Heather A. Ross

Berry extracts exert different antiproliferative effects against cervical and colon cancer cells grown in vitro.

Polyphenol-rich berry extracts were screened for their antiproliferative effectiveness using human cervical cancer (HeLa) cells grown in microtiter plates. Rowan berry, raspberry, lingonberry, cloudberry, arctic bramble, and strawberry extracts were effective but blueberry, sea buckthorn, and pomegranate extracts were considerably less effective. The most effective extracts (strawberry > arctic bramble > cloudberry > lingonberry) gave EC 50 values in the range of 25-40 microg/(mL of phenols). These extracts were also effective against human colon cancer (CaCo-2) cells, which were generally more sensitive at low concentrations but conversely less sensitive at higher concentrations. The strawberry, cloudberry, arctic bramble, and the raspberry extracts share common polyphenol constituents, especially the ellagitannins, which have been shown to be effective antiproliferative agents. However, the components underlying the effectiveness of the lingonberry extracts are not known. The lingonberry extracts were fractionated into anthocyanin-rich and tannin-rich fractions by chromatography on Sephadex LH-20. The anthocyanin-rich fraction was considerably less effective than the original extract, whereas the antiproliferative activity was retained in the tannin-rich fraction. The polyphenolic composition of the lingonberry extract was assessed by liquid chromatography-mass spectrometry and was similar to previous reports. The tannin-rich fraction was almost entirely composed of procyanidins of linkage type A and B. Therefore, the antiproliferative activity of lingonberry was caused predominantly by procyanidins.

Manipulation of S-adenosylmethionine decarboxylase activity in potato tubers

Abstract.S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.1.50) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine from putrescine and its activity is rate limiting in this pathway. Transgenic potato (Solanum tuberosum L. cv. Desiree) plants containing both sense and antisense SAMDC constructs driven by the tuber-specific patatin promoter have been generated and analysed. In sense transformants, developing tubers expressed higher steady-state levels of the SAMDC-specific transcript, had higher levels of SAMDC activity and contained significantly higher levels of spermidine than vector-transformed controls. Additionally, there was a significant shift in tuber size distribution with larger numbers of smaller tubers but no overall change in tuber yield. In developing tubers from the antisense transformed lines, there was a decrease in SAMDC transcript level, SAMDC activity and total polyamine levels. However, no obvious phenotypic effect was detected in the tuberisation physiology of the antisense lines.

Sucrolytic Enzyme Activities in Cotyledons of the Faba Bean (Developmental Changes and Purification of Alkaline Invertase)

In terms of maximum extractable catalytic activity, sucrose synthase is the predominant sucrolytic enzyme in developing cotyledons of faba bean (Vicia faba L.). Although acid invertase activity is extremely low, there is significant activity of alkaline invertase, the majority of which is extractable only with high concentrations of NaCl. Calculations of potential activity in vivo indicate that alkaline invertase is the predominant sucrolytic enzyme from 50 days after anthesis onward. However, at almost all stages of cotyledon development analyzed, the maximum extractable catalytic activities of both enzymes is in excess of the actual rate of starch deposition. Two forms of alkaline invertase were identified in developing cotyledons. The major form has been purified to homogeneity, and antibodies have been raised against it. The native protein has a molecular mass of about 238 [plus or minus] 4.5 kD. It is apparently a homotetramer (subunit molecular mass 53.4 [plus or minus] 0.9 kD). The enzyme has a pH optimum of 7.4, an isoelectric point of 5.2, and a Km[sucrose] of 10 mM and is inhibited by Tris (50% inhibition at 5 mM) and fructose (30% inhibition at 10 mM). Bean alkaline invertase is a [beta]-fructofuranosidase with no significant activity against raffinose, stachyose, trehalose, maltose, or lactose.

Hydrolytic and Phosphorolytic Enzyme Activity and Reserve Mobilisation in Sprouting Tubers of Potato (Solanum tuberosum L.)

Summary The mobilisation of reserves and changes in associated enzyme activities have been examined in sprouting potato tubers. Although considerable depletion of both starch and protein was observed there were no substantial increases in the activities of β-amylase, starch phosphorylase, invertase, protease, or aminopeptidase. Although α-amylase activity was barely detected with the assay procedures used, an increase in activity accompanied starch breakdown. The rates of starch depletion were almost identical in tissues internal and external to the vascular ring but protein degradation was intially more rapid in the latter region. Starch phosphorylase activity was higher in the external tissues and β-amylase activity higher in the internal tissues. There were no consistent differences in the activites of invertase, protease, and aminopeptidase between these zones. While the maximum catalytic activity of starch phosphorylase was just sufficient to support the observed rates of starch depletion, activities of both α- and β-amylase were in appreciable excess.

