The effects of a diet rich in inulin or wheat fibre on markers of cardiovascular disease in overweight male subjects.

BACKGROUND: Previous studies suggest that the beneficial health effects of a diet rich in whole grains could be a result of the individual fibres found in the grain. The present study aimed to investigate the influence of a diet high in either wheat fibre (as an example of an insoluble fibre) or inulin (a nondigestible carbohydrate) on markers of cardiovascular disease. METHODS: Ten male participants classified as at higher risk of cardiovascular disease [mean (SD) body mass index 30.2 (3) kg m(-2) , mean (SD) waist circumference 106.4 (7) cm, mean (SD) age 39.8 (9) years] were recruited to a randomised, controlled, cross-over study comparing the consumption of bespoke bread rolls containing either inulin, wheat germ or refined grain (control) (15 g day(-1) ) for 4 weeks with a 4-week washout period between each regime. At the end of each regime, participants underwent an oral glucose tolerance test (OGTT), measures of pulse wave velocity (PWV), 24-h ambulatory blood pressure (AMBP), plasma lipid status and markers of glucose control. RESULTS: There was no difference in measures of glucose control, lipid status, 24-h AMBP or PWV after the intervention periods and no changes compared to baseline. There was no significant difference between OGTT glucose and insulin time profiles; however, there was a significant difference in area under the curves between the wheat fibre and control interventions when comparing change from baseline (control +10.2%, inulin +4.3%, wheat fibre -2.5%; P = 0.03). CONCLUSIONS: Only limited differences between the interventions were identified, perhaps as a consequence of the amount of fibre used and intervention length. The wheat germ intervention resulted in a significant reduction in glucose area under the curve, suggesting that this fibre may aid glucose control.

Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Copper-induced LDL peroxidation investigated by 1H-NMR spectroscopy.

Oxidatively modified LDL (oLDL) is thought to play a key role in the pathogenesis of atherosclerosis. We have studied Cu(2+)-induced peroxidation reactions of LDL and have elucidated the sequence of events which subsequently occur within LDL particles by 1H-NMR spectroscopy. Studies of chloroform/methanol extracts show that LDL arachidonate is oxidised by Cu2+ at a higher rate and to a greater extent than linoleate, giving isomeric hydroperoxides with predominantly trans,trans double-bonds, whilst only cis,trans isomers were detected as intrinsic hydroperoxides in control LDL samples. These intrinsic hydroperoxides were not degraded during peroxidation, suggesting that they are not involved in the initiation of Cu(2+)-induced peroxidation. Aldehydes arising from the decomposition of hydroperoxides were also detected, as well as saturated fatty acids which were released into the external aqueous medium. Decomposition pathways of the two major isomeric hydroperoxides are discussed. Cu(2+)-induced oxidation of LDL cholesterol appears to occur only after hydroperoxide breakdown, with esterified cholesterol being oxidised to a greater extent than free cholesterol. Phospholipid hydrolysis appeared to parallel the peroxidation of arachidonic acid, and the released lysophosphatidylcholine may become associated with apoB. These results suggest that hydroperoxide breakdown (probably in phospholipids) may be a key event in the peroxidation process, leading to the oxidation of cholesterol and propagation into the core of LDL.

Transposon Tn5 target specificity: preference for insertion at G/C pairs.

The procaryotic transposon Tn5 inserts into many different sites within a single gene, but some sites (hotspots) are targeted repeatedly. Hotspots are not closely related in sequence, but most have G/C pairs at the ends of the nine base pairs duplicated by Tn5 insertion. In pBR322, the major hotspot coincides with the "-10 region" of the tet promoter. We mutated the G/C pairs at this hotspot and assayed for insertion into hotspot I, resistance to tetracycline, and plasmid supercoiling. We found that changing the G/C pairs to A/T pairs reduced the frequency of insertion into the hotspot by at least fivefold. The reduction in hotspot use caused by these G/C to A/T changes was not attributable to changes in plasmid supercoiling or tet promoter strength.

Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis.

