John Eagles

A different function for a member of an ancient and highly conserved cytochrome P450 family: From essential sterols to plant defense

CYP51 sterol demethylases are the only cytochrome P450 enzymes with a conserved function across the animal, fungal, and plant kingdoms (in the synthesis of essential sterols). These highly conserved enzymes, which are important targets for cholesterol-lowering drugs, antifungal agents, and herbicides, are regarded as the most ancient member cytochrome P450 family. Here we present a report of a CYP51 enzyme that has acquired a different function. We show that the plant enzyme AsCYP51H10 is dispensable for synthesis of essential sterols and has been recruited for the production of antimicrobial compounds (avenacins) that confer disease resistance in oats. The AsCyp51H10 gene is synonymous with Sad2, a gene that we previously had defined by mutation as being required for avenacin synthesis. In earlier work, we showed that Sad1, the gene encoding the first committed enzyme in the avenacin pathway (β-amyrin synthase), had arisen by duplication and divergence of a cycloartenol synthase-like gene. Together these data indicate an intimate evolutionary connection between the sterol and avenacin pathways. Sad1 and Sad2 lie within 70 kb of each other and are expressed specifically in the epidermal cells of the root tip, the site of accumulation of avenacins. These findings raise intriguing questions about the recruitment, coevolution, and regulation of the components of this specialized defense-related metabolic pathway.

Variation in tropane alkaloid accumulation within the solanaceae and strategies for its exploitation

Key Word Index-Datum spp.; Hyoscyamus spp.; Scopolia spp.; Solanaceae; identification; accumulation; trans- formed roots; tropane alkaloids; scopolamine; hyoscyamine. - Abstract-Petioles from over 1000 individual plants of the genera Datura, Scopolia and Hyoscyamus were analysed to establish the variation in the levels and patterns of tropane alkaloids which occur between plants. Transformed root cultures were initiated from plants of which analyses suggested extremely high or low alkaloid biosynthetic capacities, or favourable alkaloid patterns. These root cultures were then studied in detail by HPLC, GC and GC-MS. Root cultures showed substantial differences in alkaloid patterns between different species, but the quantitative differences in tropoyl esters of cc-tropine (hyoscyamine plus scopolamine) observed between different lines were less than those seen between plants. This difference is interpreted as to some extent reflecting the influence of transport and storage effects, as well as biosynthetic capacity, in determining alkaloid levels in plant petioles. In addition, there is some indication that the full biosynthetic capacity may not always be expressed in culture. Despite these limitations, the identification of plants showing favourable characteristics still, however, proved a useful first step in the development of scientifically and potentially biotechnologically interesting root cultures.

Identification of the major glucosinolate (4-mercaptobutyl glucosinolate) in leaves of Eruca sativa L. (salad rocket).

The major and structurally unique glucosinolate (GLS) in leaves of Eruca sativa L. (salad rocket) was identified as 4-mercaptobutyl GLS. Both 4-methylthiobutyl GLS and 4-methylsulfinylbutyl GLS were also present, but at lower concentrations. The 4-mercaptobutyl GLS was observed to oxidise under common GLS extraction conditions, generating a disulfide GLS that may be reduced efficiently by tris(2-carboxyethyl) phosphine hydrochloride (TCEP) to reform the parent molecule. The identities of 4-mercaptobutyl GLS and of the corresponding dimeric GLS were confirmed by LC/MS, MS/MS and NMR. Myrosinase treatment of an enriched GLS fraction or of the purified dimer GLS generated a mixture of unique bi-functional disulfides, including bis-(4-isothiocyanatobutyl) disulfide (previously identified elsewhere). TCEP reduction of the purified dimer, followed by myrosinase treatment, yielded only 4-mercaptobutyl ITC. GLS-derived volatiles generated by autolysis of fresh seedlings and true leaves were 4-mercaptobutyl ITC (from the newly identified GLS), 4-methylthiobutyl ITC (from 4-methylthiobutyl GLS) and 4-methylsulfinylbutyl ITC (from 4-methylsulfinyl-butyl GLS); no unusual bi-functional disulfides were found in fresh leaf autolysate. These results led to the conclusion that, in planta, the new GLS must be present as 4-mercaptobutyl GLS and not as the disulfide found after extraction and sample concentration. This new GLS and its isothiocyanate are likely to contribute to the unique odour and flavour of E. sativa.

