An assay for secologanin in plant tissues based on enzymatic conversion into strictosidine

The secoiridoid glucoside secologanin is the terpenoid building block in the biosynthesis of terpenoid indole alkaloids. A method for its determination in plant tissues and in cell suspension cultures has been developed. This assay is based on the condensation of secologanin with tryptamine, yielding strictosidine, in a reaction catalysed by the enzyme strictosidine synthase (STR; E.C. 4.3.3.2). Subsequently, the formation of strictosidine is quantified by high performance liquid chromatography (HPLC). STR was isolated from transgenic Nicotiana tabacum cells expressing a cDNA-derived gene coding for STR from Catharanthus roseus. The high specificity of STR for secologanin, in combination with a sensitive and selective HPLC system, allows a simple extraction of secologanin from plant tissue. The detection limit of this method is 15 ng secologanin. Using this assay, secologanin contents were determined in tissues of various plant species; Lonicera xylosteum hairy roots were found to contain 1% of secologanin on a dry weight basis. # 1998 John Wiley & Sons, Ltd.info:eu-repo/semantics/publishedVersio

Changes in complex RNA during fruit-body initiation in the fungus Schizophyllum commune

Total RNA was isolated from 4-day surface cultures of two monokaryotic strains (vegetative mycelium) and a resultant dikaryotic strain bearing fruit-body initials (fruiting mycelium) of the basidiomycete Schizophyllum commune. Single-copy DNA (scDNA) and complementary DNA (cDNA) hybridizations with these RNAs showed that both mycelial types expressed 10, 000 to 13, 000 different RNA sequences of average size. scDNA combination-hybridization experiments did not reveal differences between the RNA populations. However, cDNA cross-hybridization experiments indicated that about 5% of the complex RNA mass present in fruiting mycelium was absent in vegetative mycelium. Homologous and heterologous hybridization of total RNA with cDNA probes corresponding to more and less frequently occurring RNA sequences demonstrated that the fruiting mycelium contained about 35 specific abundant RNAs. Comparison of cell-free translation products of total RNA showed a similar difference between the abundant RNA species in vegetative and fruiting mycelium. Of about 400 polypeptides detected after two-dimensional gel electrophoresis, 18 were encoded exclusively by RNA from the fruiting dikaryon

Absence of differences in polysomal RNAs from vegetative monokaryotic and dikaryotic cells of the fungus Schizophyllum commune

Total polysomes, including free and membrane-bound polysomes, were isolated from the monokaryotic and dikaryotic mycelial cell types of the basidiomycete Schizophyllum commune grown under submerged conditions. Sucrose gradient centrifugation showed that these isolated polysomes had a size distribution as expected for polysomes functioning in vivo and thus contained intact mRNA. RNA preparations extracted from the polysomes were used to analyze the mRNA sets in the monokaryon and dikaryon. Saturation hybridization of single-copy DNA with a vast excess of polysomal RNA and cell-free translation of polysomal RNA in a wheat germ system followed by two-dimensional gel electrophoresis of the products did not reveal significant differences between the mRNA sets in both mycelial cell types. Moreover, it was found that the sequence complexities and coding properties of polysomal RNA and total (nuclear and cytoplasmic) RNA from S. commune are not detectably different. From these results and those obtained in previous studies it was concluded that the differentiation between monokaryotic and dikaryotic mycelial cell types as controlled by A and B incompatibility genes involves differential modification of polypeptides during or after mRNA translation

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH: cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants

The membrane‐bound flavoprotein NADPH:cytochrome P‐450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P‐450 mono‐oxygenases, was purified from a cell suspension culture of the higher plant Catheranthus roseus. Anti‐serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P‐450 enzymes geraniol 10‐hydroxylase and trans‐cinnamate 4‐hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P‐450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P‐450 reductases from yeast and animal species. The identity of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH‐dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N‐terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P‐450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P‐450 reductase with the corresponding enzymes from yeast and animal species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P‐450‐dependent biosynthetic pathways.info:eu-repo/semantics/publishedVersio

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacteriummediated transformation showed wide phenotypic diversity, re¯ecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg á L)1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly in¯uenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not su cient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.info:eu-repo/semantics/publishedVersio

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacteriummediated transformation showed wide phenotypic diversity, re¯ecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg á L)1 of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly in¯uenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not su cient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment.info:eu-repo/semantics/publishedVersio

Pain patterns in chronic pancreatitis : a nationwide longitudinal cohort study

Objective: Pain in chronic pancreatitis is subdivided in a continuous or intermittent pattern, each thought to represent a different entity, requiring specific treatment. Because evidence is missing, we studied pain patterns in a prospective longitudinal nationwide study. Design: 1131 patients with chronic pancreatitis (fulfilling M-ANNHEIM criteria) were included between 2011 and 2018 in 30 Dutch hospitals. Patients with continuous or intermittent pain were compared for demographics, pain characteristics, quality of life (Short-Form 36), imaging findings, disease duration and treatment. Alternation of pain pattern and associated variables were longitudinally assessed using a multivariable multinomial logistic regression model. Results: At inclusion, 589 patients (52%) had continuous pain, 231 patients (20%) had intermittent pain and 311 patients (28%) had no pain. Patients with continuous pain had more severe pain, used more opioids and neuropathic pain medication, and had a lower quality of life. There were no differences between pain patterns for morphological findings on imaging, disease duration and treatment. During a median follow-up of 47 months, 552 of 905 patients (61%) alternated at least once between pain patterns. All alternations were associated with the Visual Analogue Scale pain intensity score and surgery was only associated with the change from pain to no pain. Conclusion: Continuous and intermittent pain patterns in chronic pancreatitis do not seem to be the result of distinctly different pathophysiological entities. The subjectively reported character of pain is not related to imaging findings or disease duration. Pain patterns often change over time and are merely a feature of how severity of pain is experienced. </p