Proliferative effects of five traditional Nigerian medicinal plant extracts on human breast and bone cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plants Hunteria umbellata (HUL), Cola lepidota (CCL), Persea americana leaf (PAL), Root bark of Persea americana (RPA) and Plukenetia conophora (PCL) are used in Nigerian traditional medicine for the treatment of cancer and cancer related diseases. AIM OF THE STUDY: To scientifically evaluate the cell proliferative and apoptotic effects of the plants extracts using breast and osteocarcinoma cell lines, and also to identify the possible components via LC-MS to have a kind of chemical fingerprint. MATERIALS AND METHODS: The antiproliferative and apoptotic effects of methanolic extracts (10 µg/ml) of the five medicinal plants were subjected to in vitro evaluation using four cancer cell lines (breast-MCF-7 and BT-20; Osteocarcinoma-MG-63 and Saos-2) measured by flow cytometry. Non-tumorigenic controls MCF-12A and primary isolated osteoblasts (POB) were chosen to eliminate negative influence on healthy tissue. RESULTS: Of the five extracts RPA demonstrated a significant (P<0.05) anti-proliferative activity against estrogen receptor positive breast cancer cell lines (MCF-7). The proliferative phase was decreased by 18%, whereas, a significant increase in cell proliferation (about 27%) was observed for RPA at a concentration of 10 µg/ml. PCL, CCL, HUL and PAL did not show marked inhibition of the proliferation of cell line MCF-7. CONCLUSION: These results give suggestive evidence that the plant extracts exhibit some correlation between the claimed ethnomedicinal uses and the cell proliferative activity. RPA extract includes chemical compounds with estrogen-like activity and validates its potential use as anticancer agent, particularly against breast carcinoma; provided important information potentially helpful in drug designing and discovery. Further studies will involve the isolation of anti tumour compounds in RPA by LC-MS and detailed mechanism of anticancer activities.

How to overcome limitations in biotechnological processes - examples from hydroxynitrile lyase applications.

During the last decades, enzymes became very versatile catalysts for a variety of reactions including natural and unnatural compounds. However, many enzyme-catalysed reactions suffer from diverse restrictions because of limitations related to process parameters or the enzyme. The understanding and overcoming of those undesired side effects is therefore mandatory for the implementation of optimal process parameters. To achieve this aim, various methods from molecular biology and reaction engineering can be employed. By focusing on the hydroxynitrile lyase-catalysed synthesis of enantiopure cyanohydrins, we give an overview of strategies to improve commercially utilized enzymes and to suppress non-enzymatic reactions. Particular emphasis is placed on the necessity to combine approaches from different fields, such as enzyme engineering and reaction engineering.

Handling uncertainties in toxicity modelling using a fuzzy filter.

A fundamental concern in the Quantitative Structure-Activity Relationship approach to toxicity evaluation is the generalization of the model over a wide range of compounds. The data driven modelling of toxicity, due to the complex and ill-defined nature of eco-toxicological systems, is an uncertain process. The development of a toxicity predicting model without considering uncertainties may produce a model with a low generalization performance. This study presents a novel approach to toxicity modelling that handles the involved uncertainties using a fuzzy filter, and thus improves the generalization capability of the model. The method is illustrated by considering a data set dealing with the fathead minnow (Pimephales promelas) toxicity of 568 organic compounds.

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Flax-seed extracts with phytoestrogenic effects on a hormone receptor-positive tumour cell line.

