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K Severin

Publications and source records attributed to K Severin.

13 recordsLinked to original sources
PubMed1998

Regulatory role of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase for shikonin biosynthesis in Lithospermum erythrorhizon cell suspension cultures.

The carbon skeleton of the naphthoquinone pigment shikonin, which is produced in Lithospermum erythrorhizon Sieb. et Zucc. cell-suspension cultures, is partly derived from the isoprenoid biosynthetic pathway. The requirement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34), a key enzyme of the mevalonate route to isoprenoids, for shikonin synthesis was investigated. Conserved regions of sequences from plant HMGR genes were used to design polymerase chain reaction (PCR) primers for the cloning of a cDNA fragment from L. erythrorhizon. The resulting 443-bp clone was used as a probe for Northern analyses and hybridized to an mRNA of approx. 2.5 kb. Under shikonin-producing conditions, microsomal HMGR enzyme activity as well as mRNA level closely correlated with the accumulation of shikonin derivatives. White light, which inhibits shikonin formation, was shown to strongly suppress HMGR gene expression. The results presented here indicate that HMGR plays a significant role in the regulation of shikonin biosynthesis and that the control appears to act at the transcriptional level.

Acyl Coenzyme A↗
PubMed1997

Emergence of symbiosis in peptide self-replication through a hypercyclic network.

Symbiosis is an association between different organisms that leads to a reciprocal enhancement of their ability to survive. Similar mutually beneficial relationships can operate at the molecular level in the form of a hypercycle, a collective of two or more self-replicating species interlinked through a cyclic catalytic network. The superposition of cross-catalysis onto autocatalytic replication integrates the members of the hypercycle into a single system that reproduces through a second-order (or higher) form of nonlinear autocatalysis. The hypercycle population as a whole is therefore able to compete more efficiently for existing resources than any one member on its own. In addition, the effects of beneficial mutations of any one member are spread over the entire population. The formation of hypercycles has been suggested as an important step in the transition from inanimate to living chemistry, and a large number of hypercycles are expected to be embedded within the complex networks of living systems. But only one naturally occurring hypercycle has been well documented, while two autocatalytic chemical systems may contain vestiges of hypercyclic organization. Here we report a chemical system that constitutes a clear example of a minimal hypercyclic network, in which two otherwise competitive self-replicating peptides symbiotically catalyse each others' production.

Amino Acid Sequence↗
PubMed1997

A synthetic peptide ligase.

The preparation of synthetic molecules showing the remarkable efficiencies characteristic of natural biopolymer catalysts remains a formidable challenge for chemical biology. Although significant advances have been made in the understanding of protein structure and function, the de novo construction of such systems remains elusive. Re-engineered natural enzymes and catalytic antibodies, possessing tailored binding pockets with appropriately positioned functional groups, have been successful in catalysing a number of chemical transformations, sometimes with impressive efficiencies. But efforts to produce wholly synthetic catalytic peptides have typically resulted in compounds with questionable structural stability, let alone reactivity. Here we describe a 33-residue synthetic peptide, based on the coiled-coil structural motif, which efficiently catalyses the condensation of two shorter peptide fragments with high sequence- and diastereoselectivity. Depending on the substrates used, we observe rate enhancements of tenfold to 4,100-fold over the background, with catalytic efficiencies in excess of 10(4). These results augur well for the rational design of functional peptides.

Amino Acid Sequence↗
PubMed1997

Autocatalytic networks: the transition from molecular self-replication to molecular ecosystems.

The transition from inanimate to animate chemistry is thought to involve self-organised networks of molecular species whose collective emergent property gives rise to the overall characteristics of living systems. In the past, simple autocatalytic networks have been constructed that display basic forms of cooperative behaviour. These include reciprocal catalysis, autocratic, and hypercyclic networks. The design and emergent properties of these novel molecular networks are reviewed here.

Catalysis↗
PubMed1997

cDNA cloning and gene expression of phenylalanine ammonia-lyase in Lithospermum erythrorhizon.

Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.

Amino Acid Sequence↗
PubMed1996

A self-replicating peptide.

The production of amino acids and their condensation to polypeptides under plausibly prebiotic conditions have long been known. But despite the central importance of molecular self-replication in the origin of life, the feasibility of peptide self-replication has not been established experimentally. Here we report an example of a self-replicating peptide. We show that a 32-residue alpha-helical peptide based on the leucine-zipper domain of the yeast transcription factor GCN4 can act autocatalytically in templating its own synthesis by accelerating the thioester-promoted amide-bond condensation of 15- and 17-residue fragments in neutral, dilute aqueous solutions. The self-replication process displays parabolic growth pattern with the initial rates of product formation correlating with the square-foot of initial template concentration.

Amino Acid Sequence↗
PubMed1996

Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco.

The ubiC gene of Escherichia coli encodes chorismate pyruvatelyase, an enzyme that converts chorismate into 4-hydroxybenzoate (4HB) and is not normally present in plants. The ubiC gene was expressed in Nicotiana tabacum L. plants under control of a constitutive plant promoter. The gene product was targeted into the plastid by fusing it to the sequence for the chloroplast transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Transgenic plants showed high chorismate pyruvate-lyase activity and accumulated 4HB as beta-glucosides, with the glucose attached to either the hydroxy or the carboxyl function of 4HB. The total content of 4HB glucosides was approximately 0.52% of dry weight, which exceeded the content of untransformed plants by at least a factor of 1000. Feeding experiments with [1,7-13C2]shikimic acid unequivocally proved that the 4HB that was formed in the transgenic plants was not derived from the conventional phenylpropanoid pathway but from the newly introduced chorismate pyruvate-lyase reaction.

