Seasonal variation in food allergy to apple

The aim of the study was to investigate the possibility of a seasonal variation in reactivity to apples in 27 birch pollen allergic patients. Before and during the birch pollen season 1998, the patients were subjected to double-blind, placebo-controlled food challenges (DBPCFCs) with grated fresh Golden Delicious apple followed by an open food challenge with whole fresh apple. The clinical reactions elicited during the challenges were evaluated both by the patients and the investigators. Moreover, the skin reactivity and the in vitro reactivity to apple were evaluated by skin prick test (SPT), leukocyte histamine release (HR), measurement of specific IgE, and immunoblotting experiments. The sensitivity of the DBPCFC, when compared with the result of the open challenge, was 0.74 (14/19) before the season and 0.80 (16/20) during the season. None of the patients reacted to the blinded challenge without a subsequent reaction to the open challenge. One placebo reaction was registered both before and in season, but not in the same patient. The patient scores of the first positive challenges, and the maximal scores of each combined blinded and open challenge session, were significantly increased during the pollen season (P<0.05). The scores of the open challenge were significantly higher than the scores of the DBPCFC both before the season and during the in-season challenges (P<0.05). Specific IgE against Golden Delicious increased during season (P<0.05), while neither SPT, HR, nor immunoblotting experiments could confirm an increase in reactivity. In conclusion, the results of the oral challenge tests indicated an increase in clinical reactivity to apples during the birch pollen season in birch pollen allergic individuals.     

Purification of an extracellular cellulose-binding endoglucanase of Cellulomonas sp. ATCC 21399 by affinity chromatography on H3PO4-swollen cellulose

A cellulose-binding endoglucanase (endoglucanase A) of Cellulomonas sp. ATCC 21399 was purified to immunological homogeneity by affinity chromatography ob H(3)PO(4)-swollen cellulose. This method of purification turned out to be an easy and very gentle method for obtaining a high yield of cellulose-binding endoglucanase. The purified enzyme was immunologically homogeneous but appeared heterogeneous when analyzed by denaturing polyacrylamide gel electrophoresis. In addition to the cellulose-binding of endoglucanase A, the enzyme also had a strong affinity for Concanavaline A, indicating that the enzyme was glycosylated. Purified endoglucanase A showed an endo mode of action on carboxymethylcellulose. The enzyme could hydrolyze microcrystalline cellulose when acting alone, and the enzyme had a high specific activity on H(3)PO(4)-swollen cellulose.     

Regeneration of plants from hypocotyl protoplasts of rapessed (Brassica napus L. var. oleifera) cultivars

Protoplast cultures were prepared from hypocotyls of ten spring rapeseed cultivars. Protoplasts from all genotypes tested formed calli, and shoots were regenerated from calli of nine of the genotypes at frequencies varying from 15 to 76%. The regenerating cultivars fell into a high regenerating group (>60% and a low regenerating group <25%).     

Purification and properties of Saccharomyces cerevisiae acetolactate synthase from recombinant Escherichia coli

The yeast ilv2 gene, encoding acetolactate synthase, was subcloned in an Escherichia coli expression vector. Although a major part of the acetolactate synthase synthesized by E. coli cells harbouring this vector was packaged into protein inclusion bodies, we used these recombinant E. coli cells to produce large quantities of the yeast enzyme. The yeast acetolactate synthase was purified to homogeneity using first streptomycin and ammonium sulfate precipitations, followed by T-gel thiophilic interaction, Sephacryl S-300 gel filtration, Mono Q anion exchange, and Superose 12 gel filtration chromatography. SDS/PAGE and gel filtration of the purified enzyme showed that it is a dimer composed of two subunits, each with the molecular mass of 75 kDa. The purified yeast acetolactate synthase was further characterized with respect to pH optimum, dependence of the substrate, pyruvate, and requirements of the cofactors, thiamin diphosphate, Mg2+, and FAD.     

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.     

Characterization and immunohistochemical localization of rat salivary cobalamin-binding protein and comparison with human salivary haptocorrin

Rat saliva contains a cobalamin-binding protein that binds cobalamin as well as cobinamide. The protein binds cobalamin with an affinity constant of 8 X 10(10) l X mol-1, and it binds cobalamin over a more narrow pH range (pH 7.5-10) than does human haptocorrin. It has a Stokes radius of 2.45 nm as compared to the Stokes radius of 4.50 nm for human haptocorrin. Upon isoelectricfocusing it dissociates into four strong bands with pI between 7 and 8, while human haptocorrin dissociates into acid isoproteins. Since human haptocorrin binds to concanavalin A while rat haptocorrin does not, we suggest that rat haptocorrin lacks carbohydrate. The substance concentration of rat saliva haptocorrin is 0.04-12.9 nmol X l-1 (median 7.5 nmol X l-1, n = 9) for control animals. After stimulation with isoproterenol, a beta-adrenergic agent, the substance concentration is 46.4-96.6 nmol X l-1 (median 69.7 nmol X l-1, n = 8). Immunohistochemical studies show haptocorrin in the secretory acini of the submandibular and parotid glands of the rat. In the human submandibular gland, the protein is detected both in the mucous secretory acini and in the intercalated ducts.     

Induction of pregnancy-associated murine protein-1 in dwarf mice by human growth hormone

Pregnancy-associated murine protein-1 (PAMP-1) could not be detected in peripheral blood of female dwarf mice (genotype dw/dw of the DW strain). By contrast the normal size females of the DW strain (genotypes +/+ and +/dw) had PAMP-1 serum levels of 18.9 AU +/- 15.7 AU/ml. Following administration of biosynthetic human growth hormone (hGH) every 2 h for 52 h PAMP-1 was detected in all dwarf females at concentrations of 16.0 AU +/- 3.3 AU/ml. The albumin levels in the circulation of DW females of normal size were significantly higher (P less than 0.05) than those of DW dwarfs, and the hGH administration did not change the serum albumin levels. The present experiment adds weight to the suggestion that the PAMP-1 serum level is regulated by GH.     

Three-dimensional structure in solution of barwin, a protein from barley seed.

The solution structure of a 125-residue basic protein, barwin, has been determined using 1H nuclear magnetic resonance spectroscopy. This protein is closely related to domains in proteins encoded by wound-induced genes in plants. Analysis of the 1H nuclear Overhauser spectrum revealed the assignment of more than 1400 nuclear Overhauser effects. Twenty structures were calculated based on 676 nontrivial distance restraints, 152 torsion angle restraints (92 phi, 56 chi 1, and 4 omega for proline), and stereospecific assignments of 38 chiral centers, using distance geometry, simulated annealing, and restrained energy minimization. None of the distance restraints was violated by more than 0.5 A in any of the 20 structures, and none of the torsion angle restraints was violated by more than 1 degree in any of the structures. The RMS difference between the calculated and target interproton distance restraints is 0.033 A, and the average atomic RMS differences between the 20 structures and their geometric average are 1.23 A for backbone atoms and 1.73 A for all heavy atoms. The dominating structural feature of the protein is a well-defined four-stranded antiparallel beta-sheet, two parallel beta-sheets packed antiparallel to each other and four short alpha-helices. The binding site of barwin to the tetramer N-acetylglucosamine has been qualitatively investigated, and the dissociation constant of the complex has been determined using one-dimensional 1H nuclear magnetic resonance spectroscopy.