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Ion transport across model lipid membranes containing light-harvesting complex II: an effect of light.

The effect of light on proton transport across lipid membranes of small unilamellar liposomes containing incorporated major light-harvesting pigment-protein complex of Photosystem II (LHCII) has been studied with the application of pH-sensitive dyes entrapped inside vesicles. Proton permeability coefficient for LHCII-modified membranes was found to be about twice as high as in the case of the control pure lipid vesicles. Illumination of the samples with light absorbed by the LHCII-bound photosynthetic pigments considerably affects the kinetics of proton transport: it increases the rate and decreases the steady-state level of proton gradient across the membranes. The effect was interpreted in terms of heat-induced conformational changes of LHCII molecular structures that affect proton buffering capacity of this protein. Both the control and the LHCII-modified lipid membranes have been found to be practically impermeable to Ca(++) ions, as demonstrated by fluorescence of liposome-entrapped calcium-sensitive probe calcium crimson. The slight differences in the proton transport across the LHCII-containing membranes under the presence of Ca(++) suggest calcium binding to this antenna protein.

Animals↗

In vitro pathogenicity assay for the ergot fungus Claviceps purpurea.

The pathogenic development of the biotrophic ergot fungus Claviceps purpurea is strictly limited to the ovary of grasses. Early colonization stages occur within a defined spatio-temporal course of events, including the directed growth to the vascular tissue for nutrient supply. To characterize mutant strains with putative defects in pathogenicity, the close observation of the infection pathway is therefore indispensable. Here, we describe the establishment of a new pathogenicity assay, based on the in vitro cultivation of isolated rye ovaries. The pathogenic development of a wild-type strain of C. purpurea was compared with the infection of mature rye flowers on whole plants. Up to the sixth day post inoculation, the route of infection within the isolated ovaries was maintained and temporally equal to that seen in mature flowers. Therefore, the in vitro pathogenicity assay is an effective alternative to the whole-plant infection tests, and suitable for detailed infection studies and screening high numbers of mutants for defects in early pathogenesis.

Aniline Compounds↗

Pygeum africanum for the treatment of patients with benign prostatic hyperplasia: a systematic review and quantitative meta-analysis.

PURPOSE: To conduct a systematic review and quantitative meta-analysis of the therapeutic efficacy and tolerability of Pygeum africanum in men with symptomatic benign prostatic hyperplasia. METHODS: Studies were identified through the search of Medline (1966 to 2000), Embase, Phytodok, the Cochrane Library, bibliographies of identified trials and review articles, and contact with relevant authors and drug companies. Randomized trials were included if participants had symptomatic benign prostatic hyperplasia, the intervention was a preparation of P. africanum alone or in combination with other phytotherapeutic agents, a control group received placebo or other pharmacologic therapies for benign prostatic hyperplasia, and treatment duration was at least 30 days. Two investigators independently extracted key data on design features, subject characteristics, and therapy allocation. RESULTS: A total of 18 randomized controlled trials involving 1,562 men met the inclusion criteria and were analyzed. Many studies did not report results in a method that permitted meta-analysis. Only 1 of the studies reported a method of treatment allocation concealment, although 17 were double-blinded. The mean study duration was 64 days (range 30 to 122). Compared with placebo in 6 studies, P. africanum provided a moderately large improvement in the combined outcome of urologic symptoms and flow measures as assessed by an effect size defined by the difference of the mean change for each outcome divided by the pooled standard deviation for each outcome (-0.8 SD [95% confidence interval (CI): -1.4 to -0.3]). Summary estimates of individual outcomes were also improved by P. africanum. Men were more than twice as likely to report an improvement in overall symptoms (risk ratio = 2.1, 95% CI: 1.40 to 3.1). Nocturia was reduced by 19% and residual urine volume by 24%; peak urine flow was increased by 23%. Adverse effects due to P. africanum were mild and similar to placebo. The overall dropout rate was 12% and was similar for P. africanum (13%), placebo (11%), and other controls (8%; P = 0.4 versus placebo and P = 0.5 versus other controls). CONCLUSIONS: The literature on P. africanum for the treatment of benign prostatic hyperplasia is limited by the short duration of studies and the variability in study design, the use of phytotherapeutic preparations, and the types of reported outcomes. However, the evidence suggests that P. africanum modestly, but significantly, improves urologic symptoms and flow measures. Further research is needed using standardized preparations of P. africanum to determine its long-term effectiveness and ability to prevent complications associated with benign prostatic hyperplasia.

Fatty Alcohols↗

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures.

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H(II)) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H(II) phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H(II) phase during dehydration. The addition of CER to DOPE:DOPC:beta-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L(alpha) --> H(II) phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC:beta-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC:beta-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H(II) phase in fully hydrated mixtures and examining the influence of CER on the formation of the H(II) phase in DOPE:DOPC mixtures lacking beta-sitosterol suggested that CER facilitated the L(alpha) --> H(II) phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L(alpha) --> H(II) phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Avena↗

Light-harvesting complex II in monocomponent and mixed lipid-protein monolayers.

