Epitope specificity determines cross-protection of a SIT-induced IgG4 antibody.

BACKGROUND: The calcium-binding 2EF-hand protein Phl p 7 from timothy grass pollen is a highly cross-reactive pollen pan-allergen that can induce severe clinical symptoms in allergic patients. Recently, a human monoclonal Phl p 7-specific IgG4 antibody (mAb102.1F10) was isolated from a patient who had received grass pollen-specific immunotherapy (SIT). METHODS: We studied epitope specificity, cross-reactivity, affinity and cross-protection of mAb102.1F10 towards homologous calcium-binding pollen allergens. Sequence comparisons and molecular modelling studies were performed with ClustalW and SPADE, respectively. Surface plasmon resonance measurements were made with purified recombinant allergens. Binding and cross-reactivity of patients' IgE and mAb102.1F10 to calcium-binding allergens and peptides thereof were studied with quantitative RAST-based methods, in ELISA, basophil activation and IgE-facilitated allergen presentation experiments. RESULTS: Allergens from timothy grass (Phl p 7), alder (Aln g 4), birch (Bet v 4), turnip rape (Bra r 1), lamb's quarter (Che a 3) and olive (Ole e 3, Ole e 8) showed high sequence similarity and cross-reacted with allergic patients' IgE. mAb102.1F10 bound the C-terminal portion of Phl p 7 in a calcium-dependent manner. It cross-reacted with high affinity with Ole e 3, whereas binding and affinity to the other allergens were low. mAb102.1F10 showed limited cross-inhibition of patients' IgE binding and basophil activation. Sequence comparison and surface exposure calculations identified three amino acids likely to be responsible for limited cross-reactivity. CONCLUSIONS: Our results demonstrate that a small number of amino acid differences among cross-reactive allergens can reduce the affinity of binding by a SIT-induced IgG and thus limit cross-protection.

Carrier-bound nonallergenic Der p 2 peptides induce IgG antibodies blocking allergen-induced basophil activation in allergic patients.

BACKGROUND: More than 90% of house dust mite-allergic patients are sensitized to the major Dermatophagoides pteronyssinus allergen, Der p 2. The aim of this study was to develop and characterize an allergy vaccine based on carrier-bound Der p 2 peptides, which should allow reducing IgE- and T-cell-mediated side-effects during specific immunotherapy (SIT). METHODS: Five Der p 2 peptides (P1-P5) were synthesized and analyzed regarding IgE reactivity and allergenic activity. Lymphoproliferative and cytokine responses induced with Der p 2 and Der p 2 peptides were determined in peripheral blood mononuclear cells from mite-allergic patients. Der p 2-specific IgG antibodies induced with carrier-bound Der p 2 peptides in mice and rabbits were tested for their capacity to inhibit IgE binding and basophil activation in allergic patients. RESULTS: Of five overlapping peptides (P1-P5) covering the Der p 2 sequence, two peptides (P2 and P4) were identified, which showed no relevant IgE reactivity, allergenic activity, and induced lower Der p 2-specific T-cell activation than Der p 2. However, when coupled to a carrier, P2 and P4 induced Der p 2-specific IgG antibodies in animals, which inhibited allergic patients' IgE binding to the allergen and allergen-induced basophil activation similar as antibodies induced with Der p 2. CONCLUSIONS: Carrier-bound Der p 2 peptides should allow avoiding IgE-mediated side-effects, and because of their low potential to activate allergen-specific T cells, they may reduce late-phase side-effects during SIT. Further, these peptides may be also useful for prophylactic vaccination.

Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5.

BACKGROUND: Allergen recognition by IgE antibodies is a key event in allergic inflammation. OBJECTIVE: To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5. METHODS: In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5. RESULTS: We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5. CONCLUSION AND CLINICAL RELEVANCE: Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

Performance of multidetector computed tomography colonography compared with conventional colonoscopy.

BACKGROUND AND AIMS: This was a prospective blinded study to compare computed tomography (CT) colonography, performed with multidetector arrays CT scan (MDCT), with conventional colonoscopy for the detection of colorectal neoplasia. METHODS: Fifty patients were examined by MDCT after standard bowel preparation and rectal air insufflation in the supine and prone positions. Data sets were examined by one radiologist and one gastroenterologist blinded to the patient's history and colonoscopy results. Patients subsequently underwent colonoscopy on the same day, which served as the gold standard. RESULTS: Nine of 11 lesions >10 mm (82%), 5/15 lesions of 6-9 mm (33%), and 1/42 polyps <5 mm (3%) were detected by MDCT colonography. One false positive result for a structure larger than 10 mm was described. Nineteen of 21 patients who had no lesions during conventional colonoscopy were considered free of lesions by MDCT colonography, yielding a per patient specificity of 90%. CONCLUSION: MDCT colonography provides good data quality and has good sensitivity and specificity for the detection of colonic lesions of 10 mm or more.

