Late enzymes of vindoline biosynthesis. Acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyl-transferase

From differentiated plants of Catharanthus roseus (L.) G. Don, a specific enzyme was isolated and named acetyl-CoA : 17-O-deacetylvindoline 17-O-acetyltransferase, acting on the biosynthetic formation of the Aspidosperma type alkaloid vindoline.The enzyme shows a high selectivity towards different substrates. The acetyl-CoA-dependent transferase also catalyses the reverse reaction by hydrolysis of the 17-O-acetyl group of vindoline in the presence of free CoA. This enzyme is localized only in vindoline-containing plant parts, but was so far not detectable in cell suspension cultures of C. roseus. The enzyme allows the synthesis of labelled vindoline with high specific activity, applicable for instance as tracer for radioimmunoassays of vindoline.     

Pharmacological profile of a novel carbacyclin derivative with high metabolic stability and oral activity in the rat

A novel carbacyclin derivative (16S)-13,14-dehydro-16,20-dimethyl-3-oxa-18,18,19,19-tetradehydro- 6a- carbaprostaglandin-I2 (3-oxa-analogue) has been synthesized in order to find chemically and metabolically stable prostacyclin-mimetics with a potency equal or even superior to PGI2. The 3-oxa-analogue was found to be stabilized against beta-oxidation, a main metabolic degradation step also for chemically stable PGI2-analogues. The compound is orally available and displays a long duration of 4.5-48 h of antiaggregatory and hypotensive action. The 3-oxa-analogue inhibits ADP-induced platelet aggregation with an IC50 of 3.0 nM. Following intravenous application the 3-oxa-analogue lowers diastolic blood pressure in a dose dependent manner, the ED20 being 0.1-0.2 micrograms/kg after injection and less than or equal to 0.05 micrograms/kg/min after infusion respectively. In vivo platelet aggregation is inhibited after i.v. infusion of the 3-oxa-analogue with an IC50 of 0.037 micrograms/kg/min. As compared to Iloprost, the 3-oxa-analogue is 5-12 fold more potent with respect to in vivo hypotensive and anti-aggregatory effects. The results of the present studies indicate that the 3-oxa-analogue has a pharmacological profile comparable to prostacyclin (PGI2) and Iloprost. Due to the fact that the 3-oxa-analogue is chemically and metabolically stable, long term oral treatment can be achieved in clinical conditions in which PGI2 and Iloprost have already been shown to be therapeutically useful principles.     

A 3.6-kbp segment from the vir region of Ti plasmids contains genes responsible for border-sequence-directed production of T region circles in E. coli

The vir region of Ti plasmids is responsible for the transfer of the T region from Agrobacteria to plant cells; previous experiments suggested that formation of independent T region DNA circles is one step in this process. To study this step in Escherichia coli, we developed a binary vector system. One plasmid (= substrate) contains correctly oriented right and left borders from octopine plasmid pTiAch5. A gene with a counterselectable function (galK) was cloned between these borders. The galK gene is under control of the tac promoter-operator and the lac repressor with the laci gene also in the selection cassette. This construction allows determination of substrate plasmid mutants which have lost the selectable galK function. The second component of the system is one of a set of compatible plasmids harbouring various cloned parts from the vir region of nopaline plasmid pTiC58. A 3.6-kbp segment of the vir region turned out to be necessary and sufficient for production of substrate plasmid mutants which represented the equivalent of the T region containing a complete left border. From this vir region fragment four discrete proteins were expressed in minicells. The coding regions were mapped to a part conserved in nopaline and octopine plasmids; in the latter it appears to correspond to virC/D.     

The dopaminergic innervation of the goldfish pituitary

Summary The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.     

The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein)

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures.

Abstract: Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-³H]alkyl- sn -glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1–20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyksn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl-, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl- compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.     

Distribution of chlorpromazine and imipramine in adipose and other tissues of rats

Adipose tissue kinetics of chlorpromazine and imipramine, two drugs which are more lipophilic than thiopental, were studied in the rat. After single i.v. doses, the time-course of drug distribution was followed in adipose and various other tissues, until their concentrations in adipose tissues declined. Under these conditions the two drugs behaved almost identically. Among the tissues analyzed, the lowest concentrations were found in adipose tissue, with the exception of plasma. At its maximum concentration after about 30 minutes, total adipose tissue contained only 3 % of the dose of administered drugs. Adipose/plasma and adipose/lung concentration ratios were 2–5 and 0.05, respectively.After maximum tolerated oral doses of imipramine for 3 weeks, similar steady state concentration ratios (plasma:adipose:brain:lung 1:3:12:96) were observed. In adipose tissue the imipramine/desmethylimipramine ratio was about 1, and the desmethylimipramine steady state levels did not increase with time. Literature data indicate that many basic lipophilic drugs are not stored in adipose tissue. This is now clearly shown for chlorpromazine and imipramine, even under extreme, subchronic conditions in the case of imipramine.     

Processing of yeast mitochondrial RNA: involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5' part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5' splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation (sup-) compensating for the effect of the primary box9- mutation. The sup- mutation alters a sequence in intron 4,293 bp upstream from the box9- primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9- and sup- are compensatory base pair exchanges (G----A and C----U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.