Monomolecular layers at the air-water interface were formed directly with isolated largest light-harvesting pigment-protein complex of Photosystem II (LHC II) or out of egg yolk lecithin (EYL) liposomes containing incorporated LHC II. Pure protein monolayers showed a mean area of 1400 A2 per molecule at the air-water interface. Monolayers were deposited onto glass slides by means of Langmuir-Blodgett (LB) technique. Chlorophyll fluorescence of LHC II-LB and EYL-LHC II-LB films proved energetic coupling of chlorophyll a and b, thus indicating native conformation of LHC II within the monolayers. Scanning force microscopy (SFM) revealed ring-like structures formed in monocomponent protein layers as well as in mixed protein-lipid films. These results suggest that a structural arrangement of LHC II is favoured in a lipid environment but that the protein has itself a strong tendency for structural complex rearrangement in our system.

Chlorophyll↗

Optical imaging of cell membrane potential changes induced by applied electric fields.

We report the first imaging of the spatial distributions of transmembrane potential changes induced in nonexcitable cells by applied external electric fields. These changes are indicated by the fluorescence intensity of a charge-shift potentiometric dye incorporated in the cell plasma membrane and measured by digital intensified video microscopy.

Basidiomycota↗

Transport of lucifer yellow CH into plant vacuoles--evidence for direct energization of a sulphonated substance and implications for the design of new molecular probes.

Contrasting observations exist which indicate that in plants the fluorescent dye lucifer yellow CH (LYCH) either can be used as a tracer for endocytosis or as a substrate for an anion transporter located at the vacuolar membrane. In addition, LYCH as a disulphonated substance may represent an analogue of sulphonated or sulfated natural compounds like some flavonoids. We performed uptake experiments with LYCH into isolated rye vacuoles and observed saturable (Km = 0.3-0.6 mM) vacuolar transport and accumulation of the dye against the concentration gradient only when MgATP was present. GTP and, to a low extent, UTP could substitute for ATP, while the non-hydrolysable ATP analogue AMP-PNP did not drive LYCH uptake. Vanadate and probenecid, the latter substance is known to inhibit organic anion transport at the liver canalicular membrane, both strongly decreased the vacuolar uptake of LYCH, while bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, had no effect. Together with the fact that abolishment of the delta pH via CCCP had only a weak influence on LYCH accumulation, our results indicate that this compound is taken up into rye vacuoles by a directly energized process. Uptake of LYCH was strongly inhibited by other sulfated compounds including sulfobromophthalein and the flavones apigenin 7,4'-disulfate and luteolin 7,4'-disulfate arguing for the presence of a vacuolar transporter for structurally different sulphonated or sulfated compounds. Glucuronates like the rye-specific flavone luteolin 7-O-diglucuronide also strongly decreased uptake of the dye, whereas only a weak effect was observed in the presence of glutathione and a glutathione conjugate, suggesting that LYCH uptake is not mediated via the vacuolar glutathione conjugate pump.

ATP-Binding Cassette Transporters↗

Temperature/light dependent development of selective resistance to photoinhibition of photosystem I.

Exposure of winter rye leaves grown at 20 degrees C and an irradiance of either 50 or 250 micromol m(-2) s(-1) to high light stress (1600 micromol m(-2) s(-1), 4 h) at 5 degrees C resulted in photoinhibition of PSI measured in vivo as a 34% and 31% decrease in deltaA820/A820 (P700+). The same effect was registered in plants grown at 5 degrees C and 50 micromol m(-2) s(-1). This was accompanied by a parallel degradation of the PsaA/PsaB heterodimer, increase of the intersystem e- pool size as well as inhibition of PSII photochemistry measured as Fv/Fm. Surprisingly, plants acclimated to high light (800 micromol m(-2) s(-1)) or to 5 degrees C and moderate light (250 micromol m(-2) s(-1)) were fully resistant to photoinhibition of PSI and did not exhibit any measurable changes at the level of PSI heterodimer abundance and intersystem e- pool size, although PSII photochemistry was reduced to 66% and 64% respectively. Thus, we show for the first time that PSI, unlike PSII, becomes completely resistant to photoinhibition when plants are acclimated to either 20 degrees C/800 micromol m(-2) s(-1) or 5 degrees C/250 micromol m(-2) s(-1) as a response to growth at elevated excitation pressure. The role of temperature/light dependent acclimation in the induction of selective tolerance to PSI photoinactivation is discussed.

Chloroplasts↗

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis.

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3-100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.

Antibodies, Monoclonal↗

Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.

