Extreme variations in the ratios of non-synonymous to synonymous nucleotide substitution rates in signal peptide evolution.

Nucleotide sequences encoding signal peptides from the precursors of alpha-amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (KA) and synonymous (KS) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in KA/KS ratios has been observed; from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins, which are unrelated to those of the alpha-amylase/trypsin inhibitor family, and an average KA/KS of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints.

Barley cultivar discrimination: I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and glycoprotein blotting.

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.

Barley cultivar discrimination: II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with immobilized pH gradients.

Isoelectric focusing performed with immobilized pH gradients was found superior to other commonly used electrophoretic methods for discrimination of 55 European winter and spring barley cultivars. Hordeins, the alcohol-soluble proteins, yielded 32 different patterns, allowing identification of 22 cultivars and classification of the remaining ones into ten groups of two to eight cultivars each. Only 21 different hordein patterns were observed using horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining. Twelve cultivars exhibited unique hordein patterns, the remaining nine groups contained 2-11 cultivars. Resolution of isoelectric focusing with immobilized pH gradients was further enhanced in some cases when the patterns of urea/dithiothreitol-soluble proteins were used instead of the hordein patterns. However, evaluation was more complicated because of the larger number of protein bands detected.

Accurate measurements of coupling constants from two-dimensional nuclear magnetic resonance spectra of proteins and determination of phi-angles.

A new and simple method to measure 3JHNH alpha coupling constants of proteins by adding and subtracting traces from corresponding two-dimensional nuclear Overhauser enhanced spectroscopy and two-dimensional correlated spectroscopy cross peaks after scaling is proposed. The optimal scaling for the addition and the subtraction of the two traces is obtained by minimizing an error function. The method was proven to give accurate and precise measurements of coupling constants when tested with a series of simulated spectra. The accuracy of the method was better than 0.1 Hz for all test cases including the limiting case of J = 2.0 Hz and line-width = 11.0 Hz. The accuracy of the method was better than 0.1 Hz for all test cases including The 3JHNH alpha coupling constants were measured in two-dimensional nuclear magnetic resonance spectra of the two proteins barley serine proteinase inhibitor (CI-2) and the bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens. The experimentally measured coupling constants were used to calculate the constants in a Karplus equation to be: 3JHNH alpha = 6.7 cos2(phi-60) -1.3 cos(phi-60) +1.5. These constants are in good accordance with those obtained for basic pancreatic trypsin inhibitor (BPTI). In addition, special emphasis is given to the measurements of positive phi-angles, and to the contribution of molecular dynamics on the apparent coupling constants.

Functional reconstitution of the malate carrier of barley mesophyll vacuoles in liposomes.

The malate carrier of barley (Hordeum vulgare L.) mesophyll vacuoles was highly purified by chromatography on hydroxyapatite followed by affinity-chromatography using 5-amino-1,2,3-benzenetricarboxylic acid as ligand. The carrier, reconstituted in asolectin liposomes, had properties similar to those described previously for the carrier in intact vacuoles (Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U. and Heldt, H.W. (1985) Biochim. Biophys. Acta 806, 311-319). The apparent Km for malate uptake was 2-3 mM, and the uptake was inhibited by other carboxylic acids (preferentially tricarboxylic). The sulfhydryl reagent, p-chloromercuribenzenesulfonate, as well as the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, also inhibited malate uptake. The transport was dependent on the membrane potential with an optimum at about 35 mV.

Analysis of triallate residues in cereals and soil by gas chromatography with ion-trap detection.

Triallate residues in barley seedlings and soil samples were determined by gas chromatography with ion-trap detection. Soil was extracted with methanol on a mechanical shaker, and plants were extracted with acetonitrile in a Sorvall homogenizer. After evaporation of the organic solvents, the residue was dissolved in hexane, and plants extracts were cleaned-up on an alumina column. Gas chromatographic analysis was carried out using a BP-1 fused-silica capillary column with helium as carrier gas. To quantitate residues the total-ion chromatogram was obtained and then the selected-ion monitoring chromatograms were displayed at m/z 86 for triallate and at m/z 154 for the internal standard, methyl-(4-amino-2-chloro)-benzoate. The average recovery through the method from barley and soil samples was always higher than 80%. The limit of detection in the selected-ion mode was 0.01 mg/kg. Barley and soil samples treated with triallate were also analysed. A good agreement was observed between results obtained by this method and by gas chromatography with nitrogen-phosphorus detection.

The amino acid sequence of ferredoxin from Hordeum vulgare.

Ferredoxin from barley consists of a single polypeptide chain of 97 amino acids, four of which are cysteine. These residues, which bind the iron atoms of the active centre, are in identical positions to those of other ferredoxins. The primary structure shows considerable similarity with other plant ferredoxins.

Multielement determination in rice, wheat, and barley by instrumental neutron activation analysis.

