The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme

Toxoplasma gondii nucleoside triphosphate diphosphohydrolase (NTPDase) 1 was crystallized in an intermediate tetrameric conformation. The crystal structure is similar to that of T. gondii NTPDase3 and represents an inactive conformation as the activating disulfide bridge is not reduced and the active site cleft between the two domains of each monomer is open. However, the arrangement of the monomers within the tetramer differs from that of the inactive form of NTPDase3 and may represent an intermediate conformation on the path of the closure motion of the tetramer induced upon activation. Proteins 2013; 81:1271–1276. © 2013 Wiley Periodicals, Inc.     

Noise-Induced Inner Hair Cell Ribbon Loss Disturbs Central Arc Mobilization: A Novel Molecular Paradigm for Understanding Tinnitus

Increasing evidence shows that hearing loss is a risk factor for tinnitus and hyperacusis. Although both often coincide, a causal relationship between tinnitus and hyperacusis has not been shown. Currently, tinnitus and hyperacusis are assumed to be caused by elevated responsiveness in subcortical circuits. We examined both the impact of different degrees of cochlear damage and the influence of stress priming on tinnitus induction. We used (1) a behavioral animal model for tinnitus designed to minimize stress, (2) ribbon synapses in inner hair cells (IHCs) as a measure for deafferentation, (3) the integrity of auditory brainstem responses (ABR) to detect differences in stimulus-evoked neuronal activity, (4) the expression of the activity-regulated cytoskeletal protein, Arc, to identify long-lasting changes in network activity within the basolateral amygdala (BLA), hippocampal CA1, and auditory cortex (AC), and (5) stress priming to investigate the influence of corticosteroid on trauma-induced brain responses. We observed that IHC ribbon loss (deafferentation) leads to tinnitus when ABR functions remain reduced and Arc is not mobilized in the hippocampal CA1 and AC. If, however, ABR waves are functionally restored and Arc is mobilized, tinnitus does not occur. Both central response patterns were found to be independent of a profound threshold loss and could be shifted by the corticosterone level at the time of trauma. We, therefore, discuss the findings in the context of a history of stress that can trigger either an adaptive or nonadaptive brain response following injury.     

Increasing and decreasing functional area of the dentition (FAD) of Mammuthus primigenius

The occlusal morphology and continuous molar replacement in elephants provide a very effective functional area for grinding the biomass that is more or less abrasive. Parts of two subsequent molars contribute to the “functional area of the dentition” (FAD). The FAD size, measured in cm², is associated with age and body size. The FAD stage is indicated by the specific teeth contributing to the FAD and represents the individual age. This study concentrates on Mammuthus primigenius and compares the FAD stages, as derived from growth series, with the fossil Elephas antiquus, as well as the extant Elephas maximus and Loxodonta africana. During the life history of the taxa studied, the functional area increases simultaneously with an increase in body size, but decreases severely in senile age stages. In some senile individuals, the FAD is only about 20–50 % of the mean area of an adult animal. The reduction of the FAD beyond a specific size does not mean an immediate starvation of the animal. The general constitution of the individual and the resources of fat accumulation earlier may support the animal for some time but certainly not over a longer period. Nevertheless, the highly reduced functional area was sufficient to keep the animal alive despite its full adult body mass. A much larger FAD in all adult stages provides the energy requirements needed for all additional life functions including competition and reproduction.     

Amyloid precursor proteins are constituents of the presynaptic active zone

The amyloid precursor protein (APP) and its mammalian homologs, APLP1, APLP2, have been allocated to an organellar pool residing in the Golgi apparatus and in endosomal compartments, and in its mature form to a cell surface‐localized pool. In the brain, all APPs are restricted to neurons; however, their precise localization at the plasma membrane remained enigmatic. Employing a variety of subcellular fractionation steps, we isolated two synaptic vesicle (SV) pools from rat and mouse brain, a pool consisting of synaptic vesicles only and a pool comprising SV docked to the presynaptic plasma membrane. Immunopurification of these two pools using a monoclonal antibody directed against the 12 membrane span synaptic vesicle protein2 (SV2) demonstrated unambiguously that APP, APLP1 and APLP2 are constituents of the active zone of murine brain but essentially absent from free synaptic vesicles. The specificity of immunodetection was confirmed by analyzing the respective knock‐out animals. The fractionation experiments further revealed that APP is accumulated in the fraction containing docked synaptic vesicles. These data present novel insights into the subsynaptic localization of APPs and are a prerequisite for unraveling the physiological role of all mature APP proteins in synaptic physiology.