Cryptococcus neoformans var. neoformans is an important opportunistic fungal pathogen of patients whose immune system has been compromised due to viral infection, antineoplastic chemotherapy, or tissue transplantation. As many as 13% of all AIDS patients suffer a life-threatening cryptococcal infection at some time during the course of their HIV disease. To begin to understand the molecular basis for virulence in Cryptococcus neoformans var. neoformans serotype A, we have employed signature-tagged mutagenesis (STM) to identify mutants with altered virulence in a mouse model. The critical parameters of signature-tagged mutagenesis in C. neoformans are explored. Data are presented showing that at least 100 different strains can be mixed together in a single animal with each participating in the infection and that there is no apparent interaction between a virulent strain and an avirulent strain in our animal model. Using signature-tagged mutagenesis, we identified 39 mutants with significantly altered growth in a competitive assay. Molecular analyses of these mutants indicated that 19 (49%) contained an insertion in the actin promoter by homologous recombination from a single crossover event, creating a duplication of the actin promoter and the integration of single or multiple copies of the vector. Analysis of the chromosomal insertion sites of those mutants that did not have an integration event in the actin promoter revealed an approximately random distribution among the chromosomes. Individual challenge of the putative mutants in a mouse model revealed five hypovirulent mutants and one hypervirulent mutant.

Thiol chelation of Cu2+ by dihydrolipoic acid prevents human low density lipoprotein peroxidation.

Mono-thiols can act either as pro- or anti-oxidants during metal-catalyzed low density lipoprotein (LDL) peroxidation, however investigation of the role of vicinal thiols has been neglected. Therefore dihydrolipoic acid (DHLA), a vicinal dithiol, and lipoic acid, its oxidized form, were used to investigate Cu2+-mediated LDL peroxidation. We demonstrate here that DHLA inhibited Cu2+-dependent LDL peroxidation by chelating copper. DHLA (0-20 microM) increased lag-times of conjugated diene formation in LDL (100 microg/ml) oxidized with 5 microM Cu2+ in a concentration dependent manner, and this effect was saturated after 5 microM DHLA; enough to chelate all of the added Cu2+. In a similar fashion DHLA prevented LDL-mediated reduction of Cu2+ to Cu+. Lipoic acid had no effect in these systems. DHLA alone also reduced Cu2+, however this was inhibited when DHLA was in excess of the copper concentration. Hence there is complex formation between the two species. Copper:DHLA complex formation was further investigated and found to be dependent upon pH and the presence of oxygen. At low pH (<6), or in the absence of oxygen, the complex is stable, presumably due to vicinal thiol chelation. As the pH is increased, the carboxylate group also participates in copper chelation, this results in a less stable complex which is susceptible to oxidation, and copper is eventually released. Electron spin resonance studies demonstrate the formation of hydroxyl, but not superoxide, radicals during Cu2+-catalyzed DHLA oxidation. Thus in our LDL experiments at physiological pH, DHLA is able to either reductively inactivate Cu2+ when Cu2+ is in excess, or effectively chelate Cu2+ when DHLA is in excess. The Cu2+:DHLA complex eventually undergoes copper-catalyzed oxidation, copper is released and LDL peroxidation proceeds. DHLA, thus, has both pro- and antioxidant properties depending upon the ratio of Cu2+:DHLA and the pH. These results provide an additional mechanism of thiol-mediated formation of radicals and metal chelation.

Alpha-lipoic acid in liver metabolism and disease.