Chrysin and other leaf exudate flavonoids in the genus Pelargonium

In a chemotaxonomic survey of 57 Pelargonium species, leaf exudate flavonoids were detected in 35% of the sample, mostly in trace amounts. However, chrysin and a related C-methylflavanone were identified as major leaf surface constituents of P. crispum, and a mixture of quercetin and kaempferol mono-, and di- and trimethyl ethers of P. quercifolium. In two other species, P. fulgidum and P. exstipulatum, methylated flavones were the only lipophilic flavonoids present. This is the first report of leaf surface flavonoids from the genus Pelargonium.

Simultaneous time-varying systemic appearance of oral and hepatic glucose in adults monitored with stable isotopes

The rates (and extent) of appearance of glucose in arterialized plasma from an oral glucose load and from liver (RaO, RaH) can be estimated in humans using radioisotopes, but estimates vary among laboratories. We investigated the use of stable isotopes and undertook 22 primed intravenous infusions of D-[6,6-2H2]glucose with an oral load including D-[13C6]glucose in healthy humans. The effective glucose pool volume (VS) had a lower limit of 230 ml/kg body weight (cf. 130 ml/kg commonly assumed). This VS in Steeles one-compartment model of glucose kinetics gave a systemic appearance from a 50-g oral glucose load per 70 kg body weight of 96 +/- 3% of that ingested, which compared with a theoretical value of approximately 95%. Maris two-compartment model gave 100 +/- 3%. The two models gave practically identical RaO and RaH at each point in time and a plateau in the cumulative RaO when absorption was complete. Less than 3% of 13C was recycled to [13C3]glucose, suggesting that recycling errors were practically negligible in this study. Causes of variation among laboratories are identified. We conclude that stable isotopes provide a reliable and safe alternative to radioactive isotopes in these studies.The rates (and extent) of appearance of glucose in arterialized plasma from an oral glucose load and from liver (RaO, RaH) can be estimated in humans using radioisotopes, but estimates vary among laboratories. We investigated the use of stable isotopes and undertook 22 primed intravenous infusions ofd-[6,6-2H2]glucose with an oral load includingd-[13C6]glucose in healthy humans. The effective glucose pool volume (VS) had a lower limit of 230 ml/kg body weight (cf. 130 ml/kg commonly assumed). This VS in Steeles one-compartment model of glucose kinetics gave a systemic appearance from a 50-g oral glucose load per 70 kg body weight of 96 ± 3% of that ingested, which compared with a theoretical value of ∼95%. Maris two-compartment model gave 100 ± 3%. The two models gave practically identical RaO and RaH at each point in time and a plateau in the cumulative RaO when absorption was complete. Less than 3% of13C was recycled to [13C3]glucose, suggesting that recycling errors were practically negligible in this study. Causes of variation among laboratories are identified. We conclude that stable isotopes provide a reliable and safe alternative to radioactive isotopes in these studies.

The bioavailability of iron in different weaning foods and the enhancing effect of a fruit drink containing ascorbic acid

ABSTRACT: There is limited information on the bioavailability of Fe in infant weaning foods, mainly because of the difficulties of measuring Fe utilization directly in infants. The aim of this study was to develop a safe and relatively noninvasive method for studying Fe bioavailability (measured as percent Fe incorporation into red blood cells) in infants using 54Fe, 57Fe, and 58Fe stable isotopes. Four commonly used weaning foods were selected for study, labeled extrinsically with 57Fe- or 58Fe-enriched ferrous sulfate, and fed to five female and five male 9-mo-old fasting infants, using a multiple-dosing technique. Each food was given three times, labeled with one isotope, with a fruit juice drink containing 50 mg of ascorbic acid, and three times, labeled with a different isotope, with an ascorbic acid-free drink. Fourteen days after the last test meal, a blood sample was obtained from a heel-prick, spiked with a known amount of 54Fe, digested, and purified by ion exchange; isotopic enrichment and total Fe content were measured by quadrupole thermal ionization mass spectrometry. The proportion of administered dose of isotope circulating in the blood was calculated from an estimate of blood volume. The geometric mean bioavailability (range) was 3.0% (1.2–9.5%) in a proprietary dehydrated vegetable product, 3.0% (1.1–21.2%) in Weetabix whole-wheat breakfast cereal, 3.1% (1.2–15.4%) in wholemeal bread and 4.3% (1.7–10.3%) in baked beans. When taken with the drink containing ascorbic acid, there was a 2-fold increase in bioavailability in all foods except the vegetable meal, presumably because this was already fortified with ascorbic acid. Thus, drinks containing 50 mg of ascorbic acid, taken with a meal, can significantly improve Fe bioavailability to infants from weaning foods low in ascorbic acid.

