The TraA relaxase autoregulates the putative type IV secretion-like system encoded by the broad-host-range Streptococcus agalactiae plasmid pIP501.

The conjugative multiple antibiotic resistance plasmid pIP501 can be transferred and stably maintained in a variety of Gram-positive genera, including multicellular Streptomyces lividans, as well as in Gram-negative Escherichia coli. The 15 putative pIP501 transfer (tra) genes are organized in an operon-like structure terminating in a strong transcriptional terminator. This paper reports co-transcription of the pIP501 tra genes in exponentially growing Enterococcus faecalis JH2-2 cells, as shown by RT-PCR.

Crystallization and preliminary structure determination of the plant food allergen Pru av 2.

Thaumatin-like proteins (TLPs) have mostly been investigated in the context of their function as pathogenesis-related proteins and only in recent years have some of them been classified as allergens. Here, the purification and crystallization of the first allergenic TLP, Pru av 2, a 23.3 kDa protein isolated from ripe cherries, is reported.

Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity.

Background: Cherry allergy is often reported in the context of allergy to other fruits of the Rosaceae family and pollinosis to trees because of cross-reactive allergens. Allergic reactions to cherry are reported by 19-29% of birch pollen-allergic patients. Pru av 2, identified as a thaumatin-like protein (TLP) from sweet cherry, was recognized by the majority of cherry-allergic patients in immunoblotting.

Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin.

An anthraquinone with potent quinone reductase-inducing activity and other constituents of the fruits of Morinda citrifolia (noni).

Morinda citrifolia, commonly known as noni, has a long history of utilization throughout much of tropical Polynesia and is considered to be the second most important medicinal plant in the Hawaiian Islands. Recently, the use of noni as a dietary supplement in the United States has greatly increased. Bioassay-guided fractionation of a dichloromethane-soluble partition of a MeOH extract of noni fruits has led to the isolation of an extremely potent quinone reductase inducer, 2-methoxy-1,3,6-trihydroxyanthraquinone (1).

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice.

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion.

Altering the fatty acids in milk fat by including canola seed in dairy cattle diets.

The objective was to evaluate the effects of feeding ground canola seed on the fatty acid profile, yield, and composition of milk from dairy cows. Twenty-four multiparous Holstein cows (548.3 +/- 11.

WW domains provide a platform for the assembly of multiprotein networks.

WW domains are protein modules that mediate protein-protein interactions through recognition of proline-rich peptide motifs and phosphorylated serine/threonine-proline sites. To pursue the functional properties of WW domains, we employed mass spectrometry to identify 148 proteins that associate with 10 human WW domains. Many of these proteins represent novel WW domain-binding partners and are components of multiprotein complexes involved in molecular processes, such as transcription, RNA processing, and cytoskeletal regulation.

Two naturally occurring deletion mutants of 12S seed storage proteins in Arabidopsis thaliana.

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the alpha-subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively.

Increased levels of erucic acid in Brassica carinata by co-suppression and antisense repression of the endogenous FAD2 gene.

Erucic acid and its derivatives represent important industrial feedstock compounds, and there is an increasing demand for the production of high erucate oils in this regard. Our goal therefore, is to develop high erucic acid (HEA) Brassicaceae lines with increased proportions of erucic acid and very long-chain fatty acids (VLCFAs). We proposed that oleate availability may be a rate-limiting factor in the biosynthesis of erucic acid.

A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination.

Background: Allergy vaccines based on natural allergen extracts contain greatly varying amounts of individual allergens with different immunogenicity.

Objective: To develop a novel type of allergy vaccine for complex allergen sources that combines defined amounts of the major allergens in the form of single hybrid molecules.

Methods: A hybrid molecule was engineered by PCR-based mending and expression of the cDNAs coding for the 4 major grass pollen allergens and compared with its single components by circular dichroism analysis, T-cell proliferation, ELISA competition, and histamine release assays.

Chemical constituents of the fruits of Morinda citrifolia (Noni) and their antioxidant activity.

Purification of a n-BuOH-soluble partition of the MeOH extract of Morinda citrifolia (Noni) fruits led to the isolation of two new iridoid glucosides, 6alpha-hydroxyadoxoside (1) and 6beta,7beta-epoxy-8-epi-splendoside (2), as well as 17 known compounds, americanin A (3), narcissoside (4), asperuloside, asperulosidic acid, borreriagenin, citrifolinin B epimer a, citrifolinin B epimer b, cytidine, deacetylasperuloside, dehydromethoxygaertneroside, epi-dihydrocornin, d-glucose, d-mannitol, methyl alpha-d-fructofuranoside, methyl beta-d-fructofuranoside, nicotifloroside, and beta-sitosterol 3-O-beta-d-glucopyranoside. The structures of the new compounds were determined by spectroscopic data interpretation. Compound 4, borreriagenin, cytidine, deacetylasperuloside, dehydromethoxygaertneroside, epi-dihydrocornin, methyl alpha-d-fructofuranoside, and methyl beta-d-fructofuranoside were isolated for the first time from M.

A new yeast poly(A) polymerase complex involved in RNA quality control.

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNAMet (tRNAiMet). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p.

TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution.

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6xHis-tagged fusions and purified.

