Follow-up after colon cancer treatment in the Netherlands; a survey of patients, GPs, and colorectal surgeons.

INTRODUCTION: Follow-up to detect recurrence is an important feature of care after colon cancer treatment. Currently, follow-up visits are surgeon-led with focus on recurrence. To date, there is increasing interest for general practitioners (GPs) providing this care, as GPs might provide more holistic care. The present study assessed how surgeons, GPs, and patients evaluate current surgeon-led colon cancer follow-up and to list their views on possible future GP-led follow-up. METHODS: The study consists of a cross-sectional survey including colorectal surgeons, patients who participate or recently finished a follow-up programme, and GPs in the Netherlands. RESULTS: Eighty-seven out of 191 GPs, 113 out of 238 surgeons, and 186 out of 243 patients responded. Patients are satisfied about current surgeon-led follow-up, especially about recurrence detection and identification of physical problems (94% and 85% respectively). However, only 56% and 49% of the patients were satisfied about the identification of psychological and social problems respectively. Only 16% of the patients evaluated future GP-led follow-up positively. Regarding healthcare providers, surgeons were more positive compared to GPs; 49% of the surgeons, and only 30% of the GPs evaluated future GP-led follow-up positively (P = 0.002). Furthermore, several reservations and principle requirements for GP-led follow-up were identified. DISCUSSION: The results suggest an unfavourable view among patients and healthcare providers, especially GPs, regarding a central role for GPs in colon cancer follow-up. However, low satisfaction on psychosocial aspects in current follow-up points out a lack in care. Therefore, the results provide a justification to explore future GP-led care further.

Biotransformation of tryptamine and secologanin into plant terpenoid indole alkaloids by transgenic yeast.

A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.

Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis.

Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

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 Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting 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.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 influenced 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 sufficient, 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.

Purification and characterization of two isoforms of isopentenyl-diphosphate isomerase from elicitor-treated Cinchona robusta cells.

In Cinchona robusta (Rubiaceae) cell suspension cultures, the activity of the enzyme isopentenyl-diphosphate isomerase (isopentenyl-POP isomerase) is transiently induced after addition of a homogenate of the phytopathogenic fungus Phytophthora cinnamomi. The enzyme catalyses the interconversion of isopentenyl-POP and dimethylallyl diphosphate (dimethylallyl-POP) and may be involved in the biosynthesis of anthraquinone phytoalexins that accumulate rapidly after elicitation of Cinchona cells. From elicitor-treated C. robusta cells, two isoforms of isopentenyl-POP isomerase have been purified to apparent homogeneity in four chromatographic steps. The purified forms are monomeric enzymes of 34 kDa (isoform I) and 29 kDa (isoform II), with Km values for isopentenyl-POP of 5.1 microM and 1.0 microM, respectively. Both isoforms require Mn2+ or Mg2+ as cofactor, isoform II showing a preference for Mn2+ with maximum activity at 1.5-2 mM. Isoform I was most active in the presence of 0.5-1.5 mM Mg2+ or in the presence of 0.5 mM Mn2+. A pH optimum of 7-7.8 was found for both forms and both were competitively inhibited by geranyl diphosphate (Ki 96 microM for isoform I) and the transition state analogue 2-(dimethylamino)ethyl diphosphate. Rechromatography of purified isoforms did not indicate any interconversion of both forms. Western blot analysis, using antibodies raised against isopentenyl-POP isomerase purified from Capsicum annuum, showed the presence of both isoforms in the crude protein extracts from C. robusta cells. Isoform II was specifically induced by elicitation, non-treated cells contained low activity of this isoform. The possible role of isopentenyl-POP isomerase in the biosynthesis of anthraquinones is discussed.

Induction of ajmalicine formation and related enzyme activities in Catharanthus roseus cells: effect of inoculum density.

In Catharanthus roseus cell cultures the time courses of four enzyme activities, tryptophan decarboxylase (TDC), strictosidine synthase (SSS), geraniol-10-hydroxylase (G10H) and anthranilate synthase (AS), and alkaloid accumulation were compared under two different culture conditions (low-inoculum density and high-inoculum density on induction medium) and a control on growth medium. In growth medium a transient increase in TDC activity was first observed after which G10H reached its maximum activity; only tryptamine accumulated, no ajmalicine could be detected. Apparently, a concerted induction of enzyme activities is required for ajmalicine formation. Cells inoculated in induction medium showed such a concerted induction of AS, TDC and G10H activities. After 30 days the low-density culture had accumulated six times more ajmalicine (in mumoles/g) than the high-density culture. Thus, increase in biomass concentration (high-density cultures) did not enhance the total alkaloid production. The major differences observed in enzyme levels between high- and low-density cultures were in the AS and TDC activities, which were two to three times higher in the low-density culture, indicating that there is a positive correlation between ajmalicine formation and AS and TDC activities.