UNLABELLED: The higher soy intake in the Asian population compared to Europeans is believed to be an essential factor for the lower incidence of hormone-dependent tumours in Asia. It has already been shown that soya beans, with their ingredients genistein and daidzein from the isoflavonoid group, have protective effects on hormone-caused diseases. Lignans are another, less investigated, group of phytoestrogens. The aim of this study was to investigate the effects of flax-seed, which is typically found in Northern European diets, on the proliferation and hormone production of an estrogen receptor (ER)-positive trophoblast tumour cell line. MATERIALS AND METHODS: Trophoblast tumour cells of the cell line Jeg3 were incubated with 2 different concentrations of the isolated crude extract of flax-seed and 7 chemically partitioned extract fractions. Untreated cells were used as controls. After 48 h of stimulation, cell proliferation was measured using the BrdU method. The concentrations of hCG and progesterone produced by the trophoblast tumour cells were measured 48 h after stimulation. Extract fractions with antiproliferative effects in the BrdU- test were analysed by HPLC-MS. RESULTS: Our study showed an inhibitory influence of some of the isolated flax-seed fractions on the Jeg3 tumour cells. Proliferation of the Jeg3 cells was decreased by flax-seed fractions I, V, VI and VII in a dose-dependent manner. Inhibition of hCG production by flax-seed extracts III, V, VI and VII was also dose-dependent. Extract fractions V and VI decreased the production of progesterone by 58% to 86%. Some extract fractions showed a stimulating effect on hormone production and cell proliferation. HPLC-MS analysis showed the presence of matairesinol and biochanin A in flax-seed fraction VI. DISCUSSION: Flax-seed seems to have similar inhibitory effects to soya on hormone production and proliferation of hormone-sensitive tumour cells. Our results showed a dose-dependent inhibition by isolated flax-seed extracts on the Jeg3 cell line. Matairesinol and biochanin A seem to be useful candidates for extended tests on other tumour cell lines and normal tissues to evaluate the potential benefit of a lignan-containing therapy in hormone-dependent diseases.

Laccase-catalysed synthesis of coupling products of phenolic substrates in different reactors.

Substrate oxidation of aromatic substances by the enzyme laccase followed by a heteromolecular coupling with a co-substrate is a promising possibility for the synthesis of new compounds. To find a suitable reactor for the effective production of new compounds, the laccase-catalysed coupling of 3-(3,4-dihydroxyphenyl)propionic acid with 4-aminobenzoic acid was investigated as a model system. Based on the kinetic parameters, a mathematical model was used to predict the reaction yield and oxygen demand in a discontinuously stirred tank reactor and a continuously operated stirred tank reactor. Membrane processes were used for bubble-free aeration of the system and to recover the soluble enzyme.

The development of new methods for the recycling of chiral catalysts.

This article discusses different methods for the recycling of chiral catalysts, including heterogenization of the soluble catalyst on an insoluble inorganic or organic support, membrane filtration of homogeneously soluble catalysts, precipitation, and two-phase systems. In principle, all the methods presented enable the repeated use of a chiral catalyst without loss of activity and/or enantioselectivity. Examples will be given from laboratory and industrial processes, incuding hydrogenations, ketone reductions, epoxidations, dihydroxylations, diethylzinc additions and Diels-Alder reactions catalyzed by chemocatalysts or biocatalysts. Different approaches for cyanation, hydrogenation and epoxidation are compared. Data from industrial processes include the production of metalochlor, the production of (-)-menthol and the production of L-tert-leucine.

Prevalent vertebral deformity predicts incident hip though not distal forearm fracture: results from the European Prospective Osteoporosis Study.

