Number and Distribution of Superficial Neuromasts in Twelve Common European Cypriniform Fishes and Their Relationship to Habitat Occurrence

This paper gives the first detailed data on the number and body part related distribution of superficial neuromasts in twelve common European Cypriniform species and examines whether such anatomical variables can be related to rough scale habitat occurrence. The fishes (Barbatula barbatula, Barbus barbus, Chondrostoma nasus, Cobitis taenia, Leuciscus cephalus, Leuciscus leuciscus, Phoxinus phoxinus, Rutilus rutilus, Rhodeus sericeus, Scardinius erythrophthalmus, Tinca tinca, Vimba vimba) were classified in two generalized ‘ecological guilds’, 1) rheophilic and 2) limnophilic or indifferent, based on literature data. The total number of superficial neuromasts was consistent within each species, but differed considerably between species. Lowest numbers of superficial neuromasts were found in Barbatula barbatula (21 ± 4.9 superficial neuromasts per cm body length) (mean ± SD), highest numbers in Vimba vimba (233 ± 36.1). Both species can be classified as rheophilic. Over all no relationship was found between the total number of superficial neuromasts and large scale habitat occurrence. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Maternal effects,flight versus fecundity trade-offs,and offspring immune defence in the Speckled Wood butterfly, <Emphasis Type="Italic">Pararge aegeria</Emphasis>

Background  

Maternal condition can generate resource-related maternal effects through differential egg provisioning, and can greatly affect offspring performance. In the present study, the speckled wood butterfly Pararge aegeria (L.) was used to investigate whether (after controlling for egg size) maternal age, and increased flight during the oviposition period, resulted in changes in egg provisioning and whether this contributed to variation in offspring performance, i) early in development (egg stage and early post-hatching development), and ii) later in larval development after being exposed to the model viral pathogen system; the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV).     

Ras Homolog Enriched in Brain (Rheb) Enhances Apoptotic Signaling

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.     

The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as C. glutamicum and M. tuberculosis.Pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) activities catalyze key reactions in central metabolism. They exist as huge enzyme complexes of up to 11 MDa to convert a 2-oxoacid to an acyl-coenzyme A (CoA) derivative, which is acetyl- or succinyl-CoA, respectively (for reviews, see references 28 and 29 and references therein). The reaction requires distinct enzyme activities and involves the sequential actions of thiamine-pyrophosphate-dependent oxidative decarboxylation (E1, EC 1.2.4.2), with the concomitant transfer of the respective acyl group to a lipoamide residue. This is followed by the acyl group transfer to CoA, catalyzed by dihydrolipoyl transacylase activity (E2, EC 2.3.1.6), and, finally, the last step is dihydrolipoamide reoxidation to lipoamide by an FAD-dependent dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4), thus enabling the initiation of a new catalytic cycle. As a result, the energy of the C₁-C₂ bond of an α-oxoacid is preserved in acetyl-CoA and succinyl-CoA, respectively, and NADH.PDH and ODH are structurally closely related assemblies. Structural data for the three-dimensional organization of PDH of Bacillus stearothermophilus have culminated in the current view that the complex consists of an E2 core, to which E1 and E3 are flexibly tethered (20-22). This has similarly been disclosed for the PDH of Escherichia coli (23), as well as for components of ODH (6, 8, 18, 37). The PDH possesses specific E1p and E2p proteins, and ODH possesses specific E1o and E2o proteins, whereas the dihydrolipoyl dehydrogenase component E3 is shared by the two multienzyme complexes (28, 29). Thus, PDH and ODH complexes share one identical polypeptide plus very similar polypeptides, and they also have a similar overall quaternary structure (21, 23).Within the Gram-positives, the Corynebacterineae, such as Mycobacterium tuberculosis and Corynebacterium glutamicum, have a number of distinctive features. This includes the synthesis of mycolic acids enabling the formation of a periplasmic space as in Gram-negatives (15) and the possession of unusual glycans and lipoylated glycans in their cell wall (1). It now has become clear that also the PDH and ODH of these organisms have unique properties, with respect to their protein components, three-dimensional organization, and regulation (25, 36). There is only one E2 protein present and with the isolated protein, it is shown to reconstitute PDH activity together with E1 and E3 proteins (35). An E2 protein specific to ODH is absent in M. tuberculosis, as is the case with C. glutamicum as well. Instead, Corynebacterineae possess one large fusion protein, termed OdhA in C. glutamicum and Kgd in M. tuberculosis, consisting of an E2 domain plus an E1 domain (36). However, as a lipoylated protein in Mycobacterium, only the E2 protein, which confers PDH activity in the reconstitution assay, is known, and no ODH activity is detectable in M. tuberculosis (35). A further remarkable feature found for C. glutamicum is the formation of a mixed 2-oxoacid dehydrogenase complex, since tagged OdhA copurified with the E2, E3, and E1p proteins, and vice versa, tagged E1p copurified with the E2 and E3 proteins together with OdhA (25). Another conspicuous feature shared by the OdhA and Kgd proteins is their interaction with a small regulatory protein which contains a phosphopeptide recognition domain (FHA domain) well characterized for many eukaryotic regulatory proteins. The protein is termed OdhI for C. glutamicum and GarA for M. tuberculosis (4, 25), and the structure of OdhI has recently been resolved (3). These proteins themselves are phosphorylated by one or several serine/threonine protein kinases present in the Corynebacterineae (25, 32), and they interact in their unphosphorylated form with OdhA or Kgd, respectively, to inhibit the activity of these proteins (25, 26).Due to these remarkable features of activities and structures enabling pyruvate and 2-oxoglutarate conversion in the Corynebacterineae, we decided to study PDH and ODH as well as features of their constituent polypeptides in C. glutamicum in somewhat more detail, leading to the detection of the unprecedented structural and functional organization of these important enzyme complexes within central metabolism.     