Lipotropes alter casein gene expression in bovine mammary acinar culture.

Lipotropes (methyl group containing nutrients, including methionine and choline, folic acid, and vitamin B(12)) are essential for cell proliferation and differentiation of mammary tissues. Lipotropes interact in the supply and regulation of intracellular methyl group pools, thereby affecting synthesis and methylation of DNA. To determine the effect of lipotropes on milk protein gene expression, acini isolated from mammary tissues of lactating cows were cultured in one of three treatment media: (1) control, (2) lipotrope deficient, and (3) lipotrope supplemented.

Cloning of Brassica napus phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase1 (BnPtdIns S1)--comparative analysis of the effect of abiotic stresses on the expression of phosphatidylinositol signal transduction-related genes in B. napus.

The cloning and identification of full-length cDNA fragments coding for the Brassica napus phosphatidylinositol-specific phospholipase C2 (BnPLC2), phosphatidylinositol 3-kinase (BnVPS34) and phosphatidylinositol synthase (BnPtdIns S1) is described. In addition, two complementary fragments (120 nucleotides long) corresponding to Arabidopsis PtdIns 4-kinase (PtdIns 4-K) and PtdIns-4-phosphate 5-kinase (PtdIns4P 5-K) sequences were chemically synthesized. These, as well as the cDNA clones, were used as probes to study the corresponding steady state mRNA levels in different tissues and developmental stages of B.

Biochemical and structural insights into substrate binding and catalytic mechanism of mammalian poly(A) polymerase.

Polyadenylation of messenger RNA precursors is an essential process in eukaryotes. Poly(A) polymerase (PAP), a member of the nucleotidyltransferase family that includes DNA polymerase beta, incorporates ATP at the 3' end of mRNAs in a template-independent manner. Although the structures of mammalian and yeast PAPs are known, their mechanism of ATP selection has remained elusive.

Effect of psychotherapy and relaxation on the psychosocial and somatic course of Crohn's disease: main results of the German Prospective Multicenter Psychotherapy Treatment study on Crohn's Disease.

Objective: Few studies have been published on the influence of psychotherapy on the physical and psychosocial course of Crohn's disease (CD).

Methods: The present study, a prospective, randomized multicenter investigation conducted with 108 of 488 consecutive CD patients, was designed to investigate the influence of short-term psychodynamic therapy and relaxation in addition to a standardized glucocorticoid therapy on the somatic course of the disease as well as on patient psychosocial status. Based on the same standardized somatic treatment, the psychotherapy and control groups were compared after a 1-year treatment period and a follow-up of another year with regard to somatic course and psychosocial situation.

Distinct sequence motifs within the 68-kDa subunit of cleavage factor Im mediate RNA binding, protein-protein interactions, and subcellular localization.

Cleavage factor I(m) (CF I(m)) is required for the first step in pre-mRNA 3'-end processing and can be reconstituted in vitro from its heterologously expressed 25- and 68-kDa subunits. The binding of CF I(m) to the pre-mRNA is one of the earliest steps in the assembly of the cleavage and polyadenylation machinery and facilitates the recruitment of other processing factors. We identified regions in the subunits of CF I(m) involved in RNA binding, protein-protein interactions, and subcellular localization.

Functions for S. cerevisiae Swd2p in 3' end formation of specific mRNAs and snoRNAs and global histone 3 lysine 4 methylation.

The Saccharomyces cerevisiae WD-40 repeat protein Swd2p associates with two functionally distinct multiprotein complexes: the cleavage and polyadenylation factor (CPF) that is involved in pre-mRNA and snoRNA 3' end formation and the SET1 complex (SET1C) that methylates histone 3 lysine 4. Based on bioinformatic analysis we predict a seven-bladed beta-propeller structure for Swd2p proteins. Northern, transcriptional run-on and in vitro 3' end cleavage analyses suggest that temperature sensitive swd2 strains were defective in 3' end formation of specific mRNAs and snoRNAs.

Sequence motifs that distinguish ATP(CTP):tRNA nucleotidyl transferases from eubacterial poly(A) polymerases.

ATP(CTP):tRNA nucleotidyl transferases, tRNA maturing enzymes found in all organisms, and eubacterial poly(A) polymerases, enzymes involved in mRNA degradation, are so similar that until now their biochemical functions could not be distinguished by their amino acid sequence. BLAST searches and analysis with the program "Sequence Space" for the prediction of functional residues revealed sequence motifs which define these two protein families. One of the poly(A) polymerase defining motifs specifies a structure that we propose to function in binding the 3' terminus of the RNA substrate.

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e.

Generation of an allergy vaccine by disruption of the three-dimensional structure of the cross-reactive calcium-binding allergen, Phl p 7.

The grass pollen allergen, Phl p 7, belongs to a family of highly cross-reactive calcium-binding pollen allergens. Because Phl p 7 contains most of the disease-eliciting epitopes of pollen-derived calcium-binding allergens, hypoallergenic variants were engineered according to the x-ray crystal structure of Phl p 7 for allergy vaccination. In three recombinant variants, amino acids essential for calcium binding were mutated, and two peptides comprising the N- and C-terminal half were obtained by synthetic peptide chemistry.