Amino Acid Sequence↗
PubMed1994

Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase.

Chorismate pyruvate-lyase from Escherichia coli converts chorismate to 4-hydroxybenzoate. The enzyme was enriched 3000-fold by overexpression and chromatographic purification. It has an apparent Km value for chorismate of 6.1 microM and an isoelectric point of pH 6.45. The enzyme activity did not require metal cofactors. Promoter sequences in the 5' flanking sequences of the ubiCA operon were localized by transcription and translation of active chorismate pyruvate-lyase in vitro from different PCR fragments. Sequencing of the ubiC gene of the mutant strain AN244 revealed a G-->A transition resulting in a change from glutamic acid to lysine. A feeding experiment with [1,7-13C2]shikimate confirmed the chorismate pyruvate-lyase as the sole enzymic source of 4-hydroxybenzoate in vivo.

Bacterial Proteins↗
PubMed1993

Effect of human and recombinant IFN-alpha and IFN-beta on the sister-chromatid exchange (SCE) frequency in amniotic fluid cells in vitro.

Sister-chromatid exchange (SCE) and rates of proliferation in human amniotic fluid cells from healthy donors exposed to human IFN-alpha and IFN-beta and recombinant IFN-alpha and -beta were investigated. Amniotic fluid cells were obtained from pregnant women undergoing genetic amniocentesis. For 46 h, cells were treated with IFNs at concentrations of 10(3)-10(5) U/l. A dose-depending decrease of SCE rate with IFN-alpha and IFN-beta was observed. Our studies in amniotic fluid cells show that the mean SCE frequencies are reduced after incubation with IFN-alpha as well as with IFN-alpha. In contrast to IFN-gamma, the type I IFNs IFN-alpha and IFN-beta cause a genetic effect on DNA repair or a protection from DNA damage. Previously we had shown that a significant dose-depending increase of SCE rates was found in amniotic fluid cultures after addition of IFN-gamma. Therefore, IFN-alpha and IFN-beta (both human IFNs) and also recombinant IFN-alpha and IFN-beta, also in high doses, are neither genotoxic/clastogenic nor embryotoxic. Amniotic cells are vulnerable human cells, which may be well suited for examining the effects of agents like interferon.

Amniotic Fluid↗
PubMed1992

Ubiquinone biosynthesis. Cloning of the genes coding for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from Escherichia coli.

Chorismate pyruvate-lyase activity was detected in extracts of Escherichia coli. 4-Hydroxybenzoate was identified as the product of the enzymatic reaction by chemical derivatization and GC-MS analysis. The ubiC gene, coding for the chorismate pyruvate-lyase, was cloned and sequenced. The molecular weight of the gene product was calculated as 18,776 Da and confirmed by expression of the protein in E. coli minicells. The ubiA gene, coding for the 4-hydroxybenzoate octaprenyl transferase, was identified by sequence homology and complementation of a ubiA- strain. It is located directly downstream of ubiC in a typical operon structure.

Alkyl and Aryl Transferases↗
PubMed1990

Heat-inducible hygromycin resistance in transgenic tobacco.

We have constructed a chimaeric gene consisting of the promoter of the soybean heat shock (hs) gene Gmhsp17, 6-L, the coding region of a hygromycin phosphotransferase (hpt) gene, and the termination sequence of the nopaline synthase (nos) gene. This gene fusion was introduced into tobacco by Agrobacterium-mediated gene transfer. Heat-inducible synthesis of mRNA was shown by northern hybridization, and translation of this RNA into a functional protein was indicated by plant growth on hygromycin-containing media in a temperature-dependent fashion. One hour incubation at 40 degrees C per day, applied for several weeks, was sufficient to express the resistant phenotype in transgenic plants containing the chimaeric hs-hpt gene. These data suggest that the hygromycin resistance gene is functional and faithfully controlled by the soybean hs promoter. The suitability of these transgenic plants for selection of mutations that alter the hs response is discussed.

Amino Acid Oxidoreductases↗
PubMed1982

The antenatal use of ambroxol (bromhexine metabolite VIII) to prevent hyaline membrane disease: a controlled double-blind study.

A prospective double-blind clinical trial was carried out to determine whether ambroxol (bromhexine metabolite VIII) treatment (1000 mg/day for a period of 5 days) reduces the risk of hyaline membrane disease (HMD) in potentially premature infants. Amniocentesis was performed before the first and 24 h after the last application of ambroxol or placebo to assess the development of the total phospholipid phosphorus content, the L/S ratio, the P/S ratio, and the properties of the surface tension of the amniotic fluid after ambroxol or placebo. There were 246 infants born to 224 mothers. Of the 116 infants with less than or equal to 36 completed weeks' gestation, 56 were in the ambroxol and 60 in the placebo group. No differences between groups occurred in risk factors for HMD (diabetes, asphyxia, male sex, cesarean section). Statistically significant differences in favor of the infants in the ambroxol group were found in the HMD incidence: 23.2% in the ambroxol group compared with 41.7% in the placebo group (p less than 0.05). There was no reduction of the HMD incidence in the less than or equal to 32-week gestational age category in the ambroxol group compared with the placebo group inspite of the fact that all the examined parameters for determining lung maturity reflected a stimulatory effect of ambroxol compared with the results of the placebo group, particularly before the 33rd week of gestation. Prolonged rupture of the membranes played no protective role against HMD.

Ambroxol↗