Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier-to-autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain-carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature-sensitive baf-1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF-depleted worms and flies. We identified vaccinia-related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell-cycle-dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.     

Distribution of candidate division JS1 and other Bacteria in tidal sediments of the German Wadden Sea using targeted 16S rRNA gene PCR-DGGE

The bacterial candidate division JS1 dominates a number of 16S rRNA gene libraries from deep subseafloor sediments, yet its distribution in shallow, subsurface sediments has still to be fully documented. Sediment cores (down to 5.5 m) from Wadden Sea tidal flats (Neuharlingersieler Nacken and Gr?ninger Plate) were screened for JS1 16S rRNA genes using targeted PCR-denaturing gradient gel electrophoresis (DGGE), which also detects some other important Bacteria. Bacterial subpopulations at both sites were dominated by Gammaproteobacteria in the upper sediment layers (down to 2 m) and in deeper layers by members of the Chloroflexi. The deeper layers of Neuharlingersieler Nacken consisted of grey mud with low sulphate (0.1-10 mM), elevated total organic carbon (TOC) ( approximately 1-2%) and JS1 sequences were abundant. In contrast, the deeper sandy layers of Gr?ninger Plate, despite also having reduced sulphate concentrations, had lower TOC (<0.6%) with few detectable JS1 sequences. Results indicated that JS1 prefers muddy, shallow, subsurface sediments with reduced sulphate, whereas Chloroflexi may out-compete JS1 in shallow, sandy, subsurface sediments. Bacterial population changes at both sites ( approximately 2 m) were confirmed by cluster analysis of DGGE profiles, which correlated with increased recalcitrance of the organic matter. This study extends the biogeographical range of JS1. The presence of JS1 and Chloroflexi in Wadden Sea sediments demonstrates that subsurface tidal flats contain similar prokaryotic populations to those found in the deeper subseafloor biosphere.     

Conserved nucleotides in an RNA essential for hepatitis B virus replication show distinct mobility patterns

The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA’s biological function.     

Semen-derived amyloid fibrils drastically enhance HIV infection

Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.     

Structural Mechanism of the Interaction of Alzheimer Disease Aβ Fibrils with the Non-steroidal Anti-inflammatory Drug (NSAID) Sulindac Sulfide

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp²³-Lys²⁸ salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly³³, a residue involved in Met³⁵ oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.     

Airway Surface Dehydration Aggravates Cigarette Smoke-Induced Hallmarks of COPD in Mice

Introduction

Airway surface dehydration, caused by an imbalance between secretion and absorption of ions and fluid across the epithelium and/or increased epithelial mucin secretion, impairs mucociliary clearance. Recent evidence suggests that this mechanism may be implicated in chronic obstructive pulmonary disease (COPD). However, the role of airway surface dehydration in the pathogenesis of cigarette smoke (CS)-induced COPD remains unknown.

Objective

We aimed to investigate in vivo the effect of airway surface dehydration on several CS-induced hallmarks of COPD in mice with airway-specific overexpression of the β-subunit of the epithelial Na⁺ channel (βENaC).

Methods

βENaC-Tg mice and wild-type (WT) littermates were exposed to air or CS for 4 or 8 weeks. Pathological hallmarks of COPD, including goblet cell metaplasia, mucin expression, pulmonary inflammation, lymphoid follicles, emphysema and airway wall remodelling were determined and lung function was measured.

Results

Airway surface dehydration in βENaC-Tg mice aggravated CS-induced airway inflammation, mucin expression and destruction of alveolar walls and accelerated the formation of pulmonary lymphoid follicles. Moreover, lung function measurements demonstrated an increased compliance and total lung capacity and a lower resistance and hysteresis in βENaC-Tg mice, compared to WT mice. CS exposure further altered lung function measurements.

Conclusions

We conclude that airway surface dehydration is a risk factor that aggravates CS-induced hallmarks of COPD.     

Identification of Candidate Agents Active against N. ceranae Infection in Honey Bees: Establishment of a Medium Throughput Screening Assay Based on N. ceranae Infected Cultured Cells

Many flowering plants in both natural ecosytems and agriculture are dependent on insect pollination for fruit set and seed production. Managed honey bees (Apis mellifera) and wild bees are key pollinators providing this indispensable eco- and agrosystem service. Like all other organisms, bees are attacked by numerous pathogens and parasites. Nosema apis is a honey bee pathogenic microsporidium which is widely distributed in honey bee populations without causing much harm. Its congener Nosema ceranae was originally described as pathogen of the Eastern honey bee (Apis cerana) but jumped host from A. cerana to A. mellifera about 20 years ago and spilled over from A. mellifera to Bombus spp. quite recently. N. ceranae is now considered a deadly emerging parasite of both Western honey bees and bumblebees. Hence, novel and sustainable treatment strategies against N. ceranae are urgently needed to protect honey and wild bees. We here present the development of an in vitro medium throughput screening assay for the identification of candidate agents active against N. ceranae infections. This novel assay is based on our recently developed cell culture model for N. ceranae and coupled with an RT-PCR-ELISA protocol for quantification of N. ceranae in infected cells. The assay has been adapted to the 96-well microplate format to allow automated analysis. Several substances with known (fumagillin) or presumed (surfactin) or no (paromomycin) activity against N. ceranae were tested as well as substances for which no data concerning N. ceranae inhibition existed. While fumagillin and two nitroimidazoles (metronidazole, tinidazole) totally inhibited N. ceranae proliferation, all other test substances were inactive. In summary, the assay proved suitable for substance screening and demonstrated the activity of two synthetic antibiotics against N. ceranae.     

Enhanced anti-proliferative effects of combinatorial treatment of delta-tocopherol and resveratrol in human HMC-1 cells

The tocopherols (alpha, beta-, gamma-, and delta-tocopherol) and resveratrol are phytochemicals with alleged beneficial effects against atherosclerosis, vascular diseases and different cancers. They both can act as antioxidants, but they also modulate signal transduction and gene expression by non-antioxidant mechanisms. Here we wanted to determine whether the combined treatment of mast cells with the two compounds inhibits cell proliferation more efficiently when compared to individual treatments. Both compounds inhibit HMC-1 mastocytoma cell proliferation and reduce the activity of Protein Kinase B (PKB/Akt) by inhibiting its Ser473-phosphorylation. The combination of 50 microM delta-tocopherol and 50 microM resveratrol inhibits proliferation of HMC-1 cells more efficiently when compared to single treatments. In line with this, PKB Ser473-phosphorylation is inhibited best by delta-tocopherol and resveratrol combinatory treatment. Resveratrol acts more efficiently as an inhibitor of PKB phosphorylation than alpha-, beta-, gamma-tocopherols, whereas delta-tocopherol shows a stronger inhibition possibly as a result of its apoptotic secondary effects. Our data suggest that delta-tocopherol and resveratrol can act additively in reducing cell proliferation and PKB phosphorylation. The combination of phytochemicals with relatively broad specificity on enzymes involved in signal transduction and gene expression may increase their activity in disease prevention by modulating several different molecular targets.