the amount of FAD added. FMN, riboflavin, or 5-deazaflavin produced no chemiluminescence and had no inhibitory effect in the assay when added together with FAD. With this method, FAD could be quantitatively determined with high accuracy in perchloric acid extracts of animal tissue and bacteria.
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Ca2+ uptake by monolayer cultures of rat Kupffer cells was strongly stimulated immediately after contact with phagocytosable material, e.g. zymosan particles. The intensity of the luminol-mediated chemiluminescence following zymosan addition was dependent on extracellular Ca2+; addition of the Ca2+-ionophore A 23187 did not further increase the zymosan-elicited response. The superoxide-mediated chemiluminescence was not inhibited by indomethacin but could be suppressed by compounds known to inhibit Ca2+-calmodulin-dependent reactions. Analysis of their efficiency, however, suggests that the O-2 production is mediated by Ca2+ rather than Ca2+-calmodulin and that the inhibitors exert another, e.g. membrane-directed influence.
The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.
A method for the quantitative determination of several D-amino acids in the range of 0.05-1 nmol per assay (0.25-5 microM) is described. It is insensitive to the presence of excesses of the respective L-amino acids. The assay system employs D-amino-acid oxidase (hog kidney), peroxidase (horse radish) and luminol; the total photon output elicited by the oxidation of the D-amino acids is determined. The different reactivity of individual D-amino acids with D-amino-acid oxidase limits the applicability of the assay. Indications for the usefulness of immobilized enzymes in D-amino-acid analysers are also given.
A systematic study of the homologous series of omega-aminoalkyl-agaroses revealed differences in the affinities of 6-hydroxy-D- and 6-hydroxy-L-nicotine oxidase. In contrast to supports with nonpolar alkyl chains, omega-aminoalkyl-agarose showed high affinity towards the L-specific enzyme, while the D-specific oxidase was bound most firmly by omega-aminododecyl-agarose. 6-Hydroxy-L-nicotine oxidase could be desorbed by 1.3M NaCl only in the presence of the substrate L-6-hydroxynicotine. Using the omega-aminoalkyl-agarose, a complete separation of the enantiozymes was accomplished and an efficient purification procedure for both oxidases established.
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The induction by D,L-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Arthrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.
Kupffer cells exposed to bacterial lipopolysaccharide in vitro synthesized collagenase and released the major portion of it into the extracellular space while the intracellular level of enzyme was not altered significantly. Cycloheximide prevented the appearance of collagenase in the medium indicating de novo synthesis. Indomethacin, an inhibitor of cyclooxygenase, also blocked collagenase synthesis. In line with this observation. Kupffer cells were found to synthesize substantial amounts of prostaglandin E2 when exposed to lipopolysaccharide; concomitantly, cellular cAMP levels were increased. Indomethacin was shown to abolish the stimulated cAMP formation. Addition to the culture medium of cAMP or dibutyryladenosine 3', 5'-monophosphate as well as of prostaglandin E2 or, to a lesser extent, prostaglandin E1 allowed indomethacin-inhibited cells to resume the production of collagenase. It is proposed that in rat Kupffer cells lipopolysaccharide-elicited collagenase synthesis and excretion is mediated sequentially by stimulated production of prostaglandin E2, enhanced adenylate cyclase activity and increased intracellular cAMP levels.
A severe amniotic infection syndrome with a septic shock and a consumption coagulopathy is reported after an amniocentesis in second trimester. In literature, the risk of an amnionitis after amniocentesis amounts to 0.1%-0.4% and the risk of a severe maternal infection reaches 0.03%-0.19%. The available data show, that the clinical symptoms (rise in temperature, signs of septic shock) start 24-36 hours after the punction. The earlier treatment (evacuation of the uterus or - if necessary - hysterectomy) is accomplished, the more favourable will be the course of the disease with respect to septic shock and DIC. Determination of germs in each amniotic fluid seems to be of prognostic value; thus treatment can be started before infection becomes detrimental for the mother.
The various radiological problems in psychiatric hospitals are discussed. The patients in whom positive radiological findings are to be expected are described. The frequency of unexpected radiological findings which can be demonstrated in psychiatric patients with organic brain disease are emphasized. The equipment of radiology departments in psychiatric and regional hospitals is studied with regard to expediency and appropriate recommendations are made.
CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-14C]citrate formed in the presence of [4-14C]oxaloacetate and citrate synthase; 0.49 mumol/g cell wet wt. were found. CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-14C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mumol CoASH and 1.17 mumol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.
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