R-alpha-Lipoic acid is found naturally occurring as a prosthetic group in alpha-keto acid dehydrogenase complexes of the mitochondria, and as such plays a fundamental role in metabolism. Although this has been known for decades, only recently has free supplemented alpha-lipoic acid been found to affect cellular metabolic processes in vitro, as it has the ability to alter the redox status of cells and interact with thiols and other antioxidants. Therefore, it appears that this compound has important therapeutic potential in conditions where oxidative stress is involved. Early case studies with alpha-lipoic acid were performed with little knowledge of the action of alpha-lipoic acid at a cellular level, but with the rationale that because the naturally occurring protein bound form of alpha-lipoic acid has a pivotal role in metabolism, that supplementation may have some beneficial effect. Such studies sought to evaluate the effect of supplemented alpha-lipoic acid, using low doses, on lipid or carbohydrate metabolism, but little or no effect was observed. A common response in these trials was an increase in glucose uptake, but increased plasma levels of pyruvate and lactate were also observed, suggesting that an inhibitory effect on the pyruvate dehydrogenase complex was occurring. During the same period, alpha-lipoic acid was also used as a therapeutic agent in a number of conditions relating to liver disease, including alcohol-induced damage, mushroom poisoning, metal intoxification, and CCl4 poisoning. Alpha-Lipoic acid supplementation was successful in the treatment for these conditions in many cases. Experimental studies and clinical trials in the last 5 years using high doses of alpha-lipoic acid (600 mg in humans) have provided new and consistent evidence for the therapeutic role of antioxidant alpha-lipoic acid in the treatment of insulin resistance and diabetic polyneuropathy. This new insight should encourage clinicians to use alpha-lipoic acid in diseases affecting liver in which oxidative stress is involved.

Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line.

Exposure of the human skin to ultraviolet radiation (UVR) leads to depletion of cutaneous antioxidants, regulation of gene expression and ultimately to the development of skin diseases. Although exogenous supplementation of antioxidants prevents UVR-induced photooxidative damage, their effects on components of cell signalling pathways leading to gene expression has not been clearly established. In the present study, the effects of the antioxidants alpha-lipoic acid, N-acetyl-L-cysteine (NAC) and the flavonoid extract silymarin were investigated for their ability to modulate the activation of the transcription factors nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) in HaCaT keratinocytes after exposure to a solar UV simulator. The activation of NF-kappaB and AP-1 showed a similar temporal pattern: activation was detected 2 h after UV exposure and maintained for up to 8 h. To determine the capacity of activated NF-kappaB to stimulate transcription, NF-kappaB-dependent gene expression was measured using a reporter gene assay. The effects of the antioxidants on NF-kappaB and AP-1 activation were evaluated 3 h after exposure. While a high concentration of NAC could achieve a complete inhibition, low concentrations of alpha-lipoic acid and silymarin were shown to significantly inhibit NF-kappaB activation. In contrast, AP-1 activation was only partially inhibited by NAC, and not at all by alpha-lipoic acid or silymarin. These results indicate that antioxidants such as alpha-lipoic acid and silymarin can efficiently modulate the cellular response to UVR through their selective action on NF-kappaB activation.

Novel biologically active nonpeptidic inhibitors of myristoylCoA:protein N-myristoyltransferase.

A new class of biologically active nonpeptidic inhibitors of Candida albicans NMT has been synthesized starting from the octapeptide ALYASKLS-NH2 (2). The synthetic strategy entailed the preparation of novel protected Ser-Lys mimics 9 and 12 from (S)- or (R)-3-iodotyrosine and then grafting key enzyme recognition elements in a stepwise manner. Like 2, compounds 16, 17, and 18 are competitive Candida NMT inhibitors that bind to the peptide recognition site of the enzyme. Moreover, 16-18 have an affinity comparable to that of 2 even though they are devoid of peptide bonds. In contrast to 2, these nonpeptidic inhibitors exhibit antifungal activity.

Design and synthesis of novel imidazole-substituted dipeptide amides as potent and selective inhibitors of Candida albicans myristoylCoA:protein N-myristoyltransferase and identification of related tripeptide inhibitors with mechanism-based antifungal activity.