BACKGROUND AND AIMS: Celiac disease is caused by T-cell responses to wheat gluten-derived peptides. The presence of such peptides in other widely consumed grains, however, has hardly been studied. METHODS: We have performed homology searches to identify regions with sequence similarity to T-cell stimulatory gluten peptides in the available gluten sequences: the hordeins of barley, secalins of rye, and avenins of oats. The identified peptides were tested for T-cell stimulatory properties. RESULTS: With 1 exception, no identical matches with T-cell stimulatory gluten peptides were found in the other grains. However, less stringent searches identified 11 homologous sequences in hordeins, secalins, and avenins located in regions similar to those in the original gluten proteins. Seven of these 11 peptides were recognized by gluten-specific T-cell lines and/or clones from patients with celiac disease. Comparison of T-cell stimulatory sequences with homologous but non-T-cell stimulatory sequences indicated key amino acids that on substitution either completely or partially abrogated the T-cell stimulatory activity of the gluten peptides. Finally, we show that single nucleotide substitutions in gluten genes will suffice to induce these effects. CONCLUSIONS: These results show that the disease-inducing properties of barley and rye can in part be explained by T-cell cross-reactivity against gluten-, secalin-, and hordein-derived peptides. Moreover, the results provide a first step toward a rational strategy for gluten detoxification via targeted mutagenesis at the genetic level.

Alkaloids↗

Selective identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of different types of gluten in foods made with cereal mixtures.

The gluten toxic fractions responsible for the mucosal damage in coeliac disease (CD), so-called gliadins, hordeins, secalins and avenins from a large number (30-40) of wheat, barley, rye and oats cultivars respectively, have been mass analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Gliadin, secalin and avenin characteristic mass profiles are nearly identical amongst distinct cultivars from the corresponding cereal, while hordeins profiles show more variability depending on the particular barley cultivar. On the basis of these four distinguishable characteristic mass patterns spreading within the 20,000-40,000 Da range, MALDI-TOF-MS has permitted the direct and simultaneous visualization of gliadins, hordeins, secalins and avenins in foods elaborated with cereal mixtures of wheat, barley, rye and oats. This capacity has been demonstrated by mass analyzing foods made with these four cereals in varying ratios. Thus MALDI-TOF-MS can be preliminarily established as a unique system with the ability to discriminate the specific type of gluten toxic fractions present in food samples.

Avena↗

The ABC-like vacuolar transporter for rye mesophyll flavone glucuronides is not species-specific.

In many cases, the vacuolar uptake of secondary metabolites has been demonstrated to be strictly specific for a given compound and plant species. While most plants contain glycosylated secondary substances, few cases are known where flavonoids may also carry negative charges, e.g. as glucuronide conjugates. Vacuolar transport of glucosylated phenylpropanoid derivatives has been shown to occur by proton substrate antiport mechanisms (Klein, M., Weissenböck. G., Dufaud, A., Gaillard, C., Kreuz, K., Martinoia, E., 1996. Different energization mechanisms drive the vacuolar uptake of a flavonoid glucoside and a herbicide glucoside. J. Biol. Chem. 271, 29,666-29,671). In contrast, flavone glucuronides appearing specifically in rye mesophyll vacuoles are taken up by direct energisation utilising MgATP, strongly arguing for the presence of an ATP-binding cassette (ABC) transporter belonging to the subfamily of multidrug resistance-associated proteins (MRP) on the rye vacuolar membrane (Klein, M., Martinoia, E., Hoffmann-Thoma, G., Weissenböck, G., 2000. A membrane-potential dependent, ubiquitous ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides regulation of glucturonide uptake by glutathione and its conjugates. Plant Journal 21, 289-304). MRPs are known to transport negatively charged organic anions. Results presented here suggest that the vacuolar directly energised MRP-like glucuronate pump for plant-specific flavone glucuronides is ubiquitously present in diverse plant species since rye flavone glucuronides are taken up into vacuoles isolated from the barley mesophyll or from the broccoli stalk parenchyma representing two species which do not synthesise glucuronidated secondary compounds. According to the transport characteristics and inhibition profile observed we propose the existence of a high-capacity, uncoupler-insensitive vacuolar ABC transporter for flavone glucuronides and possibly other negatively charged organic compounds -- plant-born or xenobiotic -- irrespective of the plant's capability to endogenously produce glucuronidated compounds.

ATP-Binding Cassette Transporters↗

Cytochrome P450 monooxygenases of DIBOA biosynthesis: specificity and conservation among grasses.

DIBOA and DIMBOA are secondary metabolites of grasses which function as natural pesticides. The four maize genes BX2 through BX5 encode cytochrome P450-dependent monooxygenases that catalyse four consecutive reactions in the biosynthesis of these secondary products. Although BX2-BX5 share significant sequence homology, the four enzymes have evolved into specific enzymes each catalysing predominantly only one reaction in the pathway. In addition to these natural reactions, BX3 hydroxylates 1,4-benzoxazin-3-one and BX2 shows pCMA demethylase activity. With respect to DIBOA biosynthesis, identical enzymatic reactions have been found in rye as compared to maize, indicating early evolution of the P450 enzymes in the grasses.

Benzoxazines↗