The presence of a vertebral deformity increases the risk of subsequent spinal deformities. The aim of this analysis was to determine whether the presence of vertebral deformity predicts incident hip and other limb fractures. Six thousand three hundred and forty-four men and 6788 women aged 50 years and over were recruited from population registers in 31 European centers and followed prospectively for a median of 3 years. All subjects had radiographs performed at baseline and the presence of vertebral deformity was assessed using established morphometric methods. Incident limb fractures which occurred during the follow- up period were ascertained by annual postal questionnaire and confirmed by radiographs, review of medical records and personal interview. During a total of 40348 person-years of follow-up, 138 men and 391 women sustained a limb fracture. Amongst the women, after adjustment for age, prevalent vertebral deformity was a strong predictor of incident hip fracture, (rate ratio (RR) = 4.5; 95% CI 2.1-9.4) and a weak predictor of 'other' limb fractures (RR = 1.6; 95% CI 1.1-2.4), though not distal forearm fracture (RR = 1.0; 95% CI 0.6-1.6). The predictive risk increased with increasing number of prevalent deformities, particularly for subsequent hip fracture: for two or more deformities, RR = 7.2 (95% CI 3.0-17.3). Amongst men, vertebral deformity was not associated with an increased risk of incident limb fracture though there was a nonsignificant trend toward an increased risk of hip fracture with increasing number of deformities. In summary, prevalent radiographic vertebral deformities in women are a strong predictor of hip fracture, and to a lesser extent humerus and 'other' limb fractures; however, they do not predict distal forearm fractures.

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.

In situ generated O-glycan core 1 structure as substrate for Gal(beta 1-3)GalNAc beta-1,6-GlcNAc transferase.

beta-Galactosidase from bovine testes was used in a one pot reaction together with a recombinant beta-1,6-GlcNAc transferase for the synthesis of GlcNAc(beta 1-6)GalNAc(alpha 1-OBn) (core 6-Bn). The galactosidase, which reversibly links galactose via a (beta 1-3) linkage to N-acetylgalactosamine, provides the substrate for the GlcNAc transferase in situ. The synthesis was carried out with a yield > 90%.

Enzymatic large-scale production of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid in enzyme membrane reactors.

The enzymatic synthesis of 2-keto-3-deoxy-D-glycero-D-galacto-nonopyranulosonic acid (KDN) starting from D-mannose and pyruvic acid using Neu5Ac-aldolase has been scaled up. A repetitive batch ultrafiltration bioreactor was used for the KDN synthesis on 100 g scale with a conversion of up to 85%. Furthermore, a 440 mL pilot-scale enzyme membrane reactor (EMR) was performed for the continuous production of KDN. Conversion of mannose was 75% at a space--time yield of 375 g/(L d). KDN was advanteageously isolated by crystallization with an overall yield of 75%.

Improvement of the total turnover number and space-time yield for chloroperoxidase catalyzed oxidation.

Chloroperoxidase from Caldariomyces fumago was applied for the oxidation of indole to oxindole using hydrogen peroxide as the oxidant in aqueous t-butyl alcohol medium. Different ways of adding the oxidant, various reactor types, and the use of a hydrogen peroxide-stat were compared, resulting in a 20-fold increase of the total turnover number (ttn) and space-time yield (sty). The highest ttn of >860,000 was obtained in a fed-batch reactor, whereas the highest sty of 120 g/(L . d) was reached in a continuously operated enzyme membrane reactor. The results were compared to other enzyme systems already established for the synthesis of amino acids and carbohydrates. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 283-288, 1997.

Enzyme engineering aspects of biocatalysis: cofactor regeneration as example.

Reaction engineering is an important tool in the case of cofactor depending enzyme-catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product-specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water-soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L-tert-Leucine has been produced by reductive amination with a space-time yield of up to 366 g L(-1) d(-1) in a single continuously operated enzyme membrane reactor and a two-stage cascade. Total turnover number of the cofactor NAD(+) increased to 4230. (S)-1-Phenyl-2-propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space-time yield of 63 g L(-1) d(-1) and a total turnover number of 1350 have been reached. L-Leucine has been produced using polymer-enlarged NADH. The total turnover number was 80,000 at a space-time yield of 214 g L(-1) d(-1).

Enzymatic resolution of 1-phenyl-1,2-ethanediol by enantioselective oxidation: overcoming product inhibition by continuous extraction.

