PubMed Health⌕ Search

Biomedical subjects

M Luckner

Publications and source records attributed to M Luckner.

At least 19 recordsLinked to original sources
PubMed2002

A simple method for the identification and assay of extracellular plant beta-galactosidase.

A simple, rapid and reproducible procedure for the identification of extracellular Californian poppy (Eschscholzia californica Cham.) beta-galactosidase is described using callus cultures of seedlings from the tested plant, roots of 4-days-old seedlings of Californian poppy germinating on agar plates and cell suspension cultures cultivated from callus cultures. 6-Bromo-2-naphthyl-beta-D-galactopyranoside and p-nitrophenyl-beta-D-galactopyranoside were used as substrates for the determination of the intracellular and extracellular activities of beta-galactosidase. The extracellular beta-galactosidase activity was identified by evaluating the dye-zones in an agar medium. The enzyme from Californian poppy callus cultures or from seedling roots cultivated on agar plates supplemented with 6-bromo-2-naphthyl-galactopyranoside hydrolyzed this substrate releasing 6-bromo-2-naphthol. By simultaneous coupling with hexazonium p-rosaniline the corresponding (reddish-brown) azo-dye was formed. The agar plate method described permits rapid, simple and specific detection of plant producers of extracellular beta-galactosidase.

Cells, Cultured↗
PubMed2000

Functional replacement of the essential ESS1 in yeast by the plant parvulin DlPar13.

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Amino Acid Sequence↗
PubMed2000

Haploid plants regenerated from androgenic cell cultures of Digitalis lanata.

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.

Androgens↗
PubMed2000

Cloning and functional expression in Escherichia coli of a cDNA encoding cardenolide 16'-O-glucohydrolase from Digitalis lanata Ehrh.

A clone of cardenolide 16'-O-glucohydrolase cDNA (CGH I) was obtained from Digitalis lanata which encodes a protein of 642 amino acids (calculated molecular mass 73.2 kDa). The amino acid sequence derived from CGH I showed high homology to a widely distributed family of beta-glucohydrolases (glycosyl hydrolases family 1). The recombinant CGH I protein produced in Escherichia coli had CGH I activity. CGH I mRNA was detected in leaves, flowers, stems and fruits of D. lanata.

Amino Acid Sequence↗
PubMed1999

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress.

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/transisomerases (PPIases). The difference in pI (9.2 +/- 0.2 and 9.5 +/- 0.2, +/- SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 +/- 4 Da (D. lanataCyp18.0) and 18132 +/- 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 +/- 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 +/- SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress.

Adaptation, Physiological↗
PubMed1999

Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. - a multifunctional enzyme in steroid metabolism?

Delta(5)-3beta-Etaydroxysteroid dehydrogenase (Delta(5)-3beta-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3beta-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Delta(5)-3beta-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Delta(5)-3beta-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.

3-Hydroxysteroid Dehydrogenases↗
PubMed1998

Purification and characterization of lanatoside 15'-O-acetylesterase from Digitalis lanata Ehrh.

Lanatoside 15'-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 mumol.s-1.(g protein)-1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4-24 nmol.s-1.(g protein)-1.

Acetylesterase↗
PubMed1998

Cardenolide 16'-O-glucohydrolase from Digitalis lanata. Purification and characterization.

A three-step chromatographic procedure was developed for purification of cardenolide 16'-O-glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70 kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16'-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.

Amino Acid Sequence↗
PubMed1995

[In vitro measurement of digitalis-like compounds by inhibition of Na+ K+-ATPase: determination of the inhibitory effect].

The linked optical test for the analysis of digital-like substances with the enzyme Na+/K(+)-ATPase was investigated with regard to inhibition by components of the reaction medium. Most of the tested inorganic salts influenced the activity of the enzyme. However, the concentrations of the salts in human tissues and fluids are too small to cause measurable effects. Higher concentration of salts, which may be obtained by the preparative treatment of clinical material can influence the test. The determination of a reference value is recommended in these cases. The organic solvents DMSO and Methanol influenced the activity of the Na+/K(+)-ATPase, too. While the influence of DMSO in concentrations below 50% (v/v) was negligible, the measurement of a reference sample at higher DMSO concentrations and for methanolic samples is necessary.

Ammonium Sulfate↗
PubMed1990

Formation of Digitalis lanata Clone Lines by Shoot Tip Culture.

Propagation of DIGITALIS LANATA by shoot tip culture made possible (a) the rapid multiplication of elite plants with the formation of plant clones and (b) the long-term cultivation of these plants which under normal growth conditions would die at the end of the second vegetation period. Optimum conditions were established for the regeneration of shoots from shoot tips, for daughter shoot formation and rooting as well as for the adaptation of the regenerated plants to the open ground. Gene banks of valuable clones were built by keeping shoots at 4 degrees C on media with high sucrose concentration (maximum period of storage 2 years) or by growing juvenile clone plants in the greenhouse at temperatures preventing the induction of flowering. The clone plants were in the juvenile state even if they were derived from flowering mother plants. They showed normal growth and development.

Journal Article↗
PubMed1990

Batch Cultures of Somatic Embryos of Digitalis lanata in Gaslift Fermenters. Development and Cardenolide Accumulation.