A new class of antifungal agents has been discovered which exert their activity by blockade of myristoylCoA: protein N-myristoyltransferase (NMT; EC 2.1.3.97). Genetic experiments have established that NMT is needed to maintain the viability of Candida albicans and Cryptococcus neoformans,the two principal causes of systemic fungal infections in immunocompromised humans. Beginning with a weak octapeptide inhibitor ALYASKLS-NH2 (2, Ki = 15.3 +/- 6.4 microM), a series of imidazole-substituted Ser-Lys dipeptide amides have been designed and synthesized as potent and selective inhibitors of Candida albicans NMT. The strategy that led to these inhibitors evolved from the identification of those functional groups in the high-affinity octapeptide substrate GLYASKLS-NH2 1a necessary for tight binding, truncation of the C-terminus, replacement of the four amino acids at the N-terminus by a spacer group, and substitution of the glycine amino group with an N-linked 2-methylimidazole moiety. Initial structure-activity studies led to the identification of 31 as a potent and selective peptidomimetic inhibitor with an IC50 of 56 nM and 250-fold selectivity versus human NMT. 2-Methylimidazole as the N-terminal amine replacement in combination with a 4-substituted phenacetyl moiety imparts remarkable potency and selectivity to this novel class of inhibitors. The (S,S) stereochemistry of serine and lysine residues is critical for the inhibitory activity, since the (R,R) enantiomer 40 is 10(3)-fold less active than the (S,S) isomer 31. The inhibitory profile exhibited by this new class of NMT ligands is a function of the pKa of the imidazole substituent as illustrated by the benzimidazole analog 35 which is about 10-fold less potent than 31. The measured pKa (7.1 +/- 0.5) of 2-methylimidazole in 31 is comparable with the estimated pKa (approximately 8.0) of the glycyl residue in the high-affinity substrate 1a. Groups bulkier than methyl, such as ethyl, isopropyl, or iodo, at the imidazole 2-position have a detrimental effect on potency. Further refinement of 31 by grafting an alpha-methyl group at the benzylic position adjacent to the serine residue led to 61 with an IC50 of 40 nM. Subsequent chiral chromatography of 61 culminated in the discovery of the most potent Candida NMT inhibitor 61a reported to date with an IC50 of 20 nM and 400-fold selectivity versus the human enzyme. Both 31 and 61a are competitive inhibitors of Candida NMT with respect to the octapeptide substrate GNAASARR-NH2 with Ki(app) = 30 and 27 nM, respectively. The potency and selectivity displayed by these inhibitors are dependent upon the size and orientation of the alpha-substituent. An alpha-methyl group with the R configuration corresponding to the (S)-methyl-4-alanine in 2 confers maximum potency and selectivity. Structural modification of 31 and 61 by appending an (S)-carboxyl group beta to the cyclohexyl moiety provided the less potent tripeptide inhibitors 73a and 73b with an IC50 of 1.45 +/- 0.08 and 0.38 +/- 0.03 microM, respectively. However, these tripeptides (73a and 73b) exhibited a pronounced selectivity of 560- and 2200-fold versus the human NMT. More importantly 73a displayed fungistatic activity against C albicans with an EC50 of 51 +/- 17 microM in cell culture. Compound 73b also exhibited a similar antifungal activity. An Arf protein gel mobility shift assay for monitoring intracellular myristoylation revealed that a single dose of 200 microM of 73a or 73b produced < 50% reduction in Arf N-myristoylation, after 24 and 48 h, consistent with their fungistatic rather than fungicidal activity. In contrast, the enantiomer 73d which had an IC50 > 1000 microM against C. albicans NMT did not exhibit antifungal activity and produced no detectable reduction in Arf N-myristoylation in cultures of C. albicans. These studies confirm that the observed antifungal activity of 73a and 73b is due to the attenuation of NMT activity and that NMT represents an attractive tar

Cu2+ -induced low density lipoprotein peroxidation is dependent on the initial O2 concentration: an O2 consumption study.

Atherosclerotic plaques form in the arterial intima, where low density lipoprotein (LDL) is thought to be oxidatively modified at sites which may contain catalytic amounts of copper in the presence of low O2 tension. We have investigated O2 consumption during LDL peroxidation induced by Cu2+ ions in vitro and found two phases: a lag phase followed by a phase of rapid O2 consumption. The length of the lag phase was dependent on Cu2+ and on initial O2 concentrations; increasing either decreased the lag time; however, LDL. concentration had no effect. LDL-induced Cu2+ reduction, however, was not affected by low initial O2 concentrations, suggesting that O2 is not required for LDL-mediated reduction of Cu2+. Following the lag phase, O2 consumption was dependent upon LDL or initial O2 concentrations; Cu2+ concentrations had little effect, suggesting that the propagation phase is more dependent on the presence of LDL lipids and O2 as substrates for the reaction. In summary, LDL peroxidation takes place in the presence of Cu2+ at low O2 tension; however, the reaction is dependent upon initial O2 concentrations; increases shorten the lag phase and accelerate O2 consumption.

