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Biomedical subjects

A J Cutler

Publications and source records attributed to A J Cutler.

27 records · Page 2Linked to original sources
PubMed1987

Permeabilizing soybean protoplasts to macromolecules using electroporation and hypotonic shock.

The percentage of soybean cell culture protoplasts permeabilized by electroporation was dependent on the voltage and the number of successive pulses that were applied. Best results were obtained with two 50 milliseconds, 400 volts per centimeter pulses after which 78% of the surviving protoplasts had been permeabilized to the fluorescent dye calcein. Quantitation of the volume of extracellular fluid taken up was performed using radioactive inulin (molecular weight 5000-5500). Typically between 20 and 40 nanoliters of fluid was taken up by 10(6) protoplasts. Electroporation and hypotonic shock treatments (M Saleem, AJ Cutler 1986 J Plant Physiol 124: 11-21) were compared with respect to the volume of fluid taken up under optimum conditions. Electroporation produced 10 times more uptake than hypotonic shock treatment. In all experiments there was a direct relationship between the number of protoplasts lysed and both the amount of fluid taken up and the percentage of surviving protoplasts that were permeabilized.

Journal Article↗
PubMed1987

Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus.

The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline.

Journal Article↗
PubMed1985

Properties of a microsomal enzyme system from Linum usitatissimum (linen flax) which oxidizes valine to acetone cyanohydrin and isoleucine to 2-methylbutanone cyanohydrin.

Microsomal preparations from flax seedlings have recently been shown to convert L-valine to acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin [A. J. Cutler and E. E. Conn (1981) Arch. Biochem. Biophys. 212, 468-474]. Further details of this four-step biosynthetic sequence and also details of the analogous reactions in lotaustralin biosynthesis have been obtained. The lotaustralin precursor, 2-methylbutyraldoxime, is the best substrate for cyanide production (Vmax = 413 nmol h-1 g fresh wt-1) and inhibits the conversion of valine and isoleucine into products. Similarly, the linamarin precursor isobutyraldoxime is an excellent substrate (Vmax = 400 nmol h-1 g fresh wt-1) and also inhibits oxidation of the amino acids. The substrate specificity of the oxime-metabolizing step is low and a variety of aliphatic oximes are converted to cyanide. On the other hand, the activity of the microsomal extract is highly selective with regard to the amino acid substrate since, of the aliphatic amino acids tested, only valine and isoleucine are metabolized. We were unable to demonstrate product formation from isobutyronitrile (a linamarin precursor) but did observe detectable cyanide formation from 2-methylcyanobutane, the corresponding precursor of lotaustralin. Competition experiments showed that the biosynthesis of linamarin and lotaustralin is not likely to be catalyzed by separate enzyme systems.

Chromatography, Gas↗
PubMed1985

Isoelectric focusing of plant cell protoplasts: separation of different protoplast types.

The surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pI). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 +/- 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants.

Journal Article↗
PubMed1984

Quantitation of the delivery of liposome contents into plant protoplasts.

A procedure for the quantitation of the delivery of liposome contents into Catharanthus roseus protoplasts has been developed. The method is based on the uptake of liposome encapsulated methylumbelliferyl beta-D-glucoside and its enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free glucoside is not taken up by the protoplasts to a significant extent, the delivery of material in the nanomole range can be measured with ease.

Biological Transport↗
PubMed1981

The in vitro biosynthesis of taxiphyllin and the channeling of intermediates in Triglochin maritima.

The in vitro biosynthesis of the cyanogenic glucoside taxiphyllin has recently been demonstrated in Triglochin maritima (Hösel, W., and Nahrstedt, A. (1980) Arch. Biochem. Biophys. 203, 753-757). We have now studied in more detail the multistep conversion of tyrosine into p-hydroxymandelonitrile, the immediate precursor of taxiphyllin, catalyzed by microsomes isolated from dark-grown seedlings. The biosynthetic pathway involves N-hydroxytyrosine, p-hydroxyphenylacetaldoxime, and p-hydroxyphenylacetonitrile. In marked contrast to an analogous pathway in Sorghum bicolor, p-hydroxyphenylacetonitrile is the best substrate for cyanide production (Vmax = 224 nmol/h/g, fresh wt) and the physiological substrate tyrosine is the poorest (Vmax = 18.8 nmol/h/g, fresh wt). The substrates exhibit alkaline pH optima between 7.5 and 9, and all except tyrosine show pronounced substrate inhibition. We have found that p-hydroxyphenylacetonitrile generated in situ from tyrosine is free to equilibrate by diffusion with exogenous material. On the other hand, neither N-hydroxytyrosine nor p-hydroxyphenylacetaldoxime will readily exchange with exogenous intermediates. We consider both N-hydroxytyrosine and p-hydroxyphenylacetaldoxime to be channeled in T. maritima, whereas in S. bicolor N-hydroxytyrosine and p-hydroxyphenylacetonitrile are channeled and the aldoxime is freely exchangeable.

Carbon Radioisotopes↗
PubMed1979

Regulation of hydroxydocosanoic acid sophoroside production in Candida bogoriensis by the levels of glucose and yeast extract in the growth medium.

Cells of Candida bogoriensis produce as a major extracellular lipid 13-[(2'-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]docosanoic acid 6',6''-diacetate (Ac2Glc2HDA), the diacetylated sophoroside of 13-hydroxydocosanoic acid (HDA), along with mono- and unacetylated derivatives. The HDA glycolipid production is greater than 2 g/liter when cells are grown on a "standard" medium of 3% glucose and 0.15% yeast extract. Either lowering the glucose concentration (0.5 to 2.0% glucose, at 0.2% yeast extract) or raising the yeast extract concentration (2 to 4% yeast extract at 3% glucose) greatly decreased the yield of this glycolipid, as well as its rate of synthesis measured by [14C]acetate incorporation. Total HDA production was also depressed on the low glucose medium, as was the activity of UDP-glucose:HDA glucosyltransferase, the first enzyme involved in the synthesis of Ac2Glc2HDA from HDA. Levels of acetyl-CoA:Glc2HDA acetyltransferase were not decreased by growth on a low glucose medium, however, even under conditions in which glycolipid production was less than 4% of that found in the standard medium. Low levels of the HDA glycolipids were monitored by high pressure liquid chromatography of their p-bromophenacyl esters, formed by the action of alpha,beta-dibromoacetophenone on the sodium salt of the lipid in the presence of a crown reagent catalyst. This regulation of extracellular Ac2Glc2HDA production by the nutrient composition of the growth medium may represent an important property in the adaptation of C. bogoriensis to its natural environment, the phyllosphere.

Acetyltransferases↗
PubMedDate not supplied

A low molecular weight peptide from snow mold with epitopic homology to the winter flounder antifreeze protein.

Evidence for a small size protein (ca. 3500 kDa) exhibiting epitopic homology to the Atlantic winter flounder antifreeze protein (AFP) is found in the snow molds Coprinus psychromorbidus, Myriosclerotinia borealis, and Typhula incarnata. The protein shows strong cross-reactivity with antisera specific for the flounder AFP. Preliminary studies suggest that the protein is synthesized in response to lowering the culture temperature, and that it is membrane associated and, therefore, may function in an analogous capacity to the fish AFP. Also, the protein is shown to have antifreeze properties as determined by nuclear magnetic resonance microimaging experiments.

Agaricales↗