Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs).

A novel series of acylated omega-aminoalkyl 2-amino-2-deoxy-4-phosphono-beta-D-glucopyranosides (aminoalkyl glucosaminide 4-phosphates) was synthesized and screened for immunostimulant activity. Several of these compounds enhance the production of tetanus toxoid-specific antibodies in mice and augment vaccine-induced cytotoxic T cells against EG.7-ova target cells.

Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity.

Flavonoid and isoflavonoid glycosides are common dietary phenolics which may be absorbed from the small intestine of humans. The ability of cell-free extracts from human small intestine and liver to deglycosylate various (iso)flavonoid glycosides was investigated. Quercetin 4'-glucoside, naringenin 7-glucoside, apigenin 7-glucoside, genistein 7-glucoside and daidzein 7-glucoside were rapidly deglycosylated by both tissue extracts, whereas quercetin 3,4'-diglucoside, quercetin 3-glucoside, kaempferol 3-glucoside, quercetin 3-rhamnoglucoside and naringenin 7-rhamnoglucoside remained unchanged. The Km for hydrolysis of quercetin 4'-glucoside and genistein 7-glucoside was approximately 32+/-12 and approximately 14+/-3 microM in both tissues respectively. The enzymatic activity of the cell-free extracts exhibits similar properties to the cytosolic broad-specificity -glucosidase previously described in mammals.

Induction of the anticarcinogenic marker enzyme, quinone reductase, in murine hepatoma cells in vitro by flavonoids.

Some flavonoids induce phase II enzymes both in vivo and in vitro. We have determined the structural requirements for this activity by examining the ability of naturally-occurring flavonoids to induce the phase II enzyme, quinone reductase (NAD(P)H:quinone oxidoreductase; EC 1.6.99.2), in murine Hepalclc7 cells. Hydroxylation of the B ring is not essential for induction, since galangin and kaempferol (with 0 and 1 hydroxyl in the B ring, respectively) are better inducers than quercetin (2 B ring hydroxyls). A 2,3 double bond in the C ring is essential for induction, since taxifolin, which has the same substitution pattern as quercetin but lacks the 2,3 double bond, is not an inducer. This is supported by catechin and epicatechin, which do not possess the 2,3 double bond and are also not inducers. A 3-hydroxyl group increases the activity but is not essential for induction, since apigenin is an inducer but kaempferol (which has the same structure as apigenin but possesses a 3-hydroxyl group) is more effective. The data show that, of the flavonoids, the flavonols are the most effective inducers of quinone reductase activity in Hepa1c1c7 cells (kaempferol approximately galangin > quercetin > myricetin approximately apigenin (a flavone)) and that flavanols and flavans are ineffective.

Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA.

A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium. Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA. The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv. tabaci. Residues involved at the active site of the mouse ODC are conserved in the plant enzyme. The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life. Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme. This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

Tropane alkaloid production in transformed root cultures of brugmansia Candida.

Transformed root cultures of BRUGMANSIA CANDIDA were established by infection with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones with different growth index and tropane alkaloid pattern and content were obtained and two were examined in depth. The alkaloid content and pattern changed during the time course. At 21 days of culture clone 1 revealed an alkaloid spectrum dominated by 3alpha-acetoxytropane (about 50% of the total alkaloid) and hyoscyamine (about 25%), with a ratio of hyoscyamine to scopolamine of 11.2. In clone 40 this ratio was 1.5 and the content of 3alpha-acetoxytropane was low (2%). The maximum concentrations of hyoscyamine were obtained at 3 weeks of culture, and were 700 and 500 microg/g FW in clone 1 and 40, respectively.

Factors Affecting the Growth and Hyoscyamine Production during Batch Culture of Transformed Roots of Datura stramonium.