Alpha- and gamma-tocotrienols are metabolized to carboxyethyl-hydroxychroman derivatives and excreted in human urine.

Limited information is available regarding metabolism of vitamin E forms, especially the tocotrienols. Carboxyethyl-hydroxychromans (alpha- and gamma-CEHC) are human urinary metabolites of alpha- and gamma-tocopherols, respectively. To evaluate whether tocotrienols are also metabolized and excreted as urinary CEHC, urine was monitored following tocotrienol supplementation. Complete (24 h) urine collections were obtained for 2 d prior to (baseline), the day of, and 2 d after human subjects (n = 6) ingested tocotrienol supplements. The subjects consumed 125 mg gamma-tocotrienyl acetate the first week, then the next week 500 mg; then 125 mg alpha-tocotrienyl acetate was administered the third week, followed by 500 mg the fourth week. Urinary alpha- and gamma-CEHC were measured by high-performance liquid chromatography with electrochemical detection. Urinary gamma-CEHC levels rose about four- to sixfold in response to the two doses of gamma-tocotrienol and then returned to baseline the following day. Significant (P < 0.0001) increases in urinary alpha-CEHC were observed only following ingestion of 500 mg alpha-tocotrienyl acetate. Typically, 1-2% of alpha-tocotrienyl acetates or 4-6% of gamma-tocotrienyl acetates were recovered as their respective urinary CEHC metabolites. A gamma-CEHC excretion time course showed an increase in urinary gamma-CEHC at 6 h and a peak at 9 h following ingestion of 125 mg gamma-tocotrienyl acetate. In summary, tocotrienols, like tocopherols, are metabolized to CEHC; however, the quantities excreted in human urine are small in relation to dose size.

Mutations that affect Tn5 insertion into pBR322: importance of local DNA supercoiling.

The major hot spot of transposon Tn5 insertion in plasmid pBR322 (hot spot I) is in the promoter for the tetracycline resistance gene (tet). We made a series of pBR322 derivatives with mutations in and around this promoter and assayed their effects on insertion of Tn5 into hot spot I. Those mutations which reduced transcription from the tet promoter also reduced the frequency of insertion into hot spot I. Transcription and translation of tet are thought to cause the formation of paired domains of negative and positive supercoiling in pBR322. An amber codon in tet, 345 base pairs from hot spot I, decreases the negative supercoiling of the DNA segment containing hot spot I because it terminates translation of tet prematurely. We report here that this amber mutation also reduces insertion into hot spot I. These results suggest that the ability of Tn5 to insert into its major hot spot in pBR322 depends directly on negative supercoiling of the target DNA.

Development of positive selectable markers for the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in approximately 10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

Formation of supercoiling domains in plasmid pBR322.

Twin domains of positive and negative supercoiling are thought to form in DNA molecules whenever free rotation of a transcription complex around the DNA helix is impeded. Evidence for these domains has come from findings with Escherichia coli strains that are deficient in DNA topoisomerase I (top mutants) or that have been treated with DNA gyrase inhibitors. Plasmid pBR322 is highly supercoiled in these strains, whereas some of its deletion derivatives are not. The studies of pBR322 derivatives presented here show that high negative supercoiling in top strains requires translation as well as transcription of the first 98 codons of the tet gene and does not require the divergently transcribed amp gene. The N-terminal region of the TetA protein is thought to insert into the inner membrane. Our results favor models in which supercoiling domains are created when DNA segments are anchored to a large cellular structure via coupled transcription, translation, and membrane insertion of a nascent protein.

Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein.

