Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.     

Identification and characterization of Hedgehog modulator properties after functional coupling of Smoothened to G15

The seven-transmembrane receptor Smoothened (Smo) transduces the signal initiated by Hedgehog (Hh) morphogen binding to the receptor Patched (Ptc). We have reinvestigated the pharmacological properties of reference molecules acting on the Hh pathway using various Hh responses and a novel functional assay based on the coexpression of Smo with the alpha subunit of the G15 protein in HEK293 cells. The measurement of inositol phosphate (IP) accumulation shows that Smo has constitutive activity, a response blocked by Ptc which indicates a functional Hh receptor complex. Interestingly, the antagonists cyclopamine, Cur61414, and SANT-1 display inverse agonist properties and the agonist SAG has no effect at the Smo-induced IP response, but converts Ptc-mediated inactive forms of Smo into active ones. An oncogenic Smo mutant does not mediate an increase in IP response, presumably reflecting its inability to reach the cell membrane. These studies identify novel properties of molecules displaying potential interest in the treatment of various cancers and brain diseases, and demonstrate that Smo is capable of signaling through G15.     

24,25-dihydroxyvitamin D3 binds to catalase

There is increasing evidence that the vitamin D metabolite, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) has endocrine actions. In the current work, we report that an endogenous binding protein for 24,25(OH)2D3 is catalase, based on sequence analysis of the isolated protein. An antibody (Ab 365) generated against equivalent protein recognized bovine catalase and a 64 kDa band in subcellular fractions of chick intestine. A commercially available anti-catalase antibody reduced specific [3H]24,25(OH)2D3 binding in subcellular fractions of chick intestine by greater than 65%, relative to the same fractions treated with an unrelated antibody (Ab 099). The same commercially available anti-catalase was able to block the inhibitory actions of 24,25(OH)2D3 on 32P uptake in isolated intestinal epithelial cell suspensions. We subsequently characterized binding of steroid to commercially available catalase, and found that between 0 and 5 nM of enzyme added to subcellular fraction P2 (20,000g, 10-min post-nuclear pellet) resulted in a linear increase in the amount of [3H]24,25(OH)2D3 specifically bound. Additional studies indicated that 25(OH)D3 was an effective competitor for binding, whereas 1,25(OH)2D3 only poorly displaced [3H]24,25(OH)2D3. Saturation analyses with added catalase yielded a physiologically relevant affinity constant (KD=5.6+/-2.7 nM) and a Bmax=209+/-34 fmols/mg protein, comparable to previous studies using purified basal lateral membranes or vesicular fractions. Moreover, in a study on subcellular fractions isolated from chickens of varying ages, we found that in females, both specific [3H]24,25(OH)2D3 binding and catalase activity increased from 7- to 58-week-old birds, whereas in males, elevated levels of both parameters were expressed in preparations of 7- and 58-week-old birds. The data suggest that signal transduction may occur through modulation of hydrogen peroxide production.     

A critical analysis of the biological impacts of plasticizers on wildlife

This review provides a critical analysis of the biological effects of the most widely used plasticizers, including dibutyl phthalate, diethylhexyl phthalate, dimethyl phthalate, butyl benzyl phthalate and bisphenol A (BPA), on wildlife, with a focus on annelids (both aquatic and terrestrial), molluscs, crustaceans, insects, fish and amphibians. Moreover, the paper provides novel data on the biological effects of some of these plasticizers in invertebrates, fish and amphibians. Phthalates and BPA have been shown to affect reproduction in all studied animal groups, to impair development in crustaceans and amphibians and to induce genetic aberrations. Molluscs, crustaceans and amphibians appear to be especially sensitive to these compounds, and biological effects are observed at environmentally relevant exposures in the low ng l⁻¹ to µg l⁻¹ range. In contrast, most effects in fish (except for disturbance in spermatogenesis) occur at higher concentrations. Most plasticizers appear to act by interfering with the functioning of various hormone systems, but some phthalates have wider pathways of disruption. Effect concentrations of plasticizers in laboratory experiments coincide with measured environmental concentrations, and thus there is a very real potential for effects of these chemicals on some wildlife populations. The most striking gaps in our current knowledge on the impacts of plasticizers on wildlife are the lack of data for long-term exposures to environmentally relevant concentrations and their ecotoxicity when part of complex mixtures. Furthermore, the hazard of plasticizers has been investigated in annelids, molluscs and arthropods only, and given the sensitivity of some invertebrates, effects assessments are warranted in other invertebrate phyla.     

