Body odor evoked potentials: a new method to study the chemosensory perception of self and non‐self in humans

A new method will be presented which allows the perception of body odors in humans to be studied objectively. The analysis of body odor‐evoked potentials was used to investigate if and how the human brain is able to differentiate self from non‐self body odor for the first time. Six subjects (three females) participated in two experimental sessions. In each session, two body odors (axillary hair) were presented within an olfactory oddball paradigm. One of the odors was collected from the subject and the other from an odor donor of the same sex. In the first session the subjects' attention was distracted to a secondary task (passive paradigm), in the second session the subjects were asked to actively differentiate the odors (active paradigm). For the EEG recordings the odors were presented within a constantly flowing airstream. The results show that the subjects could hardly differentiate the body odors subjectively. However, it could be demonstrated that the central nervous processing of one's own odor was faster than the processing of the chemosensory non‐self signal. Moreover, in the active paradigm, the potentials appeared to be larger when the subjects perceived their own body odor. The conclusion is reached that the measurement of chemosensory event‐related potentials (CSERP) is the method of choice for the investigation of HLA‐associated body odors. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.     

Première découverte d’un squelette de Channidae (Poisson téléostéen) dans le Miocène inférieur d’Illerkirchberg, près d’Ulm (Wurtemberg, Allemagne)

A skeleton of a Channid fish is reported for the first time from the European Lower Miocene. Because of the lack of any appropriate diagnostic character, it is nevertheless impossible to determine if it belongs either to the Asiatic genusChanna Scopoli or to the African one,Parachanna Teugels &Daget. For this reason, its palaeobiogeographical significance remains unclear. Otoliths (sagitta) that very likely belong to the same Channid species have been described from Illerkirchberg asChanna elliptica (vonSalis). They are compared to the sagitta from recent species of the generaChanna (Scopoli) andParachanna (Teugels &Daget). Their morphological characters are intermediate between those characterizing the sagitta of both genera.     

Activation of the Small GTPase Ral in Platelets

Ral is a ubiquitously expressed Ras-like small GTPase which is abundantly present in human platelets. The biological function of Ral and the signaling pathway in which Ral is involved are largely unknown. Here we describe a novel method to measure Ral activation utilizing the Ral binding domain of the putative Ral effector RLIP76 as an activation-specific probe. With this assay we investigated the signaling pathway that leads to Ral activation in human platelets. We found that Ral is rapidly activated after stimulation with various platelet agonists, including α-thrombin. In contrast, the platelet antagonist prostaglandin I₂ inhibited α-thrombin-induced Ral activation. Activation of Ral by α-thrombin could be inhibited by depletion of intracellular Ca²⁺, whereas the induction of intracellular Ca²⁺ resulted in the activation of Ral. Our results show that Ral can be activated by extracellular stimuli. Furthermore, we show that increased levels of intracellular Ca²⁺ are sufficient for Ral activation in platelets. This activation mechanism correlates with the activation mechanism of the small GTPase Rap1, a putative upstream regulator of Ral guanine nucleotide exchange factors.     

Cerebral Asymmetry of fMRI-BOLD Responses to Visual Stimulation

Hemispheric asymmetry of a wide range of functions is a hallmark of the human brain. The visual system has traditionally been thought of as symmetrically distributed in the brain, but a growing body of evidence has challenged this view. Some highly specific visual tasks have been shown to depend on hemispheric specialization. However, the possible lateralization of cerebral responses to a simple checkerboard visual stimulation has not been a focus of previous studies. To investigate this, we performed two sessions of blood-oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) in 54 healthy subjects during stimulation with a black and white checkerboard visual stimulus. While carefully excluding possible non-physiological causes of left-to-right bias, we compared the activation of the left and the right cerebral hemispheres and related this to grey matter volume, handedness, age, gender, ocular dominance, interocular difference in visual acuity, as well as line-bisection performance. We found a general lateralization of cerebral activation towards the right hemisphere of early visual cortical areas and areas of higher-level visual processing, involved in visuospatial attention, especially in top-down (i.e., goal-oriented) attentional processing. This right hemisphere lateralization was partly, but not completely, explained by an increased grey matter volume in the right hemisphere of the early visual areas. Difference in activation of the superior parietal lobule was correlated with subject age, suggesting a shift towards the left hemisphere with increasing age. Our findings suggest a right-hemispheric dominance of these areas, which could lend support to the generally observed leftward visual attentional bias and to the left hemifield advantage for some visual perception tasks.     

Value of Dynamic Susceptibility Contrast Perfusion MRI in the Acute Phase of Transient Global Amnesia

Purpose

Transient global amnesia (TGA) is a transitory, short-lasting neurological disorder characterized by a sudden onset of antero- and retrograde amnesia. Perfusion abnormalities in TGA have been evaluated mainly by use of positron emission tomography (PET) or single-photon emission computed tomography (SPECT). In the present study we explore the value of dynamic susceptibility contrast perfusion-weighted MRI (PWI) in TGA in the acute phase.