     

Characterization of an ntrX Mutant of Neisseria gonorrhoeae Reveals a Response Regulator That Controls Expression of Respiratory Enzymes in Oxidase-Positive Proteobacteria

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.     

Role of Herpes Simplex Virus VP11/12 Tyrosine-Based Motifs in Binding and Activation of the Src Family Kinase Lck and Recruitment of p85, Grb2, and Shc

Previous studies have shown that the abundant herpes simplex virus 1 (HSV-1) tegument protein VP11/12, encoded by gene UL46, stimulates phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling: it binds the Src family kinase (SFK) Lck, is tyrosine phosphorylated, recruits the p85 subunit of PI3-kinase, and is essential for the activation of Akt during HSV-1 infection. The C-terminal region of VP11/12 contains tyrosine-based motifs predicted to bind the SH2 domains of SFKs (YETV and YEEI), p85 (YTHM), and Grb2 (YENV) and the phosphotyrosine-binding (PTB) domain of Shc (NPLY). We inactivated each of these motifs in the context of the intact viral genome and examined effects on binding and activation of Lck and recruitment of p85, Grb2, and Shc. Inactivating the p85, Grb2, or Shc motif reduced (p85) or eliminated (Grb2 and Shc) the interaction with the cognate signaling molecule without greatly affecting the other interactions or activation of Lck. Inactivating either SFK motif had only a minor effect on Lck binding and little or no effect on recruitment of p85, Grb2, or Shc. In contrast, inactivation of both SFK motifs severely reduced Lck binding and activation and tyrosine phosphorylation of VP11/12 and reduced (p85) or eliminated (Grb2 and Shc) binding of other signaling proteins. Overall, these data demonstrate the key redundant roles of the VP11/12 SFK-binding motifs in the recruitment and activation of SFKs and indicate that activated SFKs then lead (directly or indirectly) to phosphorylation of the additional motifs involved in recruiting p85, Grb2, and Shc. Thus, VP11/12 appears to mimic an activated growth factor receptor.     

X-Ray Absorption Near-Edge Structure (XANES) Spectroscopy Study of the Interaction of Silver Ions with Staphylococcus aureus,Listeria monocytogenes,and Escherichia coli

Silver ions are widely used as antibacterial agents, but the basic molecular mechanism of this effect is still poorly understood. X-ray absorption near-edge structure (XANES) spectroscopy at the Ag LIII, S K, and P K edges reveals the chemical forms of silver in Staphylococcus aureus and Escherichia coli (Ag⁺ treated). The Ag LIII-edge XANES spectra of the bacteria are all slightly different and very different from the spectra of silver ions (silver nitrate and silver acetate), which confirms that a reaction occurs. Death or inactivation of bacteria was observed by plate counting and light microscopy. Silver bonding to sulfhydryl groups (Ag-S) in cysteine and Ag-N or Ag-O bonding in histidine, alanine, and dl-aspartic acid was detected by using synthesized silver-amino acids. Significantly lower silver-cysteine content, coupled with higher silver-histidine content, in Gram-positive S. aureus and Listeria monocytogenes cells indicates that the peptidoglycan multilayer could be buffering the biocidal effect of silver on Gram-positive bacteria, at least in part. Bonding of silver to phosphate groups was not detected. Interaction with DNA or proteins can occur through Ag-N bonding. The formation of silver-cysteine can be confirmed for both bacterial cell types, which supports the hypothesis that enzyme-catalyzed reactions and the electron transport chain within the cell are disrupted.