The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.

Conjugative plasmid transfer is one of the most important mechanisms for the spread of antibiotic resistance genes and thereby the emergence of multiple resistant pathogenic bacteria. pIP501 is a 30,599-bp plasmid with the broadest known host range for a conjugative plasmid originating from grampositive (Gϩ) bacteria. pIP501 can self-transfer to a variety of Gϩ bacteria, including pathogens and nosocomial pathogens such as streptococci, staphylococci, enterococci, listeria, multicellular Streptomyces lividans, and also to gram-negative (GϪ) Escherichia coli (32). The pIP501 tra region is organized in an operon comprising almost half of the plasmid genome. It bears 15 genes, all of which are putatively involved in conjugative plasmid transfer. Cotranscription of all 15 tra genes was shown by reverse transcription-PCR in Enterococcus faecalis JH2-2 (33). The tra genes are transcribed throughout the growth cycle of E. faecalis, and their expression level remains constant independent of the growth phase. The pIP501 tra operon is negatively autoregulated at the transcriptional level by the first gene product of the operon, the TraA relaxase (33). The TraA relaxase was biochemically characterized as the enzyme attacking a specific dinucleotide within oriT, thereby initiating the directed transfer of the plasmid single strand to the recipient. The TraA relaxase and its amino-terminal relaxase domain TraAN 246 (the first 246 amino-terminal amino acids) were shown to bind to oriT and to the tra operon promoter, P tra , which partially overlaps with oriT (30, 33).