Modeling the hypothalamus–pituitary–adrenal system: homeostasis by interacting positive and negative feedback

The hypothalamus–pituitary–adrenal (HPA) system is closely related to stress and the restoration of homeostasis. This system is stimulated in the second half of the night, decreases its activity in the daytime, and reaches the homeostatic level during the late evening. In this paper, we derive and discuss a novel model for the HPA system. It is based on three simple rules that constitute a principle of homeostasis and include only the most substantive physiological elements. In contrast to other models, its main components include, apart from the conventional negative feedback ingredient, a positive feedback loop. To validate the model, we present a parameter estimation procedure that enables one to adapt the model to clinical observations. Using this methodology, we are able to show that the novel model is capable of simulating clinical trials. Furthermore, the stationary state of the system is investigated. We show that, under mild conditions, the system always has a well-defined set-point, which reflects the clinical situation to be modeled. Finally, the computed parameters may be interpreted from a physiological point of view, thereby leading to insights about diseases like depression, obesity, or diabetes.     

Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

A multi-mycotoxin immunoassay—using the MultiAnalyte Profiling (xMAP) technology—is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability.     

Roles of the Redox-Active Disulfide and Histidine Residues Forming a Catalytic Dyad in Reactions Catalyzed by 2-Ketopropyl Coenzyme M Oxidoreductase/Carboxylase

NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC), an atypical member of the disulfide oxidoreductase (DSOR) family of enzymes, catalyzes the reductive cleavage and carboxylation of 2-ketopropyl-coenzyme M [2-(2-ketopropylthio)ethanesulfonate; 2-KPC] to form acetoacetate and coenzyme M (CoM) in the bacterial pathway of propylene metabolism. Structural studies of 2-KPCC from Xanthobacter autotrophicus strain Py2 have revealed a distinctive active-site architecture that includes a putative catalytic triad consisting of two histidine residues that are hydrogen bonded to an ordered water molecule proposed to stabilize enolacetone formed from dithiol-mediated 2-KPC thioether bond cleavage. Site-directed mutants of 2-KPCC were constructed to test the tenets of the mechanism proposed from studies of the native enzyme. Mutagenesis of the interchange thiol of 2-KPCC (C82A) abolished all redox-dependent reactions of 2-KPCC (2-KPC carboxylation or protonation). The air-oxidized C82A mutant, as well as wild-type 2-KPCC, exhibited the characteristic charge transfer absorbance seen in site-directed variants of other DSOR enzymes but with a pK_a value for C87 (8.8) four units higher (i.e., four orders of magnitude less acidic) than that for the flavin thiol of canonical DSOR enzymes. The same higher pK_a value was observed in native 2-KPCC when the interchange thiol was alkylated by the CoM analog 2-bromoethanesulfonate. Mutagenesis of the flavin thiol (C87A) also resulted in an inactive enzyme for steady-state redox-dependent reactions, but this variant catalyzed a single-turnover reaction producing a 0.8:1 ratio of product to enzyme. Mutagenesis of the histidine proximal to the ordered water (H137A) led to nearly complete loss of redox-dependent 2-KPCC reactions, while mutagenesis of the distal histidine (H84A) reduced these activities by 58 to 76%. A redox-independent reaction of 2-KPCC (acetoacetate decarboxylation) was not decreased for any of the aforementioned site-directed mutants. We interpreted and rationalized these results in terms of a mechanism of catalysis for 2-KPCC employing a unique hydrophobic active-site architecture promoting thioether bond cleavage and enolacetone formation not seen for other DSOR enzymes.     

Phylogenomics changes our understanding about earwig evolution

Earwigs are one of the comparatively species-poor insect orders. Although various aspects of the phylogeny of this lineage are poorly understood, before the present study, there was a general consensus that Dermaptera comprises two major lineages: the paraphyletic Protodermaptera or ‘lower earwigs’ and the monophyletic Epidermaptera or ‘higher earwigs’, which are nested within the former. Our phylogenomic study based on the analysis of 3247 nuclear single-copy genes reverses these relationships by placing monophyletic Protodermaptera within paraphyletic Epidermaptera. This phylogenetic reversal among the major earwig lineages is not contradicted by morphological arguments but results in far-reaching reinterpretations of the dermapteran ground plan. Within Dermaptera, Apachyidae form the sister group to the remaining earwigs which might imply that social behaviour is not part of the earwig ground plan. Our results corroborate the monophyly of Eudermaptera within Epidermaptera and the paraphyly of several traditional families. The monophyly of Protodermaptera is supported by molecular and morphological evidence, although the exact position of Karschiellidae which were not included in the molecular dataset cannot be determined.     

The separation of leukotrienes and hydroxyeicosatetraenoic acid metabolites of arachidonic acid by high performance liquid chromatography (HPLC)

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.     

Anostracans in dark sections of Saudi Arabian caves

Anostracans were found living in ephemeral pools in the dark sections of three caves on the As Summan Plateau in Saudi Arabia. Branchipus schaefferi Fischer, 1834 occurred alone in one while it cohabited with Streptocephalus torvicornisbucheti Daday, 1910 in a second cave; fairy shrimps were observed but not collected from the third. None of the specimens demonstrated any of the types of morphological changes typically associated with cave adapted species. This is likely due to continuing colonization of the pools during flooding events. This revised version was published online in August 2006 with corrections to the Cover Date.