Exogenous application of pokeweed antiviral protein (PAP), a ribosome-inhibiting protein found in the cell walls of Phytolacca americana (pokeweed), protects heterologous plants from viral infection. A cDNA clone for PAP was isolated and introduced into tobacco and potato plants by transformation with Agrobacterium tumefaciens. Transgenic plants that expressed either PAP or a double mutant derivative of PAP showed resistance to infection by different viruses. Resistance was effective against both mechanical and aphid transmission. Analysis of the vacuum infiltrate of leaves expressing PAP showed that it is enriched in the intercellular fluid. Analysis of resistance in transgenic plants suggests that PAP confers viral resistance by inhibiting an early event in infection. Previous methods for creating virus-resistant plants have been specific for a particular virus or closely related viruses. To protect plants against more than one virus, multiple genes must be introduced and expressed in a single transgenic line. Expression of PAP in transgenic plants offers the possibility of developing resistance to a broad spectrum of plant viruses by expression of a single gene.

Aldehydes from metal ion- and lipoxygenase-induced lipid peroxidation: detection by 1H-n.m.r. spectroscopy.

The modification of lipoproteins by reactive aldehydes formed via lipid peroxidation is thought to be a key process in the pathogenesis of atherosclerosis. We show that 1H-n.m.r. spectroscopy can readily be used to detect a variety of different aldehydes resulting from peroxidation of liposomes induced by Fenton's reagent or lipoxygenase, and aldehydes arising from copper-induced reactions of low-density lipoprotein. There is a clear contrast between the major aldehydic products arising from metal-ion- and lipoxygenase-induced reactions.

A rapid method for the extraction and determination of vitamin E metabolites in human urine.

A method for the direct extraction and routine analysis of the vitamin E metabolites gamma- and alpha-carboxyethyl hydroxychroman (gamma- and alpha-CEHC) from human urine has been developed. A relatively small sample volume (5 ml) can be used and, after enzymatic hydrolysis of the conjugated forms and acidification, the metabolites are extracted with diethyl ether. Recovery of alpha- and gamma-CEHC was compared to that of trolox, used as an internal standard, added to 24-h urine collections from vitamin E-unsupplemented volunteers. Various solvent conditions were initially tested; acidification and ether extraction gave the highest recovery. It was found that after addition and extraction from urine, trolox, alpha- and gamma-CEHC are recovered to a similar extent, hence trolox is viable as an internal standard. The samples were analyzed by both GC and HPLC with electrochemical detection (ECD). HPLC-ECD was found to give higher selectivity and higher sensitivity compared to GC or HPLC with UV detection at 290 nm. The HPLC-ECD detection limit was 10 fmol, linearity (r(2) > 0.98) was achieved in the range of 40 to 200 fmol, which was found to be optimal for 24-h urines from unsupplemented subjects. Inter-sample variability was typically 2-5%. This greater sensitivity and selectivity means that vitamin E metabolites can be analyzed even in unsupplemented subjects. It is also possible to measure unconjugated forms of the metabolites. Typically these were found to represent approximately 10% of the total alpha- and gamma-CEHC. This method can be used routinely for the determination of vitamin E metabolites in urine. The new extraction and detection methods described are relatively quick, less laborious, and more cost-effective than previously available methods.

The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection.

Transgenic plants expressing the coat protein (CP) of alfalfa mosaic virus (AIMV) are resistant to infection by AIMV. A mutation was introduced into the second amino acid of the cDNA for the CP of AIMV. Three different transgenic tobacco lines expressing the mutant CP and two different transgenic tobacco lines expressing the wild-type CP at similar levels were challenged with AIMV virions and viral RNA. Whereas the lines expressing the wild-type CP were highly resistant to infection by AIMV virions and viral RNA, the lines expressing the mutant CP were susceptible to infection by both. The binding affinity of the mutant and the wild-type CPs for the 3' terminal protein binding site on AIMV RNAs was similar, as determined by electrophoretic mobility shift assay. A mixture of AIMV genomic RNAs 1-3 was infectious on the plants expressing the mutant CP but not on vector control plants or plants expressing the wild-type CP, indicating that the mutant CP can activate the AIMV genomic RNAs for infection. These results demonstrate that the second amino acid of the AIMV CP is critical for protection from AIMV but not for the initial interaction between the AIMV RNA and CP, suggesting that this initial interaction does not play a major role in CP-mediated protection.