The gene for trypsin inhibitor CMe is regulated in trans by the lys 3a locus in the endosperm of barley (Hordeum vulgare L.)

Summary A cDNA encoding trypsin inhibitor CMe from barley endosperm has been cloned and characterized. The longest open reading frame of the cloned cDNA codes for a typical signal peptide of 24 residues followed by a sequence which is identical to the known amino acid sequence of the inhibitor, except for an Ile/Leu substitution at position 59. Southern blot analysis of wheat-barley addition lines has shown that chromosome 3H of barley carries the gene for CMe. This protein is present at less than 2%–3% of the wild-type amount in the mature endosperm of the mutant Risø 1508 with respect to Bomi barley, from which it has been derived, and the corresponding steady state levels of the CMe mRNA are about I%. One or two copies of the CMe gene (synonym Itc1) per haploid genome have been estimated both in the wild type and in the mutant, and DNA restriction patterns are identical in both stocks, so neither a change in copy number nor a major rearrangement of the structural gene account for the markedly decreased expression. The mutation at the lys 3a locus in Risø 1508 has been previously mapped in chromosome 7 (synonym 5H). A single dose of the wild-type allele at this locus (Lys 3a) restores the expression of gene CMe (allele CMe-1) in chromosome 3H to normal levels.     

Wheat Inhibitors of Heterologous alpha-Amylases : Characterization of Major Components from the Monomeric Class

The four major components of the wheat monomeric α-amylase inhibitors (WMAI) from wheat, Triticum aestivum, endosperm have been isolated and characterized. Two of them, WMAI-1 and WMAI-2, are highly active against the α-amylase from the insect Tenebrio molitor and their N-terminal amino acid sequences indicate that they are closely related to each other (86% identical residues) and to the other members of the family (subunits of dimeric and tetrameric α-amylase inhibitors and trypsin inhibitors). WMAI-1, which is identical to the previously described 0.28 inhibitor, is encoded by a gene located in the short arm of chromosome 6D and WMAI-2 by a gene in the short arm of chromosome 6B. Components 3 and 4, which have blocked N-terminal residues, have identical internal amino acid sequences and are a separate class of proteins with respect to WMAI-1 and WMAI-2, although their amino acid composition and apparent molecular weights are quite similar. Their inhibitory activity versus α-amylases is either unstable during the purification process or due to contamination with other inhibitors.     

Preservation of Xanthomonas campestris on agar slopes: effects on xanthan production

Xanthomonas campestris NRRL B-1459 and a variant E2, when preserved on agar slopes (transferred monthly) over 11 months did not deteriorate in their ability to produce xanthan in quantity and quality, as determined by culture in 500-ml baffled flasks. Variations between 8 and 14% (with respect to the average) in the final xanthan concentration were observed for the E2 and B-1459 strains, respectively. A wide range of final viscosities was obtained; these were consistent with the changes in gum concentration. Differences were more likely associated with differences in fermentation kinetics rather than being inherent to the strains. The rheological quality of both polysacharides was relatively constant throughout the time of culture maintenance. Preservation of these bacteria on agar slopes was an adequate method, in contrast to previous reports. In the period studied, strain E2 produced higher gum titres and slightly lower gum quality compared to strain B-1459. Correspondence to: E. Galindo     

On the origin of Spanish two-rowed barleys

To investigate the phylogenetic origin of Spanish two-rowed barleys, we studied 44 accessions of old land-races both morphologically and biochemically to ascertain their similarity with 51 entries of old cultivars and land-races of widespread origin across Europe. They were also compared with 20 accessions of Hordeum spontaneum from the Mediterranean basin and other regions of its distribution range, 14 accessions of Moroccan cultivated six-rowed barley land-races, and different six-rowed Spanish and two-and six-rowed European cultivars. CM-(trypsin inhibitors and subunits of the barley tetrameric -amylase inhibitor) proteins and hordeins, all of which are endosperm proteins, were used as biochemical markers. The appearance of separate clusters of the Spanish barleys in the numerical classifications for both protein systems as a result of the existence of characteristic gene combinations that do not exist in entries from other origins permitted us to postulate the existence of local ancestors for most of the Spanish two-rowed barleys studied, and, therefore, a possible in situ domestication.     