The growth and hyoscyamine production of transformed roots of DATURA STRAMONIUM (cell line DS 1) were investigated in batch culture experiments at three different temperatures and in media of varying total ion composition and sucrose level. Growth rate was not greatly affected by the level of Gamborg's B5 salts or the sucrose concentration. Growth rates of roots in a range of media were similar at 25 degrees C (doubling time T (d) 1.2-1.6 days) and at 30 degrees C (T (d) 1.2-1.7 days) but roots grew slower at 20 degrees C (T (d) 1.9-3.5 days). The total root yield on a fresh weight basis was higher in full strength B5 media compared with half strength B5 media at all sucrose levels tested. However, the dry weight content of the roots increased significantly (4-20%) as the sucrose level in the medium was raised (2-10%) at each of the three temperatures. At 20 degrees C, the hyoscyamine content of the roots was higher than that at 25 degrees C or 30 degrees C at all sucrose levels tested and was unaffected by increasing sucrose levels. However, increasing the level of sucrose (1-5%) in either half or full strength B5 media at 25 degrees C or 30 degrees C increased the hyoscyamine content of the roots by up to eight fold. At 20 degrees C, the total yield of hyoscyamine per flask or per unit of sucrose supplied was also higher than at 25 degrees C or 30 degrees C with sucrose levels up to 5%. The highest rates of hyoscyamine production by cell line DS 1 were obtained with full strength B5 medium with 5% sucrose at either 20 degrees C or 25 degrees C. Under these conditions, rates of hyoscyamine formation of up to 7.4 mg/l/d were observed under batch culture conditions. The amount of hyoscyamine released into the culture medium was much greater at 30 degrees C (up to 14% of the total) than at either 20 degrees C or 25 degrees C (up to 4% of the total).

The formation of 3 alpha- and 3 beta-acetoxytropanes by Datura stramonium transformed root cultures involves two acetyl-CoA-dependent acyltransferases.

Tropine (tropan-3 alpha-ol) is an intermediate in the formation of hyoscyamine. An acyltransferase activity that can acetylate tropine using acetylcoenzyme A as cosubstrate has been found in transformed root cultures of Datura stramonium. A further acyltransferase activity that acetylates pseudotropine (tropan-3 beta-ol) with acetyl-coenzyme A is also present. These two activities can be partially resolved by anion-exchange chromatography, some fractions containing only the pseudotropine-utilizing activity. The basic properties of these two enzymes are reported and their roles in forming the observed alkaloid spectrum of D. stramonium roots discussed.

Strategies for the genetic manipulation of alkaloid-producing pathways in plants.

Increasingly, as a result of recent biochemical work, there exists a realistic possibility of taking a molecular genetic approach to the manipulation of alkaloid-producing pathways in plant tissue cultures. In the pathways forming indole alkaloids in CATHARANTHUS ROSEUS, tropane alkaloids in DATURA and HYOSCYAMUS species, and nicotine in NICOTIANA species, recent studies have identified a number of key enzymes and at least some of the factors that regulate their levels of activity. Such knowledge contributes the basis upon which it has become feasible to design a strategy by which the flux in these pathways may be enhanced at the genomic level. This review presents a summary of the state-of-the-art pertaining to these pathways and discusses the strategy to be adopted for a molecular approach to their manipulation, together with some of the pitfalls that may arise when trying to alter their natural regulation.

Growth and hyoscyamine production of 'hairy root' cultures of Datura stramonium in a modified stirred tank reactor.

The growth and hyoscyamine production of transformed roots of Datura stramonium have been examined in a modified 14-1 stirred tank reactor in both batch and continuous fermentations on media containing half or full strength Gamborg's B5 salts and at three different temperatures. Under a range of conditions, roots grown on half strength B5 salts with 3% w/v sucrose had a higher dry matter content (up to 8.3% w/w) and a higher hyoscyamine content (up to 0.52 mg.g-1 wet weight) than roots grown on full strength B5 salts with the same level of sucrose (up to 4.6% w/w dry matter and up to 0.33 mg hyoscyamine g-1 wet weight). Growth at 30 degrees C was initially faster than at either 25 degrees C or 35 degrees C and by day 12, the drained weight of roots in the fermentor at 30 degrees C was about fourfold greater than at 25 degrees C and twice that at 35 degrees C. The ultimate hyoscyamine levels attained (approximately 0.5 mg.g-1 wet weight) were similar at both 25 degrees C and 30 degrees C but some 40% lower at 35 degrees C. Final packing densities of 70% w/v were achieved for roots after 37 days growth at 25 degrees C and the highest production rate of 8.2 mg hyoscyamine 1(-1) per day was obtained for roots grown at 30 degrees C. In continuous fermentation at 25 degrees C, the release of hyoscyamine into the culture medium was low (less than 0.5% w/w of the total) but was up to sevenfold higher in fermentors operated at 30 degrees C or 35 degrees C.