Eating disorders: a role for dipeptidyl peptidase IV in nutritional control.

Dipeptidyl peptidase IV (DPP IV), a serine protease with broad tissue distribution and known activity in serum, has been postulated to modulate nutrition control by modification or inactivation of peptide hormones operating in the enteroinsular axis. We hypothesized that changes of DPP IV activity in serum are related to the nutrition status of patients with eating disorders. Serum DPP IV activity was measured in 52 patients (28 with anorexia nervosa and 24 with bulimia nervosa) in four consecutive weekly analyses. Simultaneously, the number of CD26 (DPP IV)-positive peripheral blood lymphocytes was counted. The same analyses were carried out in 28 healthy female volunteers. In week 1 and throughout the observation period, DPP IV activity in the sera of patients with anorexia nervosa and, to a lesser extent, those with bulimia nervosa was elevated in comparison to that of healthy controls (week 1: means = 92.8 U/L for anorexia-nervosa patients and 89.3 U/L for bulimia-nervosa patients versus 74.7 U/L for healthy control subjects, P = 0.014; weeks 1-4: 91.8 U/L for anorexia-nervosa patients and 86.2 U/L for bulimia-nervosa patients versus 77.6 U/L for healthy controls, P < 0.001). We assume that the increase in DPP IV serum activity will increase the turnover of distinct peptide hormones with known effects on nutrition control and susceptibility to degradation by DPP IV. The potential impact of an increase in DPP IV activity in serum on satiety and nutrition control contributes to previously reported implications for immune function.

RNA editing by base deamination: more enzymes, more targets, new mysteries.

The posttranscriptional modification of messenger RNA precursors (pre-mRNAs) by base deamination can profoundly alter the physiological function of the encoded proteins. The recent identification of tRNA-specific adenosine deaminases (ADATs) has led to the suggestion that these enzymes, as well as the cytidine and adenosine deaminases acting on pre-mRNAs (CDARs and ADARs), belong to a superfamily of RNA-dependent deaminases. This superfamily might have evolved from an ancient cytidine deaminase. This article reviews the reactions catalysed by these enzymes and discusses their evolutionary relationships.

[The effects of psychotherapy on Crohn's disease patients--results of a randomized multicenter study]. / Die Wirksamkeit psychotherapeutischer Massnahmen bei Morbus Crohn. Ergebnisse einer randomisierten, multizentrischen Studie.

In a prospective multicenter study of Crohn disease patients, the influence of psychotherapy on the course of the disease and on psychosocial variables (anxiety, depression, life satisfaction and data of the PSKB) was studied. Psychodynamic oriented psychotherapy was provided in addition to a standardized medical treatment and took place during the first year of the two-year observation period. 108 of 488 patients were recruited and randomly assigned to the psychotherapy and the control group. 84 patients completed the somatic and 81 the psychosocial follow up. 23% of the control group and 30% of the psychotherapy group showed episode-free courses, 29% and 17% respectively underwent surgery (worst outcome group). The ranking and comparisons of the disease course showed no significant difference (p = 0.125) between psychotherapy and control group. The psychosocial variables also showed no differences between these groups. Subjectively, the patients report favourable effects to psychotherapy.

Crystal structure of mammalian poly(A) polymerase in complex with an analog of ATP.

In eukaryotes, polyadenylation of pre-mRNA plays an essential role in the initiation step of protein synthesis, as well as in the export and stability of mRNAs. Poly(A) polymerase, the enzyme at the heart of the polyadenylation machinery, is a template-independent RNA polymerase which specifically incorporates ATP at the 3' end of mRNA. We have solved the crystal structure of bovine poly(A) polymerase bound to an ATP analog at 2.5 A resolution. The structure revealed expected and unexpected similarities to other proteins. As expected, the catalytic domain of poly(A) polymerase shares substantial structural homology with other nucleotidyl transferases such as DNA polymerase beta and kanamycin transferase. The C-terminal domain unexpectedly folds into a compact domain reminiscent of the RNA-recognition motif fold. The three invariant aspartates of the catalytic triad ligate two of the three active site metals. One of these metals also contacts the adenine ring. Furthermore, conserved, catalytically important residues contact the nucleotide. These contacts, taken together with metal coordination of the adenine base, provide a structural basis for ATP selection by poly(A) polymerase.

Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations.

Glycinebetaine (betaine) affords osmoprotection in bacteria, plants and animals, and protects cell components against harsh conditions in vitro. This and a compelling body of other evidence have encouraged the engineering of betaine production in plants lacking it. We have installed the metabolic step for oxidation of choline, a ubiquitous substance, to betaine in three diverse species, Arabidopsis, Brassica napus, and tobacco (Nicotiana tabacum), by constitutive expression of a bacterial choline oxidase gene. The highest levels of betaine in independent transgenics were 18.6, 12.8, and 13 micromol g(-1) dry weight, respectively, values 10- to 20-fold lower than the levels found in natural betaine producers. However, choline-fed transgenic plants synthesized substantially more betaine. Increasing the choline supplementation further enhanced betaine synthesis, up to 613 micromol g(-1) dry weight in Arabidopsis, 250 micromol g(-1) dry weight in B. napus, and 80 micromol g(-1) dry weight in tobacco. These studies demonstrate the need to enhance the endogenous choline supply to support accumulation of physiologically relevant amounts of betaine. A moderate stress tolerance was noted in some but not all betaine-producing transgenic lines based on relative shoot growth. Furthermore, the responses to stresses such as salinity, drought, and freezing were variable among the three species.

Position-dependent inhibition of the cleavage step of pre-mRNA 3'-end processing by U1 snRNP.

The 3' ends of most eukaryotic pre-mRNAs are generated by 3' endonucleolytic cleavage and subsequent polyadenylation. 3'-end formation can be influenced positively or negatively by various factors. In particular, U1 snRNP acts as an inhibitor when bound to a 5' splice site located either upstream of the 3'-end formation signals of bovine papilloma virus (BPV) late transcripts or downstream of the 3'-end processing signals in the 5' LTR of the HIV-1 provirus. Previous work showed that in BPV it is not the first step, 3' cleavage, that is affected by U1 snRNP, but rather the second step, polyadenylation, that is inhibited. Since in HIV-1 the biological requirement is to produce transcripts that read through the 5' LTR cleavage site rather than being cleaved there, this mechanism seemed unlikely to apply. The obvious difference between the two examples was the relative orientation of the 3'-end formation signals and the U1 snRNP-binding site. In vitro assays were therefore used to assess the effect of U1 snRNP bound at various locations relative to a cleavage/polyadenylation site on the 3' cleavage reaction. U1 snRNP was found to inhibit cleavage when bound to a 5' splice site downstream of the cleavage/polyadenylation site, as in the HIV-1 LTR. U1 snRNP binding at this location was shown not to affect the recruitment of multiple cleavage/polyadenylation factors to the cleavage substrate, indicating that inhibition is unlikely to be due to steric hindrance. Interactions between U1A, U1 70K, and poly(A) polymerase, which mediate the effect of U1 snRNP on polyadenylation of other pre-mRNAs, were shown not to be required for cleavage inhibition. Therefore, U1 snRNP bound to a 5' splice site can inhibit cleavage and polyadenylation in two mechanistically different ways depending on whether the 5' splice site is located upstream or downstream of the cleavage site.

The properties of a tRNA-specific adenosine deaminase from Drosophila melanogaster support an evolutionary link between pre-mRNA editing and tRNA modification.

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.

Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed.

High-erucic acid (HEA) Brassica napus cultivars are regaining interest in industrial contexts. Erucic acid and its derivatives are important renewable raw materials utilized in the manufacture of plastic films, in the synthesis of Nylon 13,13, and in the lubricant and emollient industries. Theoretically, the highest level of erucic acid that can be achieved by means of classical breeding is 66 mol%; however, using new approaches on the basis of genetic engineering, it might be possible to develop a B. napus cultivar containing levels of erucic acid significantly above 66 mol% (>80 mol%). In an attempt to increase the amounts of very-long-chain fatty acids (VLCFAs), and erucic acid in particular, in Canadian HEA B. napus cultivars, we have focused on two targets using a transgenic approach. We examined both the role/function of the Arabidopsis thaliana FAE1 (fatty acid elongase) gene by expressing it under the control of the seed-specific napin promoter in B. napus germplasm with analysis of the changes in VLCFA content in the seed oil of transgenic lines, and the performance of the yeast SLC1-1 (sphingolipid compensation mutant) in B. napus cv. Hero transgenic progeny in the field. Here, we report analyses of the contents of 22:1, total VLCFAand oil in the seed oil, as well as seed yield of the field-grown FAE1 and SLC1-1 B. napus cv. Hero progeny.

Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.

We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.

An adenosine deaminase that generates inosine at the wobble position of tRNAs.

Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis. The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I(34) in tRNAs is described. The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs. Each subunit is encoded by an essential gene (TAD2 and TAD3), indicating that I(34) is an indispensable base modification in elongating tRNAs. These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

Editing of messenger RNA precursors and of tRNAs by adenosine to inosine conversion.

The double-stranded RNA-specific adenosine deaminases ADAR1 and ADAR2 convert adenosine (A) residues to inosine (I) in messenger RNA precursors (pre-mRNA). Their main physiological substrates are pre-mRNAs encoding subunits of ionotropic glutamate receptors or serotonin receptors in the brain. ADAR1 and ADAR2 have similar sequence features, including double-stranded RNA binding domains (dsRBDs) and a deaminase domain. The tRNA-specific adenosine deaminases Tad1p and Tad2p/Tad3p modify A 37 in tRNA-Ala1 of eukaryotes and the first nucleotide of the anticodon (A 34) of several bacterial and eukaryotic tRNAs, respectively. Tad1p is related to ADAR1 and ADAR2 throughout its sequence but lacks dsRBDs. Tad1p could be the ancestor of ADAR1 and ADAR2. The deaminase domains of ADAR1, ADAR2 and Tad1p are very similar and resemble the active site domains of cytosine/cytidine deaminases.

Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP.

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.

Lipotrope deficiency inhibits cell growth and induces programmed cell death in human breast cancer cell line MCF-7.

To determine the effects of lipotrope modification on breast cancer cell growth and cell death, the human breast cancer cell line MCF-7 was assigned to grow in one of three lipotrope treatment media for four days. The treatment media included lipotrope-control medium (LCM), containing all required lipotropes; lipotrope-deficient medium (LDM), lacking all lipotropes but supplying homocysteine instead; and lipotrope-additive medium (LAM), containing twice as much of each lipotrope as LCM. Cell count and [3H]thymidine incorporation into DNA revealed that LDM slowed cell growth and inhibited cell proliferation in the MCF-7 cell line. Gel electrophoresis showed significant DNA degradation with the appearance of fragments in LDM-treated cells, whereas the DNA in LCM and LAM cells was largely intact. The LDM group displayed more apoptotic bodies as detected by in situ immunohistochemistry. The gene expression level of bcl-2 was lower in cells treated with LDM than in those treated with LCM and LAM, whereas p53 gene expression did not appear different among the three treatment groups. It is concluded that lipotrope deficiency inhibits cell growth and induces programmed cell death in the human breast cancer cell line MCF-7.

Tad1p, a yeast tRNA-specific adenosine deaminase, is related to the mammalian pre-mRNA editing enzymes ADAR1 and ADAR2.

We have identified an RNA-specific adenosine deaminase (termed Tad1p/scADAT1) from Saccharomyces cerevisiae that selectively converts adenosine at position 37 of eukaryotic tRNAAla to inosine. The activity of purified recombinant Tad1p depends on the conformation of its tRNA substrate and the enzyme was found to be inactive on all other types of RNA tested. Mutant strains in which the TAD1 gene is disrupted are viable but lack Tad1p enzyme activity and their tRNAAla is not modified at position A37. Transformation of the mutant cells with the TAD1 gene restored enzyme activity. Tad1p has significant sequence similarity with the mammalian editing enzymes which act on specific precursor-mRNAs and on long double-stranded RNA. These findings suggest an evolutionary link between pre-mRNA editing and tRNA modification.

Human pre-mRNA cleavage factor Im is related to spliceosomal SR proteins and can be reconstituted in vitro from recombinant subunits.

Four polypeptides of 25, 59, 68, and 72 kDa copurify with the activity of human cleavage factor Im (CF Im) involved in pre-mRNA 3' end processing. We report here the cloning of the 25 and 68 kDa subunits and the reconstitution of functional CF Im25/68 from these two polypeptides. Several lines of evidence indicate that CF Im exists in at least two different forms. The 68 kDa polypeptide has a domain organization reminiscent of spliceosomal SR proteins. Analysis of the kinetics of the cleavage reaction indicates that interaction of CF Im with the RNA is one of the earliest steps in the assembly of the 3' end processing complex and facilitates the recruitment of other processing factors.