Alkaloid Production in Relation to Differentiation in Cell and Tissue Cultures of Tabernaemontana pandacaqui.

Alkaloid production in TABERNAEMONTANA PANDACAQUI cultures is dependent on the degree of differentiation. At high levels of production the major alkaloid found was 3 S-hydroxyvoacangine. This alkaloid has not previously been isolated from any plant. Micropropagation of T. PANDACAQUI was quick and highly efficient.

Thermospray Liquid Chromatography/Mass Spectrometry (TSP LC/MS) Analysis of the Alkaloids from Cinchona in vitro Cultures.

The alkaloids from CINCHONA LEDGERIANA shoot cultures and from CINCHONA ROBUSTA shoot cultures and a compact globular structure (CGS) culture were analyzed by thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Because of the relative stability of the alkaloids under TSP discharge ionization conditions, a protonated molecule was observed in the mass spectra with hardly any fragmentation. When the reference compounds were available, the knowledge of the molecular mass and of the retention time was sufficient to identify most of the alkaloids. HPLC with UV photodiode-array detection complemented LC/MS perfectly by providing information about the aromatic part of the alkaloids (structure and substitution pattern). New alkaloids detected in CINCHONA IN VITRO cultures were 5-methoxytryptamine and corynantheal. In order to determine whether 5-methoxytryptamine was a precursor of the methoxylated quinolines, this indole was incubated with secologanin and several CINCHONA ROBUSTA crude protein extracts. Under all conditions tested, the coupling of 5-methoxytryptamine with secologanin remained unsuccessful. Only tryptamine condensed with secologanin to yield strictosidine. These results indicate that CINCHONA cells are able to methoxylate simple indoles like tryptamine and that 5-methoxytryptamine is very likely not used for the subsequent biosynthesis of the methoxylated quinolines.

Characterization of a Suspension Culture of Tabernaemontana elegans on Growth, Nutrient Uptake, and Accumulation of Indole Alkaloids*.

A suspension culture of TABERNAEMONTANA ELEGANS was characterized on growth, nutrient uptake (carbohydrate, nitrogen, and phosphate), and on accumulation of indole alkaloids. Besides tryptamine seven indole alkaloids were isolated and identified: vobasine, vobasinol, perivine, isositsirikine, apparicine, tubotaiwine, and O-acetylvallesamine. In spite of the presence of vobasinol as major product, only trace amounts of dimeric indole alkaloids could be detected. Alkaloid formation started at the early stationary phase.

Accumulation of indole alkaloids in a suspension culture of Tabernaemontana divaricata.

Cell suspension cultures of TABERNAEMONTANA DIVARICATA were found to produce relatively large amounts of indole alkaloids. For their isolation an ion-pair DCCC method was used in combination with preparative TLC. The alkaloids were identified as tabernaemontanine, perivine, vobasine, voaphylline, voaphylline-hydroxyindolenine, vallesamine, apparicine, 16-hydroxy-16,22-dihydroapparicine, pericyclivine, tubotaiwine, 19- S-heyneanine, and coronaridine. Voaphylline, the main alkaloid, was produced during growth and early stationary phase and reached a maximum of 23 mg/l at day 19 of the growth cycle. After this maximum voaphylline was rapidly metabolized. Apparicine, vobasine, and coronaridine reached their maximum levels at a later stage of the growth cycle. Tubotaiwine accumulation showed a similar profile as that of voaphylline. In light-grown cells the total production was about 2 times higher than in dark-grown cells, with respective main products voaphylline and apparicine.

Assay of tryptophan decarboxylase from Catharanthus roseus plant cell cultures by high-performance liquid chromatography.

An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures. It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column. Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium. Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold. When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days. The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C.

A method for the quantitative determination of anthraquinones and alkaloids in cell and tissue cultures of cinchona sp.

A procedure is described for the extraction of both anthraquinones and alkaloids from tissue culture material of CINCHONA species. Using this extraction method, it is possible to quantitatively determine the two groups of secondary metabolites produced in CINCHONA tissue cultures.