Examination of triterpenoids produced by callus and cell suspension cultures of Glycyrrhiza glabra

Callus and cell suspension cultures of Glycyrrhiza glabra failed to produce detectable amounts of glycyrrhizin, the major oleanane-type triterpene glycoside of the thickening root, or of its 11 —deoxoderivative. However, betulinic acid, a lupane-type triterpene, which was found in the root bark, and a small amount of -amyrin, a possible precursor of oleanane-type triterpenes, were detected in cell suspension cultures in addition to lupeol, a fundamental form of lupane-type triterpenes. These findings suggest that the absence of glycyrrhizin in undifferentiated cultured cells may be partly due to interruption of the later reactions leading to the synthesis of glycyrrhizin from a triterpenoid intermediate.     

Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures

In Coptis japonica cell cultures an alternative pathway has been discovered which leads from (S)-tetrahydrocolumbamine via (S)-canadine to berberine. The two enzymes involved have been partially purified. (S)-Tetrahydrocolumbamine is stereospecifically transformed into (S)-canadine under formation of the methylenedioxy bridge in ring A. This new enzyme was named (S)-canadine synthase. (S)-Canadine in turn is stereospecifically dehydrogenated to berberine by an oxidase, (S)-canadine oxidase (COX), which was partially purified (25-fold). This enzyme has many physical properties in common with the already known (S)-tetrahydroprotoberberine oxidase from Berberis but grossly differs from the latter enzyme in its cofactor requirement (Fe) and its substrate specificity. Neither (S)-norreticuline nor (S)-scoulerine serves as substrate for the Coptis enzyme, while both substrates are readily oxidized by the Berberis enzyme. The four terminal enzymes catalyzing the pathway from (S)-reticuline to berberine are housed in Berberis as well as in Coptis in smooth vesicles with a density of =1.14 g/ml. These vesicles have been enriched and characterized by electron microscopy.     

The effect of temperature on recombination activity in testes of rodents

An extractable enzyme system capable of catalyzing recombination in vitro was described in murine spermatocytes [Hotta et al. (1985) Chromosoma 93, 140-151]. The system is specific to meiosis, its activity increasing 400-fold between the premeiotic S-phase and mid-pachytene. The present study examines the effect of temperature on this system since the elevation of testicular temperature is one of the major factors causing impairment of testicular function. A strong depression of in vitro recombination activity occurred immediately after raising the testicular temperature in vivo by translocating the testes into the abdominal cavity (cryptorchid). The in vitro study also showed that the extract from spermatocytes preferred lower temperatures (30-32 degrees C) than somatic cells (37 degrees C) for maximal activity of recombination. These results suggest that the strong depression of recombination activity may be an important factor which causes degeneration of testes by heat.     

Identification of Arabidopsis subtilisin-like serine protease specifically expressed in root stele by gene trapping

Xylem plays a role not only in the transport of water and nutrients but also in the regulation of growth and development through the transport of biologically active substances. In addition to mineral salts, xylem sap contains hormones, organic nutrients and proteins. However, the physiological functions of most of those substances remain unclear. To explore genes involved in xylem sap production, we identified Arabidopsis genes expressed in the root stele of the root hair zone from gene-trap lines by randomly inserting the β-glucuronidase gene into the genome. Among 26 000 gene-trap lines, we found that 10 lines had β-glucuronidase (GUS) staining predominantly in the root stele of the root hair zone and no GUS staining in the shoots. Of these 10 lines, 2 lines showed that gene-trap tags inserted into the promoter region of the same gene, denoted Arabidopsis thaliana subtilase 4.12( AtSBT4.12 ). Analysis of AtSBT4.12 promoter using an pAtSBT4.12 ::β-glucuronidase transgenic line showed that the AtSBT4.12 gene was expressed only in the root stele of the root hair zone. AtSBT4.12 expression in roots was increased by application of methyl jasmonate. Subtilase proteins are commonly detected in proteomic analyses of xylem sap from various plant species, including Brassica napus , a relative of Arabidopsis . These results suggest that AtSBT4.12 may be a protein localized in the apoplast of root stele including xylem vessel and involved in stress responses in Arabidopsis roots.     

Properties of yeast debranching enzyme and its specificity toward branched cyclodextrins.

Debranching enzyme was purified from Saccharomyces cerevisiae by DEAE-cellulose, omega-aminobutyl agarose and hydroxyapatite column chromatography. The activity of the eluent was monitored by the iodine-staining method which detects both the direct and indirect debranching enzymes. The elution profiles at every step showed a single peak with no shoulder. The crude and the purified enzyme preparations gave a single activity band with the same mobility on PAGE. The crude product produced 80% glucose compared to reducing sugar from glycogen-phosphorylase-limited dextrin while the partially purified and purified preparations produced 100% glucose. The activity of the purified enzyme was characterized and compared with that of the rabbit muscle enzyme by using various branched cyclodextrins as substrates. Both enzymes hydrolyzed 6-O-alpha-D-glucosyl cyclodextrins to glucose and cyclodextrins, but did not act on 6-O-alpha-maltosyl cyclomaltoheptaose. The yeast enzyme gave rise to glucose as a sole reducing sugar from 6-O-alpha-maltotriosyl cyclomaltoheptaose and 6-O-alpha-maltotetraosyl cyclomaltoheptaose, indicating that maltosyl and maltotriosyl transfers, respectively, had occurred, prior to the action of amylo-1,6-glucosidase. 6-O-alpha-D-Glucosyl cyclomaltoheptaose and 6-O-alpha-D-glucosyl cyclomalto-octaose, respectively, were better substrates than glycogen-phosphorylase-limited dextrin for the yeast and muscle enzymes. The yeast enzyme released glucose at a similar rate from 6-O-alpha-maltotriosyl cyclomaltoheptaose as from 6-O-alpha-maltotetraosyl cyclomaltoheptaose, but considerably lower rates than that from limit dextrin. The yeast debranching enzyme appears to be exclusively oligo-1,4----1,4-glucantransferase-amylo-1,6-glucosidase and does not have isoamylase.