INAA has been used for the determination of Na, Mg, Al, Cl, K, Sc, Cr, Mn, Fe, Co, Cu, Zn, As, Se, Br, Rb, Sr, Mo, and W in grains of rice, wheat, and barley, which were collected from different plant fields in Iraq. Samples and standards were irradiated in the IRT-5000 reactor, at neutron fluxes of 2 x 10(13) cm-2.s-1 and 3.2 x 10(11) cm-2.s-1. Interferences of photopeaks with each other were considered, and reaction interferences were calculated and determined experimentally. Accuracy of our method was assessed by the analysis of IAEA standards Wheat Flour and Bovine liver. A good agreement has been achieved between the present results and recommended values. The precision and detection limit were determined for all elements in all types of grain.

The isolation of high molecular weight DNA from wheat, barley and rye for analysis by pulse-field gel electrophoresis.

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals--hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.

Radioimmunoassay of nivalenol in barley.

Antibodies against nivalenol (NIV) tetraacetate (Tetra-Ac-NIV) were prepared by immunizing rabbits with a hemisuccinate derivative of 8-hydroxy-3,4,7,15-tetraacetyl-12, 13-epoxytrichothece-9-en conjugated to bovine serum albumin. A radioimmunoassay system with one of these sera was developed to measure NIV contamination in barley. The detection limit for Tetra-Ac-NIV was about 0.5 ng/ml. The relative cross-reactivities of the antiserum with Tetra-Ac-NIV, acetyl T-2 toxin, and scirpenol triacetate, which were determined by the competitive radioimmunoassay, were 1, 0.78, and 0.56, respectively. Other derivatives showed no cross-reactivity. For the determination of NIV in a barley sample, NIV was extracted from the sample with acetonitrile-water (7:3), defatted with hexane, and then acetylated with acetic anhydride to form Tetra-Ac-NIV. The reaction mixture was loaded onto a C18 cartridge to remove excess reagents and impurities. Tetra-Ac-NIV was eluted from the cartridge with 50% methanol in water, and the eluate was subjected to radioimmunoassay. Analysis of six naturally contaminated barley samples for NIV revealed that radioimmunoassay results agreed well with gas chromatographic analyses.

Production and characterization of monoclonal antibodies to nivalenol tetraacetate and their application to enzyme-linked immunoassay of nivalenol.

Three monoclonal antibodies were obtained by the fusion of mouse myeloma cells with splenocytes isolated from BALB/c mice that had been immunized with 8-hydroxy-3,4,7,15-tetraacetyl-nivalenol hemiglutarate covalently bound to bovine serum albumin. These anti-nivalenol tetraacetate monoclonal antibodies were of the IgG type and highly specific to nivalenol tetraacetate, with an apparent association constant of about 10(8)M-1. The relative cross-reactivities of one monoclonal antibody with nivalenol tetraacetate, acetyl T-2 toxin, and scirpenol triacetate were found to be 1.0, 0.02 and 0.03, respectively. Other derivatives showed no cross-reactivity at all. An indirect enzyme-linked immunosorbent assay (ELISA) based on the competitive binding principle was developed using the antibody from clone D18.102.59. The sensitivity of the system was about 0.1 ng of nivalenol tetraacetate per assay. Comparison of nivalenol levels detected in naturally contaminated barley samples by competitive indirect ELISA and gas chromatography (GC) showed good agreement, indicating that the antibody is useful for the measurement of nivalenol in naturally contaminated cereals and grains.

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley.

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.

Histochemical localization of cysteine-rich proteins by tissue printing on nitrocellulose.

A rapid technique for the histochemical localization of cysteine-rich proteins in plant tissues was developed. It is based on the immediate transfer of proteins to nitrocellulose membranes when a fresh cut organ is pressed against the membrane surface. The print was labeled for cysteine-rich proteins by reduction and alkylation of cysteinyl residues with dansylated iodoacetamide [N-iodoacetyl-N'-(-5-sulfo-1-naphthyl)ethylenediamine]. The S-carboxymethylated proteins were visualized by their fluorescence when excited with 360 nm light.

The primary structure of a 4.0-kDa photosystem I polypeptide encoded by the chloroplast psaI gene.

Partial amino acid sequences have been determined for a 4.0-kDa photosystem I polypeptide from barley. A comparison with the sequence of the chloroplast genome of Nicotiana tabacum and Marchantia polymorpha identified the polypeptide as chloroplast-encoded. We designate the corresponding gene psaI and the polypeptide PSI-I. The barley chloroplast psaI gene was sequenced. The gene encodes a polypeptide of 36 amino acid residues with a deduced molecular mass of 4008 Da. The 4.0-kDa polypeptide is N-terminally blocked with a formyl-methionine residue. Plasma desorption mass spectrometry established that the polypeptide is not post-translationally processed except for possible conversion of a methionine residue into methionine sulfone. The hydrophobic 4.0-kDa polypeptide is predicted to have one membrane-spanning alpha-helix and is homologous to transmembrane helix E of the D2 reaction center polypeptide of photosystem II.

Correlation of some published amino acid sequences for photosystem I polypeptides to a 17 kDa LHCI pigment-protein and to subunits III and IV of the core complex.