Somatic embryos of DIGITALIS LANATA STRAIN VII were successfully grown as batch cultures in gaslift fermenters. Embryo development and formation of cardenolides in the embryos resembled those of cultures grown in shake flasks. Both processes depended on the developmental stage of the embryos at the time point of inoculation, the homogeneity of the embryo structures, the density of the inoculum, the composition of the nutrient medium, the intensity of irradiation, and the composition of the gas mixtures used for agitation of the embryo suspension. The cultures contained 0.7&-1.0 (micro,mol digitoxin equivalents g (-1) dry wt. (2.6-6.5 micromol digitoxin equivalents l (-1)) after a cultivation period of 28 d.

Journal Article↗
PubMed1990

Cardenolides in Digitalis lanata Cells Transformed with Ti-Plasmids.

Crown galls were induced by transformation of leaves, leaf discs, and shoots of the plant DIGITALIS LANATA with the AGROBACTERIUM TUMEFACIENS strains C58 pTi C58, B6S3 pTi B6S3, and A136 pTi A6NCtmr-338::Tn5. Integration of plasmid DNA in the genome of D. LANATA was demonstrated by hybridization experiments. The transformed cells synthesized opines and showed hormone-autotrophic growth. The crown galls formed on leaves of D. LANATA plants contained digitoxigenin derivatives (up to 0.8 muimol digitoxin equivalents g (-1) dry weight). Transformed cell lines derived from the crown galls built cardenolides IN VITRO (ca. 0.03 mumol digitoxin equivalents g (-1) dry weight). The rate of cardenolide biosynthesis IN VITRO did not decrease during a cultivation period of 12 months.

Journal Article↗
PubMed1988

[EDLS (endogenous digitalis-like substance(s))--detection, chemistry, and physiologic function].

Human beings and higher animals contain compounds which interact with the Na+/K+-ATPase of the heart muscle and other organs like the cardiac glycosides, and bind to cardiac glycoside-specific antibodies [endogenous digitalis-like substance(s), EDLS]. EDLS cause increased natriuresis. The level of EDLS of the blood is raised under physiological stress situations (e.g., pregnancy and delivery and at certain pathophysiological conditions (e.g., hypertony). The EDLS are low molecular compounds. As yet their chemical structure is unknown.

Adenosine Triphosphatases↗
PubMed1985

Cyclopeptine synthetase activity in surface cultures of Penicillium cyclopium.

Cyclopeptine synthetase, the key enzyme of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium forms cyclo-(anthranoyl-phenylalanyl) from anthranilic acid, L-phenylalanine, the methyl group of L-methionine and ATP. The following in vitro measurable partial activities of the enzyme system were followed during the development of P. cyclopium: anthranilic acid and L-phenylalanine adenylyltransferase activities, and the ability for thioester-binding of L-phenylalanine to the enzyme protein. These activities became measurable at the beginning of the idiophase and reached a maximum 6 days after inoculation, i.e., the pattern of activity was similar to that of the other enzymes participating in the biosynthesis of the benzodiazepine alkaloids indicating that the activities of all enzymes of the pathway were coordinatedly expressed. Inhibitor experiments indicated that 48-55 h after inoculation a preprotein of anthranilic acid adenylyltransferase was formed, which later on became activated by a hitherto unknown mechanism.

Benzodiazepinones↗
PubMed1984

Nuclear inheritance of the biosynthesis of cyclopenin and cyclopenol in Penicillium cyclopium.

Balanced heterokarions were grown from Penicillium cyclopium aux-glu 1, a glutamic acid auxotroph producing benzodiazepine alkaloids of the cyclopenin-cyclopenol group, and P. viridicatum aux-met 1, a methionine auxotroph forming these alkaloids in traces only. In contrast to the hyphae of the parent strains, the hyphae of the heterokarions were dark orange-brown and grew well on media without the auxotrophic factors. In surface cultures they synthesized the benzodiazepine alkaloids cyclopenin and cyclopenol in amounts similar to those formed by the hyphae of P. cyclopium aux-glu 1. From the monokariotic conidiospores of the heterokarions homokariotic daughter strains were obtained which were similar to the parent strains in every respect. Hence no exchange of features of cyclopenin-cyclopenol biosynthesis took place between the parent strains at the stage of the heterokarion. This result indicates that the formation of cyclopenin and cyclopenol in P. cyclopium aux-glu 1 and the nearly complete lack of biosynthesis of these compounds in P. viridicatum aux-met 1 is encoded within the nucleus and is not influenced by plasmic genetic material.

Anti-Bacterial Agents↗
PubMed1979

Channelling of exogenous phenylalanine to the sites of storage and the sites of alkaloid and protein biosynthesis in Penicillium cyclopium.

Externally applied L-phenylalanine rapidly equilibrates with the cytosolic pool(s) in hyphae of emerged cultures of Penicillium cyclopium. If not incorporated into protein it is accumulated in the so called expandable pool, which is presumably localized in the vacuolar compartment. At high concentrations of exogenous L-phenylalanine practically all of the amino acid needed for protein synthesis comes from the extracellular source, contrary to alkaloid synthesis which under all conditions recruits more than 90% of the required L-phenylalanine from intracellular sources. Two pathways of alkaloid labelling can be distinguished, by which externally applied L-phenylalanine reaches the sites of alkaloid synthesis: (1) a direct way from cytosolic pool (primary labelling) and (2) an indirect way via the expandable pool (secondary labelling).

Alkaloids↗