Organic Matter Accumulation and Community Change at the Peatland–Upland Interface: Inferences from 14C and 210Pb Dated Profiles

Peatland-margin habitats with organic matter accumulation of 40–150 cm make up a significant but poorly quantified portion of Canada’s boreal forest region. Spanning the transition between non-wetland forest and fen proper, these ecosystems represent a zone of complex environmental and vegetation change, yet little is known about their ecological function or development. We here use vegetation and macrofossil analysis, traditional ¹⁴C, bomb-spike ¹⁴C, and ²¹⁰Pb dating to investigate the development, organic matter accumulation, and recent vegetation history of peat margin communities at two sites in central Saskatchewan, Canada. Although similar in general shape, bomb-spike ¹⁴C and ²¹⁰Pb chronologies show limited agreement in three of the four profiles examined, with ²¹⁰Pb generally producing younger ages than ¹⁴C. Peat initiation and long-term organic matter accumulation at the Old Black Spruce (OBS) transect were probably driven mainly by the dynamics of Sphagnum, whereas at the Sandhill Fen (SF) transect, they were controlled by water level fluctuations in the neighboring fen. Bryophyte macrofossils suggest a recent drying of the vegetation surface at both sites, most likely triggered by regional drought in the late 1950s and 1960s. At OBS, the shift from Sphagnum- to feather moss-dominated communities continued in the 1990s, possibly reflecting effects of direct disturbance on local drainage patterns. Overall, our results suggest that community composition and C dynamics of peat-margin swamps respond dynamically to climatic and hydrologic fluctuations. However, uncertainties regarding the reliability of different chronologies limit our ability to link observed community changes to specific causal events. Author Contributions  IEB conceived/designed study, performed research, analyzed data, wrote paper. JSB conceived/designed study, wrote paper. CS performed research, analyzed data, wrote paper. RKW performed research and analyzed data. CMP performed research and wrote paper.     

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Here we study ATP synthase from human ρ⁰ (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF₁ inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF₁ still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in ρ⁰ cells and not to the dimeric form. This supports previous suggestions that IF₁ plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.     

Transfer RNA processing in archaea: Unusual pathways and enzymes

Transfer RNA (tRNA) molecules are highly conserved in length, sequence and structure in order to be functional in the ribosome. However, mostly in archaea, the short genes encoding tRNAs can be found disrupted, fragmented, with permutations or with non-functional mutations of conserved nucleotides. Here, we give an overview of recently discovered tRNA maturation pathways that require intricate processing steps to finally generate the standard tRNA from these unusual tRNA genes.     

Molecular and electrophysiological characterization of GFP-expressing CA1 interneurons in GAD65-GFP mice

The use of transgenic mice in which subtypes of neurons are labeled with a fluorescent protein has greatly facilitated modern neuroscience research. GAD65-GFP mice, which have GABAergic interneurons labeled with GFP, are widely used in many research laboratories, although the properties of the labeled cells have not been studied in detail. Here we investigate these cells in the hippocampal area CA1 and show that they constitute ～20% of interneurons in this area. The majority of them expresses either reelin (70±2%) or vasoactive intestinal peptide (VIP; 15±2%), while expression of parvalbumin and somatostatin is virtually absent. This strongly suggests they originate from the caudal, and not the medial, ganglionic eminence. GFP-labeled interneurons can be subdivided according to the (partially overlapping) expression of neuropeptide Y (42±3%), cholecystokinin (25±3%), calbindin (20±2%) or calretinin (20±2%). Most of these subtypes (with the exception of calretinin-expressing interneurons) target the dendrites of CA1 pyramidal cells. GFP-labeled interneurons mostly show delayed onset of firing around threshold, and regular firing with moderate frequency adaptation at more depolarized potentials.     

Targeting the Neurokinin Receptor 1 with Aprepitant: A Novel Antipruritic Strategy

Background

Chronic pruritus is a global clinical problem with a high impact on the quality of life and lack of specific therapies. It is an excruciating and frequent symptom of e.g. uncurable renal, liver and skin diseases which often does not respond to conventional treatment with e.g. antihistamines. Therefore antipruritic therapies which target physiological mechanisms of pruritus need to be developed. Substance P (SP) is a major mediator of pruritus. As it binds to the neurokinin receptor 1 (NKR1), we evaluated if the application of a NKR1 antagonist would significantly decrease chronic pruritus.

Methods and Findings

Twenty hitherto untreatable patients with chronic pruritus (12 female, 8 male; mean age, 66.7 years) were treated with the NKR1 antagonist aprepitant 80 mg for one week. 16 of 20 patients (80%) experienced a considerable reduction of itch intensity, as assessed by the visual analog scale (VAS, range 0 to 10). Considering all patients, the mean value of pruritus intensity was significantly reduced from 8.4 VAS points (SD +/−1.7) before treatment to 4.9 VAS points (SD +/−3.2) (p<0.001, CI 1.913–5.187). Patients with dermatological diseases (e.g. atopic diathesis, prurigo nodularis) had the best profit from the treatment. Side-effects were mild (nausea, vertigo, and drowsiness) and only occurred in three patients.

Conclusions

The high response rate in patients with therapy refractory pruritus suggests that the NKR1 antagonist aprepitant may indeed exhibit antipruritic effects and may present a novel, effective treatment strategy based on pathophysiology of chronic pruritus. The results are promising enough to warrant confirming the efficacy of NKR1 antagonists in a randomized, controlled clinical trial.