Photosystem I (PSI) in barley consists of at least 11 polypeptides of which three have apparent sizes of 15-19 kDa. Two of these polypeptides (subunits III and IV) are constituents of the core complex (CCI), the third is a component of the light-harvesting complex (LHCI). After fractionation of PSI into its CCI and LHCI components, each of the polypeptides has been isolated and its N-terminal region sequenced. We conclude that the gene sequence published for subunit IV of spinach [(1988) FEBS Lett. 237, 108-112] is not that of subunit IV but rather that of the 17 kDA LHCIc pigment protein. We confirm that the published sequence for subunit III [(1988) Curr. Genet. 14, 511-518] is indeed that of subunit III; seemingly conflicting identifications, based on apparent sizes on SDS-PAGE, of which polypeptides are subunits III and IV are probably explained by subunit III's electrophoretic migration rate being dependent on the solvent.

Compositional and digestibility changes in sprouted barley and canola seeds.

Barley and canola seeds were sprouted over a 5 day period, in laboratory conditions under room temperature (22 degrees C) and room lighting. Following initial hydration, seeds were kept moist by wetting the germination trays at 9 a.m., 1 p.m. and 6 p.m. daily. A parallel germination experiment using 200 g quantities of seeds in petri dishes was conducted. Starting from the second day of germination, and every day, dishes of germinating seeds were removed, oven-dried, weighed and milled for proximate and chemical analysis. Seeds from the main germination experiment were fed in a digestibility trial to Wistar rats. Results indicated that sprouting was associated with depletion of many nutrients in both barley and canola, the major losses being in respect of dry matter, gross energy and triglycerides. In barley (but not in canola) sprouting was associated with significant increases in crude fiber and diglyceride content. In canola, there were significant losses in lipid content and increases in phytosterol and phospholipid content. Digestibility data showed an enhancement in digestibility of nutrients in barley but not in canola, implying that sprouting improved nutritional quality of barley but not canola.

Chemical test for mammalian feces in grain products: collaborative study.

A collaborative study was conducted to validate the use of the AOAC alkaline phosphatase method for mammalian feces in corn meal, 44.B01-44.B06, for 7 additional products: brown rice cream, oat bran, grits, semolina, pasta flour, farina, and barley plus (a mixture of barley, oat bran, and brown rice). The proposed method determines the presence of alkaline phosphatase, an enzyme contained in mammalian feces, by using phenolphthalein diphosphate as the enzyme substrate in a test agar medium. Fecal matter is separated from the grain products by specific gravity differences in 1% test agar. As the product is distributed on liquid test agar, fecal fragments float while the grain products sink. The alkaline phosphatase cleaves phosphate radicals from phenolphthalein diphosphate, generating free phenolphthalein, which produces a pink to red-purple color around the fecal particles in the previously colorless medium. Collaborators' recovery averages ranged from 21.7 particles (72.3%) for oat bran to 25.3 particles (84.3%) for semolina at the 30 particle spike level. Overall average background was 0.4 positive reactions per food type. The collaborators reported that the method was quick, simple, and easy to use. The method has been approved interim official first action for all 7 grain products.

The regional distribution of selenium in Greek cereals.

The selenium content of hard and soft wheat, barley, oats, rye and corn grown in approximately 100 different locations of Greece has been determined fluorimetrically. The mean values +/- SD for these cereal types were 0.29 +/- 0.19, 0.21 +/- 0.12, 0.16 +/- 0.10, 0.14 +/- 0.10, 0.19 +/- 0.10 and 0.12 +/- 0.08 ppm Se (dry weight basis), respectively. Based on data for selenium in corn from 96 different locations, a geobotanic map of Greece for the selenium in soil available for uptake by plants was prepared. Macedonia, West Epirus, south-east Thessaly, north-east Sterea Hellas and the Aegean Islands produce corn deficient or low in selenium, but only sporadic selenium-deficiency diseases in animals have been observed in many of these areas, probably because the farm animals are given mixed food or they are free to graze. No areas with toxic levels of selenium in soil were found.

Intracellular thionins of barley. A second group of leaf thionins closely related to but distinct from cell wall-bound thionins.

Leaf thionins of barley have been identified as a novel class of cell wall proteins, toxic to plant pathogenic fungi, and possibly involved in the defense mechanism of plants (Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V., and Apel, K., (1988) EMBO J. 7, 1559-1565). In the present work a second subfraction of thionins has been detected within the leaf cell, mainly in the vacuole. Thionins of both groups are closely related to each other. They are toxic to phytopathogenic fungi as well as to plant protoplasts, they share similar amino acid sequences, and their synthesis in etiolated seedlings of barley is down-regulated by light. Despite these similarities each of the two subfractions of thionins could be clearly distinguished by its subcellular distribution. In ultrathin sections of embedded etiolated leaf material, cell wall thionins could be immunogold labeled specifically by an antiserum raised against a fusion protein of Escherichia coli beta-galactosidase and the 15,000 Mr precursor polypeptide of thionins. This antiserum did not react with intracellular thionins. Inversely, intracellular thionins were recognized specifically by an anti-serum raised against soluble leaf thionins. The possible function of intracellular thionins as part of a defense mechanism has been discussed.