The corticotropin-releasing factor (CRF) 3 receptor family is involved in the regulation of the hypothalamic-pituitary-adrenal stress axis in mammals (1-3). A large body of evidence points to a major role of the receptors in mediating CRF effects in anxiety and depressive disorders and in stress-associated pathologies. Two types of CRF receptors are known, the CRF 1 and the CRF 2 receptors. The CRF 1 receptor is expressed mainly in the pituitary and central nervous system and binds CRF with high affinity. It mediates adrenocorticotrophic hormone release from the anterior pituitary and is involved in the endocrine, autonomic, and cognitive responses to stress stimuli. The CRF 2 receptors are expressed in the central nervous system but also in the periphery including skeletal muscle cells, cardiac myocytes, and cells of the gastrointestinal tract. Three splice variants of CRF 2 receptors have been described: CRF 2(a) , CRF 2(b) , and CRF 2(c) receptors. They bind CRF with low and the urocortins 1-3 with high affinity. The CRF 2 receptors are involved in the regulation of feeding behavior (4) and in recovery from a stress response (5). It is likely that they are also involved in modulating anxiety-related behavior.The CRF receptors belong to the small subgroup of GPCRs (5-10%) possessing putative N-terminal signal peptides. These peptides are believed to be cleaved-off after mediating the ER targeting/insertion process (6, 7). The majority (90 -

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Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

The corticotropin-releasing factor receptor type 2a (CRF 2(a) R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF 1 R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF 2(a) R in comparison with the CRF 1 R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G s protein, G i -mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF 1 R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.The family of corticotropin-releasing factor (CRF) 3 receptors encompasses two subtypes, the CRF 1 R and CRF 2 R (1, 2). The CRF 1 R is expressed mainly in the anterior pituitary and plays a central role in the regulation of the hypothalamic-pituitaryadrenal stress axis in mammals (3). It binds CRF with high affinity and mediates ACTH release from the pituitary leading to cortisol biosynthesis in the adrenal cortex. A large body of evidence points to a major role of the receptor in mediating CRF effects in anxiety and depressive disorders (4 -6).In the case of the CRF 2 R, three splice variants have been described as follows: the CRF 2(a) R, CRF 2(b) R, and CRF 2(c) R. All splice variants bind CRF with low affinity and the urocortins 1-3 with high affinity. They are involved in the regulation of feeding behavior (7) and in recovery from a stress response (8).It is likely that they are also involved in modulating anxietyrelated behavior.Both the CRF 1 R and the CRF 2(a) R usually couple to the G s / adenylyl cyclase system and consequently increase cytosolic cAMP as a second messenger. However, a promiscuous coupling behavior was described previously in particular for the CRF

The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.