Characterisation of a cDNA encoding fructokinase from potato (Solanum tuberosum L.)

Summary Using an in vitro transcription/translation system, functional evidence has been obtained that a cDNA clone isolated from a potato stolon tip library encodes the enzyme fructokinase. Expression of the gene has been determined in a range of potato tissues at different developmental stages. The gene is shown to be present in low copy number in the potato genome and in the genome of related non-tuberising species.

Facile assessment of cDNA constructs for expression of functional antibodies in plants using the potato virus X vector

Antibodies have been expressed in plants to confer novel traits such as virus resistance or altered phenotype. However, not every antibody is suitable for plant expression, and successful intracellular expression of antibody fragments depends primarily on their amino acid sequence in a way that is as yet unpredictable. Therefore it is desirable to assess different constructs before embarking on the production of transgenic plants. We have used a transient expression system based on potato virus X to compare different cDNA constructs for expression and stability of antibody variable gene fragments in plants. Constructs contained an anti-plant enzyme (granule-bound starch synthase I) scFv sequence derived from a naive phage display library together with different combinations of sequences encoding the human IgG κ constant domain, a murine IgG secretory signal sequence, or the endoplasmic reticulum retention signal peptide KDEL. The results obtained with the potato virus X vector correlated with those from Agrobacterium-mediated stable transformation of potato. The best expression levels were obtained by incorporating sequences that target scFv to the lumen of the endoplasmic reticulum and the secretory pathway. The anti-enzyme scFv retained activity during storage of potato tubers for more than five months. The results demonstrate the utility of the potato virus X vector for the analysis and comparison of many scFv with different epitope specificities or sequence modifications. Evaluation of scFv by transient expression from the PVX vector should aid progress in selection of functional scFv for applications in plant biotechnology.

Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture

Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties.

Partial purification and characterization of fructokinase from developing taproots of sugar beet (Beta vulgaris)

Abstract Fructokinase (FK) has been purified from developing sugar beet ( Beta vulgaris L.) taproots by ion exchange chromatography and gel filtration. One major isoform was identified. The protein appears to be a dimer ( M r 38 000). Kinetically, the purified sugar beet fructokinase has a pH optimum of 8.5 and a high specificity for fructose ( K m = 0.068 mM). The enzyme can utilise a range of nucleotide triphosphates, although ATP is the most effective. Sugar beet fructokinase is inhibited by fructose concentrations in excess of 0.6 mM. Fructose-6-phosphate and Mg ADP are also inhibitory, but at relatively high concentrations. K + at 10 mM stimulates activity by 30%. Fructokinase activity and the level of FK protein remain high throughout taproot development. Tissue-blots showed that high levels of FK protein were associated with conducting tissues.

Limit dextrinase in barley cultivars of differing malting quality: activity, inhibitors and limit dextrin profiles

Abstract Free and total limit dextrinase (LD) activity was measured in four barley varieties of differing malting quality. LD activity was not detected until 2 days after completion of steeping, at which point, total LD activity was 4–5 fold higher in Static and Chariot than in Optic or Hart. Free LD rose to higher levels in Static and Chariot than in Optic and Hart during malting. In addition, the proportion of free to total LD approached 35% in Static and Chariot whereas it did not reach 15% for Optic and Hart. Lower free LD activity was not reflected in a higher total content of branched dextrins in hot water extracts of the different varieties, but may have contributed to the persistence of branched dextrins of higher degree of polymerisation persisting in the hot water extracts of Hart and Optic. The variation in the proportion of free to total LD activity between varieties was not explained by differences in total LD activity and may be related to the presence of inhibitors of LD. Protein extracts made from the malts following steeping inhibited exogenous partially purified LD activity by >80%. After 3 days germination, Chariot, Optic and Static lost much of their inhibitory activity whereas it was retained in Hart. The increase in free LD activities during malting appeared to mirror the disappearance in inhibitory activity. Inhibitors of LD activity were also present in hot water extracts of Chariot and Hart malts. Chariot lost inhibitory activity after 3 days malting and hot water extracts from Chariot after 4 days malting caused apparent activation of LD. By contrast, hot water extracts from Hart retained inhibitory activity up to 5 days malting. These findings are discussed with reference to the availability and effectiveness of LD during malting.

Potato tuber sugar content during development and storage (10°C): possible predictors of storage potential and the role of sucrose in storage hexose accumulation

SummaryReducing sugar and sucrose contents of potato tubers (Solanum tuberosum L.) from six cultivars were monitored both during the growing season and after a four months storage period at 10°C. Significant correlations were found between tuber sugar contents (sucrose, reducing and total) measured at harvest and the reducing sugar content after storage. Similarly, in five of the six cultivars, the sucrose loss and the corresponding reducing sugar gain during storage were significantly correlated.