Expression of phenolic and tyrosyl ring iodothyronine deiodinases in Xenopus laevis oocytes is dependent on the tissue source of injected poly(A)+ RNA

The phenolic (5' position) and tyrosyl (5 position) ring deiodinases which catalyze the peripheral metabolism of thyroid hormones have proven difficult to purify and characterize biochemically. The present studies used Xenopus laevis oocytes as an in vivo translational assay system for detecting and quantitating mRNA for these enzymes. The injection of poly(A)+ RNA prepared from a human term placenta induced 5-deiodinase activity in oocytes. The expressed activity increased for up to 96 h after injection, was proportional to the amount of RNA injected, and manifested a Michaelis-Menten constant (Km) for T3 of 1.6 nM. In oocytes injected with poly(A)+ RNA prepared from rat liver, anterior pituitary gland, or brown adipose tissue, 5-deiodinase activity could not be demonstrated. The injection of poly(A)+ RNA from 15-day-old chick embryonic liver induced both 5'- and 5-deiodinase activity, with the 5'-deiodinase activity being sensitive to inhibition by 6-n-propyl-2-thiouracil. X. laevis oocytes can thus be induced to express either phenolic or tyrosyl ring deiodinase activity, or both, by the microinjection of poly(A)+ RNA prepared from selected tissues. These findings demonstrate that the types of deiodinase activity present in different organs represent tissue specific patterns of mRNA expression and strongly suggest that the enzymes responsible for types I and III deiodinase activity are encoded by different mRNAs.     

Biosynthesis of monoterpenes. Stereochemical implications of acyclic and monocyclic olefin formation by (+)- and (-)-pinene cyclases from sage

(+)-Pinene cyclase from sage (Salvia officinalis) catalyzes the isomerization and cyclization of geranyl pyrophosphate to (+)-alpha-pinene and (+)-camphene, and to lesser amounts of (+)-limonene, myrcene, and terpinolene, whereas (-)-pinene cyclase from this tissue catalyzes the conversion of the acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene, and to lesser quantities of (-)-limonene, myrcene, and terpinolene. The bicyclic products of these enzymes (pinene and camphene) are derived via the cyclization of the cisoid, anti-endo-conformers of the bound, tertiary allylic intermediates (3R)-linalyl pyrophosphate [+)-pinene cyclase) and (3S)-linalyl pyrophosphate [-)-pinene cyclase). When challenged with either enantiomer of linalyl pyrophosphate or with neryl pyrophosphate (cis-isomer of geranyl pyrophosphate) as substrate, both pinene cyclases synthesize disproportionately high levels of acyclic olefins (myrcene and ocimene) and monocyclic olefins (limonene and terpinolene), compared with the product mixtures generated from the natural geranyl precursor. Resolution of the limonene derived from linalyl pyrophosphate and neryl pyrophosphate demonstrated that this monocyclic olefin was formed via conformational foldings in addition to the cisoid,anti-endo-pattern. These results indicate that the alternate substrates are ionized by the cyclases prior to their achieving the optimum orientation for bicyclization. In the case of geranyl pyrophosphate, a preassociation mechanism is suggested in which optimum folding of the terpenyl chain precedes the initial ionization step.     

The roles of calcium and manganese ions in the in vitro conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene by lentil root membranes

Ca²⁺ and Mn²⁺ activate the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) by root microsomes of Vicia lens as they do in other similar systems. The preparation of microsomes in the presence of Mn²⁺ greatly increases their ability to convert ACC into ethylene, without addition of Mn²⁺ in the reaction mixture. Ca²⁺ does not have this property. The effect could not be attributed to Mn²⁺ entrapping into membrane vesicles (sonication followed by repelleting had no effect) but, possibly, in part to Mn²⁺-mediated binding to microsomes of a soluble factor favouring the conversion of ACC to C₂H₄. Although no direct correlation could be established in vitro between ethylene-forming-enzyme (EFE) and peroxidase activities, some soluble peroxidases might be this soluble factor. Mn²⁺ favoured attachment to membranes of some peroxidase activity from the soluble fraction and from commercial HRP and lipoxygenase. This binding effect of Mn₂₊ cannot be readily distinguished from its role in the generation of a chain of free radicals and in redox mechanisms.     

Histochemical localization of carbonic anhydrase in the testis and epididymis of the rabbit.

The distribution of carbonic anhydrase (CA) was studied in the testis and epididymis of mature, male rabbits using a cobalt precipitation method. CA was found only in the endothelium of the capillaries in the testis. The epididymal duct was divided into initial, middle and terminal segments. Strong cytoplasmic CA was present in the apical cells in the initial and middle segments. Vacuoles with CA staining in the membranes were found in the principal cells in the middle segment. Intensely stained basal cells were present in the terminal segment. In the last part of the terminal segment and the first of the ductus deferens the basolateral cell membranes were also stained. The function of the enzyme is discussed especially in relation to acidification of the epididymal fluid and facilitation of CO2 diffusion.     

Isolation of secretory cells from plant glandular trichomes and their use in biosynthetic studies of monoterpenes and other gland products.

The natural products that accumulate in or exude from plant glandular trichomes are biosynthesized by secretory cells located at the apex of the trichome. To investigate the formation of glandular trichome constituents in several species of mints (Lamiaceae), a new procedure was developed for isolating large numbers of highly purified secretory cells. In this method, the leaf surface is gently abraded with glass beads in a way that fragments the glandular trichomes and yields clusters of intact secretory cells. The isolated, intact secretory cells and cell-free preparations derived from them are very active in monoterpene biosynthesis and provide useful starting materials for the purification of several key enzymes of monoterpene metabolism. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of whole cell and cell-free systems for biosynthetic studies of plant natural products found in glandular trichomes.     

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm

The nucleotide sequence and the 5 flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria the P. littoralis rbcL gene is more closely related to that of a -purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996–4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599–608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.     

Two-compartment model for rabbit skin organ culture

Summary A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d. The present study was financially supported by grants of Duphar B. V. (Weesp, Netherlands), the European Community, and the Dutch animal welfare organizations Samenwerkingsverband van de Nederlandse Vereniging tot Bescherming van Dieren en de Nederlandse Bond tot Bestrijding van de Vivisectie, Anti-Vivisectie Stichting en Stichting Schoonheid Zonder Wreedheid.