Rye chromosome arm 3RS encodes a homodimeric inhibitor of insect α-amylase

A new inhibitor of insect -amylase, designated RDAI-1, has been purified from rye (Secale cereale L.) endosperm. RDAI-1 is homologous to wheat homodimeric inhibitors. This homology is supported by their similar N-terminal amino-acid sequences, inhibitory activities towards amylases from Tenebrio molitor (Coleoptera) and human saliva, and aggregative properties in gel-filtration chromatography. The gene encoding RDAI-1, IdhaR1, is located on the short arm of chromosome 3R, which is homoeologous with wheat chromosome arms 3BS and 3DS, where the genes for homodimeric inhibitors have been previously mapped.     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

Three inhibitor types from wheat endosperm are differentially active against α-amylases of Lepidoptera pests

Crude α-amylase preparations from seven Lepidoptera pests were susceptible to inhibition by salt-soluble proteins of bread wheat (Triticum aestivum L.) endosperm. Protein fractions that corresponded to tetrameric, dimeric, and monomeric wheat α-amylase inhibitors, were decreasingly effective against the insect α-amylase activity. To further confirm these results, purified inhibitors were tested against an α-amylase preparation fromEphestia kuehniella (Zeller). This preparation showed decreased activity when increasing amounts of an heterotetrameric inhibitor (reconstituted from its isolated subunits WTAI-CM2, -CM3 and -CM16) were assayed. Activity was only partially inhibited by homodimeric (WDAI-1, synonym 0.53; WDAI-2, synonym 0.19) and monomeric (WMAI-1, synonym 0.28) inhibitors.     

ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins.

Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2-stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.     

Effect of brain serotonin on the arterial pressure and plasma renin in normal and hypertensive rats]

The effects of changes in brain serotonin content after injections of p-chlorophenylalanine (p-CPA), L-5-hydroxytryptophan (L-5HTP) and 5-6-dihydroxytryptamine (5-6DHT) on the mean arterial pressure (MAP), plasma renin activity (PRA) and peripheral levels of atrial natriuretic peptide (ANP) have been studied in normal and hypertensive (2K:1C model) male Wistar rats. The p-CPA (250 mg/kg) and L-5HTP (200 mg/kg) were injected i.p., while 5-6 DHT (15 micrograms/animal in 10 mu/animal vehicle) was injected into lateral brain ventricles. The effects were studied 24 h after the p-CPA injection, 2 h after L-5HTP and 10 or 20 days after 5-6DHT administration. The fall in brain serotonin produced by p-CPA and 5-6DHT did not modify the MAP values in the normal and hypertensive rat model, whereas the increase induced after L-5HTP injection only caused a slight decrease in arterial pressure in normotensive animals. The ARP experimented remarkable rises in the normal and hypertensive rats, these values increasing after L-5HTP and falling after p-CPA and 5-6 DHT injections. Similar changes are detected in the normal group after administration of these substances related to serotoninergic brain activity. The ANP levels rose after renal artery constriction, and they are not affected by the above mentioned substances. Only p-CPA and 5-6DHT reduced a low decrease in the ANP levels 10 days after their administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Members of the alpha-amylase inhibitors family from wheat endosperm are major allergens associated with baker's asthma

We have identified the major antigens or IgE binding components from wheat flour. Thirty-five sera from patients with baker's asthma were used to analyze the reaction with wheat salt-soluble proteins. We found a 15 kDa SDS-PAGE band which reacted with all sera tested. Purified members of the alpha-amylase inhibitor family, which are the main components of the 15 kDa band, were recognized by specific IgE when tested with a pool of reactive sera. Immunodetection after two-dimensional electrophoretic fractionation of crude inhibitor preparations from wheat endosperms also detected several inhibitor subunits as major low-molecular-weight allergens.