Th cells act via two synergistic pathways to promote antiviral CD8+ T cell responses

The mechanisms of how Th cells promote CD8(+) T cell responses during viral infections are largely unknown. In this study, we unraveled the mechanisms of T cell help for CD8(+) T cell responses during vaccinia virus infection. Our results demonstrate that Th cells promote vaccinia virus-specific CD8(+) T cell responses via two interconnected synergistic pathways: First, CD40L expressed by activated CD4(+) T cells instructs dendritic cells to produce bioactive IL-12p70, which is directly sensed by Ag-specific CD8(+) T cells, resulting in increased IL-2Rα expression. Second, Th cells provide CD8(+) T cells with IL-2, thereby enhancing their survival. Thus, Th cells are at the center of an important communication loop with a central role for IL-2/IL-2R and bioactive IL-12.     

Evolutionary Gain of Function for the ER Membrane Protein Sec62 from Yeast to Humans

Because of similarity to their yeast orthologues, the two membrane proteins of the human endoplasmic reticulum (ER) Sec62 and Sec63 are expected to play a role in protein biogenesis in the ER. We characterized interactions between these two proteins as well as the putative interaction of Sec62 with ribosomes. These data provide further evidence for evolutionary conservation of Sec62/Sec63 interaction. In addition, they indicate that in the course of evolution Sec62 of vertebrates has gained an additional function, the ability to interact with the ribosomal tunnel exit and, therefore, to support cotranslational mechanisms such as protein transport into the ER. This view is supported by the observation that Sec62 is associated with ribosomes in human cells. Thus, the human Sec62/Sec63 complex and the human ER membrane protein ERj1 are similar in providing binding sites for BiP in the ER-lumen and binding sites for ribosomes in the cytosol. We propose that these two systems provide similar chaperone functions with respect to different precursor proteins.     

Function and regulation of the mitochondrial Sirtuin isoform Sirt5 in Mammalia

Sirtuins are a family of protein deacetylases that catalyze the nicotinamide adenine dinucleotide (NAD⁺)-dependent removal of acetyl groups from modified lysine side chains in various proteins. Sirtuins act as metabolic sensors and influence metabolic adaptation but also many other processes such as stress response mechanisms, gene expression, and organismal aging. Mammals have seven Sirtuin isoforms, three of them – Sirt3, Sirt4, and Sirt5 – located to mitochondria, our centers of energy metabolism and apoptosis initiation. In this review, we shortly introduce the mammalian Sirtuin family, with a focus on the mitochondrial isoforms. We then discuss in detail the current knowledge on the mitochondrial isoform Sirt5. Its physiological role in metabolic regulation has recently been confirmed, whereas an additional function in apoptosis regulation remains speculative. We will discuss the biochemical properties of Sirt5 and how they might contribute to its physiological function. Furthermore, we discuss the potential use of Sirt5 as a drug target, structural features of Sirt5 and of an Sirt5/inhibitor complex as well as their differences to other Sirtuins and the current status of modulating Sirt5 activity with pharmacological compounds.     

Characterization of Phleum pratense pollen extracts by 2-D DIGE and allergen immunoreactivity

The allergen content of standardized pollen material is crucial for an effective diagnosis and treatment. However, variations in IgE reactivities of allergic patients to different preparations of Phleum pratense pollen have been reported. In order to define and directly compare the allergen composition of pollen preparations provided by different suppliers, a comprehensive proteome analysis of three different timothy grass pollen extracts was performed. More than 140 proteins were annotated comprising the pollen proteome/allergome in a global 2-D map. With regard to the individual pollen preparations, several major differences in the overall protein composition were detected that also affected known Phleum allergens and their isoforms. Importantly, these differences were also reflected at the level of antibody reactivities in 1-D and 2-D immunoblots. As a consequence, it is suggested that the observed differences should be taken into consideration aiming for a standardized diagnosis and therapy of grass pollen allergies as recommended by international medical agencies.     

Time‐resolved quantitative proteome profiling of host–pathogen interactions: The response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells

Staphylococcus aureus is a versatile Gram‐positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse‐chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on‐membrane digestion, and high‐sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof‐of‐principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.     

Ligand Depletion in vivo Modulates the Dynamic Range and Cooperativity of Signal Transduction

Biological signal transduction commonly involves cooperative interactions in the binding of ligands to their receptors. In many cases, ligand concentrations in vivo are close to the value of the dissociation constant of their receptors, resulting in the phenomenon of ligand depletion. Using examples based on rotational bias of bacterial flagellar motors and calcium binding to mammalian calmodulin, we show that ligand depletion diminishes cooperativity and broadens the dynamic range of sensitivity to the signaling ligand. As a result, the same signal transducer responds to different ranges of signal with various degrees of cooperativity according to its effective cellular concentration. Hence, results from in vitro dose-response analyses cannot be applied directly to understand signaling in vivo. Moreover, the receptor concentration is revealed to be a key element in controlling signal transduction and we propose that its modulation constitutes a new way of controlling sensitivity to signals. In addition, through an analysis of the allosteric enzyme aspartate transcarbamylase, we demonstrate that the classical Hill coefficient is not appropriate for characterizing the change in conformational state upon ligand binding to an oligomeric protein (equivalent to a dose-response curve), because it ignores the cooperativity of the conformational change for the corresponding equivalent monomers, which are generally characterized by a Hill coefficient . Therefore, we propose a new index of cooperativity based on the comparison of the properties of oligomers and their equivalent monomers.