Oxidations of alcohols by alcohol dehydrogenases often suffer from low conversions and slow reaction rates due to severe product inhibition. This can be overcome by continuous product extraction, because only the concentrations, but not the kinetic parameters, can be changed. As a consequence, it is favorable to apply a differential circulation reactor with continuous product extraction, where only a small amount of product is formed per cycle. The product is then directly extracted using a microporous hydrophobic hollow fiber membrane. This results in an increase of the relative activity of the dehydrogenase at a given conversion. The reaction investigated is the kinetic resolution of racemic 1-phenyl-1,2-ethanediol by glycerol dehydrogenase (GDH). The resulting oxidation product, 2-hydroxyacetophenone, causes a strong product inhibition. Additionally, it reacts in a chemical reaction with the cofactor lowering its active concentration. Because the GDH needs beta-nicotinamide adenine dinucleotide (NAD(+)) as a cofactor, lactate dehydrogenase is used to regenerate NAD(+) from NADH by reducing pyruvate to (L)-lactate. A conversion of 50% with respect to the racemate and an enantiomeric excess >99% of the (S)-enantiomer was reached.

Recombinant soluble beta-1,4-galactosyltransferases expressed in Saccharomyces cerevisiae. Purification, characterization and comparison with human enzyme.

beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.

CMP-N-acetyl neuraminic-acid synthetase from Escherichia coli: fermentative production and application for the preparative synthesis of CMP-neuraminic acid.

In an optimized sorbitol/yeast extract/mineral salt medium up to 12 U/l CMP-N-acetyl-neuraminic-acid (Neu5Ac) synthetase was produced by Escherichia coli K-235 in shake-flask culture. A colony mutant of this strain, E. coli K-235/CS1, was isolated with improved enzyme formation: in shake flasks with a yield of up to 20.8 U/l and 54 mU/mg protein in the cell extract. With this strain 26500 U CMP-Neu5Ac synthetase was produced with a high specific activity (0.128 U/mg) by fed-batch fermentation on 230-l scale. On a 10-1 scale the enzyme yield was 191 U/l culture medium. The enzyme was partially purified by precipitation with polyethyleneglycol resulting in a three- to fourfold enrichment and a recovery rate of more than 80%; most of the CTP hydrolysing enzymes were removed. The native synthetase was deactivated completely by incubation at 45 degrees C for 10 min, but could be stabilized remarkably by glycerol and different salts. The enzyme was used for the preparative synthesis of CMP-Neu5Ac with a conversion yield of 87% based on CTP.

Scaled-up expression of human alpha 2,6(N)sialyltransferase in Saccharomyces cerevisiae.

Expression of recombinant full length human alpha 2,6(N)sialyltransferase has been scaled-up in S. cerevisiae in a 150-l bioreactor yielding 47 U at a concentration of 0.31 U/l. The protein specific activity as measured in reconstituted yeast lyophilisate was 0.8 mU/mg protein. The recombinant enzyme exhibited similar Michaelis constants as previously determined for the native rat enzyme. By immunoblotting the enzyme was shown to be heterogeneous by size (44-48 kD) and N-glycosylated. We conclude that recombinant alpha 2,6(N)sialyltransferase expressed in S. cerevisiae is retained in the endoplasmic reticulum as a fully active enzyme.

Large-scale production of a soluble human beta-1,4-galactosyltransferase using a Saccharomyces cerevisiae expression system.

We report in this communication the first large-scale heterologous expression of a glycosyltransferase in yeast. A soluble form of a human beta-1,4-galactosyltransferase (EC 2.4.1.38) was expressed using a Saccharomyces cerevisiae expression system. Fermentation technology afforded the means to increase the expression level of the beta-1,4-galactosyltransferase up to a concentration of 700 mU/liter. The enzyme was produced at a scale of 200 units. The recombinant soluble enzyme was purified 766-fold to a specific activity of approx. 2 U/mg using a purification protocol based on sequential affinity chromatography on N-acetylglucosaminyl- and alpha-lactalbumin-Sepharose, respectively. This study demonstrates that heterologous expression of a glycosyltransferase is possible on a large scale and offers an alternative to natural sources like human breast milk or bovine colostrum.