Methods

From a MRI report database we identified TGA patients who underwent MRI including PWI in the acute phase and compared these to control subjects. Quantitative perfusion maps (cerebral blood flow (CBF) and volume (CBV)) were generated and analyzed by use of Signal Processing In NMR-Software (SPIN). CBF and CBV values in subcortical brain regions were assessed by use of VOI created in FIRST, a model-based segmentation tool in the Oxford Centre for Functional Magnetic Resonance Imaging of the Brain (FMRIB) Software Library (FSL).

Results

Five TGA patients were included (2 men, 3 women). On PWI, no relevant perfusion alterations were found by visual inspection in TGA patients. Group comparisons for possible differences between TGA patients and control subjects showed significant lower rCBF values bilaterally in the hippocampus, in the left thalamus and globus pallidus as well as bilaterally in the putamen and the left caudate nucleus. Correspondingly, significant lower rCBV values were observed bilaterally in the hippocampus and the putamen as well as in the left caudate nucleus. Group comparisons for possible side differences in rCBF and rCBV values in TGA patients revealed a significant lower rCBV value in the left caudate nucleus.

Conclusions

Mere visual inspection of PWI is not sufficient for the assessment of perfusion changes in TGA in the acute phase. Group comparisons with healthy control subjects might be useful to detect subtle perfusion changes on PWI in TGA patients. However, this should be confirmed in larger data sets and serial PWI examinations.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.     

Psychosocial Stress Increases Salivary Alpha-Amylase Activity Independently from Plasma Noradrenaline Levels

Salivary alpha-amylase activity (sAA) and plasma noradrenaline (NA) concentrations are often considered to be surrogate markers of sympathetic activation in response to stress. However, despite accumulating evidence for a close association between sAA and noradrenaline and other indicators of sympathetic activity, reliability and generality of this relation remains unclear. We employed the Trier Social Stress Test (TSST) in order to directly compare the responses in sAA and NA to psychological stress in healthy volunteers (n = 23). The TSST significantly increased sAA and NA plasma levels with no significant differences in females and males. However, when subjects were divided according to their NA responses into low versus high responders, both groups did not significantly differ in their sAA before, during or after stress exposure. These data suggest that in response to acute psychological stress both plasma NA levels and sAA reflect sympathetic activity, however seemed to increase independently from each other.     

Effects of Drought,Pest Pressure and Light Availability on Seedling Establishment and Growth: Their Role for Distribution of Tree Species across a Tropical Rainfall Gradient

Tree species distributions associated with rainfall are among the most prominent patterns in tropical forests. Understanding the mechanisms shaping these patterns is important to project impacts of global climate change on tree distributions and diversity in the tropics. Beside direct effects of water availability, additional factors co-varying with rainfall have been hypothesized to play an important role, including pest pressure and light availability. While low water availability is expected to exclude drought-intolerant wet forest species from drier forests (physiological tolerance hypothesis), high pest pressure or low light availability are hypothesized to exclude dry forest species from wetter forests (pest pressure gradient and light availability hypothesis, respectively). To test these hypotheses at the seed-to-seedling transition, the potentially most critical stage for species discrimination, we conducted a reciprocal transplant experiment combined with a pest exclosure treatment at a wet and a dry forest site in Panama with seeds of 26 species with contrasting origin. Establishment success after one year did not reflect species distribution patterns. However, in the wet forest, wet origin species had a home advantage over dry forest species through higher growth rates. At the same time, drought limited survival of wet origin species in the dry forest, supporting the physiological tolerance hypothesis. Together these processes sort species over longer time frames, and exclude species outside their respective home range. Although we found pronounced effects of pests and some effects of light availability on the seedlings, they did not corroborate the pest pressure nor light availability hypotheses at the seed-to-seedling transition. Our results underline that changes in water availability due to climate change will have direct consequences on tree regeneration and distributions along tropical rainfall gradients, while indirect effects of light and pests are less important.     

Heterologous Expression of Bartonella Adhesin A in Escherichia coli by Exchange of Trimeric Autotransporter Adhesin Domains Results in Enhanced Adhesion Properties and a Pathogenic Phenotype

Human-pathogenic Bartonella henselae causes cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin (TAA) Bartonella adhesin A (BadA), which is modularly constructed, consisting of a head, a long and repetitive neck-stalk module, and a membrane anchor. BadA is involved in bacterial autoagglutination, binding to extracellular matrix proteins and host cells, and in proangiogenic reprogramming. The slow growth of B. henselae and limited tools for genetic manipulation are obstacles for detailed examination of BadA and its domains. Here, we established a recombinant expression system for BadA mutants in Escherichia coli allowing functional analysis of particular BadA domains. Using a BadA mutant lacking 21 neck-stalk repeats (BadA HN23), the BadA HN23 signal sequence was exchanged with that of E. coli OmpA, and the BadA membrane anchor was additionally replaced with that of Yersinia adhesin A (YadA). Constructs were cloned in E. coli, and hybrid protein expression was detected by immunoblotting, fluorescence microscopy, and flow cytometry. Functional analysis revealed that BadA hybrid proteins mediate autoagglutination and binding to collagen and endothelial cells. In vivo, expression of this BadA construct correlated with higher pathogenicity of E. coli in a Galleria mellonella infection model.