Studies on calcium absorption from milk using a double-label stable isotope technique

Calcium absorption was measured in ten male volunteers from skimmed milk, Ca-enriched skimmed milk or watercress (Nasturtium officinale) soup. The foods were labelled extrinsically with 30 mg 44Ca. Shortly after consuming the labelled meal, each subject was given an intravenous injection of 3 mg 42Ca. Fractional absorption from the oral dose was determined from plasma and urine samples collected 24-72 h later, using fast atom bombardment mass spectrometry to measure isotope ratios. The values for urine and plasma were in good agreement. Mean percentage absorption was 45.5 (SEM 1.9)% from the skimmed milk, 35.7 (SEM 4.7)% from the Ca-enriched milk and 27.4 (SEM 1.9)% from the watercress soup. The effect of consuming 568 ml (1 pint) Ca-enriched milk each day for 4 weeks on the efficiency of absorption of Ca was studied. Although there was no statistically significant difference between Ca absorption before and after the supplementation period, the results were considered to be somewhat inconclusive due to the small number of subjects and wide individual variation in Ca absorption.

The measurement of exchangeable pools of zinc using the stable isotope 70Zn

The present study was designed to assess the feasibility of using small doses of a stable isotope of Zn to follow plasma kinetics over a 10 d period and, hence, make deductions about Zn turnover and body pool sizes. At the beginning of the 10 d metabolic balance, two adults, consuming their habitual diet, were given an intravenous injection of 70Zn. There was a fourfold difference in the administered dose between the two subjects (0.445 and 2.078 mg). Blood samples were taken at regular intervals and plasma enrichment with 70Zn measured by thermal ionization mass spectrometry. Urine and faeces were collected and analysed for Zn and 70Zn. Kinetic analysis of the plasma 70Zn decay by several different methods was undertaken. It was apparent from both deconvolution analysis of the short-term (0-90 min) decay data and four-compartment modelling of the longer-term (0-24 h) data that isotopic Zn very rapidly equilibrates with the plasma Zn and with a rapidly exchanging non-plasma pool, probably located within the liver. This latter pool appears to contain less than 10 mg Zn and the peak of isotope enrichment occurs at about 20 min post injection. The later decay of plasma Zn enrichment appears to be dictated by exchange with a much larger pool of approximate size 350 mg.

Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species

Abstract The extracellular anionic polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) contains l -mannose, l -rhamnose, d -glucose, and d -glucuronic acid in the molar ratios 1.0:4.5:3.1:2.3. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide indicated terminal non-reducing rhamnose and mannose, (1→4)-linked rhamnose, (1→3)- and (1→3,1→4)-linked glucose, and (1→4)-linked glucuronic acid to be present in the ratios 1.0:0.8:2.1:2.2:2.0:2.2. Partial acid hydrolysis and base-catalysed β-elimination gave a series of oligosaccharides that were isolated as their alkylated alditol derivatives by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and 1H-n.m.r. spectroscopy. The repeating unit 1, excluding O-acyl groups, is proposed.

True fractional calcium absorption in Chinese children measured with stable isotopes (42Ca and 44Ca)

True fractional Ca absorption (TFCA) was compared in children with different habitual Ca intakes using a double-label stable-isotope technique. Chinese children aged 7 years from Hongkong (n22) and Jiangmen (n12) participated in the study. An oral administration of 8 mg 44Ca in 100 g chocolate milk was given shortly after an intravenous injection of 0.75 mg 42Ca. Ca isotopic ratios were determined in urine samples collected 24 h later using thermal-ionization mass spectrometry. There was no significant difference in TFCA between Jiangmen and Hongkong children (P = 0.16). TFCA of a lower-Ca-intake group (Ca < or = 500 mg/d, n19) with mean Ca intake 359 mg/d was 63.1 (SD 10.7)% and that of a higher-Ca-intake group (Ca > 500 mg/d, n15) with mean Ca intake 862 mg/d was 54.8 (SD 7.3)%; the difference in TFCA was significant (P = 0.016). Serum levels of 25-hydroxycholecalciferol of the children were adequate (33.7 (SD 7.7) ng/ml). The present study indicates that growing children accustomed to a low-Ca diet appear to be able to enhance their absorptive capacity. If it is assumed that dietary Ca absorption by Chinese children resembles their TFCA from a single meal of chocolate milk, then the recommended dietary allowance (RDA) for Ca for Chinese children would be lower than the US RDA (800 mg/d), which is based on an estimated 40% Ca absorption as reported for Caucasian children. A comparative absorption study is necessary to determine whether there is any difference in TFCA between Caucasian and Chinese children.