Alkaloid Production by Transformed Root Cultures of Cinchona ledgeriana.

Transformed root cultures of CINCHONA LEDGERIANA have been generated by infecting shoots cultured IN VITRO with AGROBACTERIUM RHIZOGENES LBA9402. These root cultures grow axenically in the absence of antibiotics or exogenous plant growth regulators in media containing Gamborg B5 salts at half or full strength with 3% (w/v) sucrose as the carbon source. They show a 6- to 8-fold increase in fresh weight in 28 days. Transformation has been confirmed by Southern blotting using [ (32)P]-labelled fragments from both the T (L)- and T (R)-DNA of the Ri plasmid. The cultures are shown to synthesise a range of quinoline alkaloids, of which quinine, cinchonidine, and quinidine are the major components. The level of these alkaloids changes with the age of the cultures, reaching a maximum at about 50 microg/g fresh weight after 45 days. In addition, the roots contain quinamine and a number of other, unidentified, indole alkaloids, one of which is the major alkaloid present. Only about 1% of the total alkaloid present is released to the medium.

Production of Hyoscyamine by 'Hairy Root' Cultures of Datura stramonium.

'Hairy root' cultures of DATURA STRAMONIUM were established following infection of aseptic leaves with AGROBACTERIUM RHIZOGENES. Transformation was confirmed by Southern blotting using [ (32)P]-labelled fragments of the T-DNA as probes. The transformed cultures grew in the absence of added phytohormones and cell mass increased 55-fold during 28 days incubation. Hyoscyamine was a major component of the alkaloid fraction and accounted for at least 0.3% of the dry matter, comparable to pot-grown plants from which the cultures were initiated. Production of hyoscyamine followed growth during the first 15 days of incubation, but continued to increase during the early part of stationary phase. The alkaloid was retained almost entirely in the root tissue. The effects of medium composition and pH on growth and hyoscyamine production are reported.

Quinoline Alkaloid Production by Transformed Cultures of Cinchona ledgeriana.

A culture of CINCHONA LEDGERIANA, transformed with AGROBACTERLUM TUMEFACIENS A6 capable of growing and producing quinoline alkaloids in medium free of exogenous phytohormones has been obtained. Unlike the untransformed culture of this species, addition of ZR in combination with either IAA or IBA to medium did not affect alkaloid production. Growing the transformed culture in the dark, however, produced a marked enhancement of alkaloid accumulation, up to 50 times that of cultures grown in the light. This dark-dependent accumulation was not confined to any particular time in the growth cycle, although the extent of the stimulatory effect increased the longer cultures were kept in darkness. Blue light was detrimental to alkaloid accumulation but in red or green light the level of accumulation was equivalent to that in the dark. Alternating cultures for several 28-day periods between light and dark conditions resulted in alternate periods of low and higfralkaloid productivity, indicating this was not an adaption effect. These findings correlate to previously reported differences in key enzymes from the biosynthetic pathway. Increasing the phosphate or nitrate concentration above that of Gamborg B5, increasing sucrose above 2% (w/v), replacing sucrose with glucose or adding tryptophan or casamino acids to the medium resuited in lower alkaloid yields by dark-grown cultures of the transformed line.

A spectrophotometric assay for strictosidine synthase.

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.

Radioimmunoassay for the quantitative determination of quinine in cultured plant tissues.

The development of a radioimmunossay for the determination of picogram amounts of quinine is described. The antiserum, raised against a conjugate of quinine and bovine serum albumin, is highly specific to the CINCHONA quinoline alkaloids having the 8S configuration and will not cross-react to any detectable extent with the 8R series. Neither will it cross-react with quininone or cinchonidinone, the common 9-keto precursors. Using this assay a number of clones of C. LEDGERIANA have been screened for their quinine content.