Structural Forms and Transitions for the Complex of Mercury(II) with Poly(dG-dC)

Abstract

Infrared spectroscopy was used to study hydrated double-helical poly(dG-dC) complexed with varying amounts of mercury (II). For one Hg(II) per ten nucleotide residues (r = 0.1), the B structure was stabilized and the B* structure was absent at high hydration. The Z structure did not form as hydration was reduced. For r = 0.2, the B and Z structures coexisted at high hydration and the transition to total Z structure was broad as hydration was reduced. Hg(II) was bound exclusively to the guanine residues probably at N 3 orN7forr < 0.25. The cytosine residue did not protonate (at N3) as Hg(II) was bound to guanine. The addition of NaCl together with Hg(II) reduced the binding of Hg(II), stabilized the B structure at the highest hydration and caused a sharp transition between the B and Z structures as hydration was lowered. Hydration with D,0 stabilized the Z structure for poly(dG-dC) complexed with HgCl₂.     

Twenty‐Four‐Hour and Annual Variation in Onset of Epistaxis in Osler Disease

Osler disease is an autosomal dominant disorder of the fibrovascular tissue characterized by arteriovenous malformations with multi‐systemic haemorrhages. Recurrent epistaxis is the predominant symptom in more than 90% of patients. Recent studies showed circadian and seasonal patterns in the onset of nosebleeds, similar to acute cardiovascular events, such as myocardial infarction and stroke. The aim of this study was to determine whether such patterns would also apply to the onset of epistaxis in patients with Osler disease. In all, 110 patients with Osler disease who were under treatment for recurrent epistaxis at the University Hospital of Mannheim were requested to complete a questionnaire addressing the intensity and frequency of epistaxis according to the classification of Bergler et al., as well as circadian and circannual rhythmicity in the occurrence of epistaxis according to visual analogue scales (VAS). More than half of the patients claimed to experience daily to weekly episodes of recurrent epistaxis. The occurrence of epistaxis showed a biphasic 24 h pattern, with a primary peak in the morning (05∶00–8∶00 h) and smaller secondary peaks in the evening (17∶00–20∶00 h and 21∶00–00∶00 h). No significant seasonal variation was found in the onset of epistaxis. However, a slight tendency, with a peak in winter months, was observed. Similar to other chronobiological studies on nosebleeds, this study showed that the 24 h pattern and seasonal tendency in the onset of epistaxis even applied to patients with Osler disease. Further investigations are necessary to determine the pathological mechanism underlying this phenomenon.     

Sexual Dimorphism in Juvenile Hormone Synthesis by Corpora Allata and in Juvenile Hormone Acid Methyltransferase Activity in Corpora Allata and Accessory Sex Glands of Some Lepidoptera

Summary

The kinetic profiles of vitellin accumulation in the oyster ovary during oocyte growth and the effects in vivo and in vitro of estradiol-17β (E₂) on vitellin formation were examined in this study. The relative vitellin content measured by an enzyme-linked immunosorbent assay (ELISA) shows an apparent increase as the oocyte develops. Immunoblotting of the vitellin using anti-vitellin indicated that two main bands (179 and 110 kD), which begin to accumulate at an early stage of maturation, become pronounced during oocyte growth. Meanwhile, the major peak of the intact form of vitellin (530 kD) in gel filtration also enlarges with oocyte growth, supporting the results of immunoblot analysis and vitellin determination. E₂ treatment in vivo causes significant increases in oocyte diameter and vitellin content in the female oyster. A similar trend was observed in ovarian tissues cultured in the presence of E₂. It is concluded that E₂ is one of the major factors which control the vitellogenesis in the oyster and that the ovary is undoubtedly the site of synthesis of vitellin.     

Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material1

Fluorescent proteins (FPs) were developed for live-cell imaging and have revolutionized cell biology. However, not all plant tissues are accessible to live imaging using confocal microscopy, necessitating alternative approaches for protein localization. An example is the phloem, a tissue embedded deep within plant organs and sensitive to damage. To facilitate accurate localization of FPs within recalcitrant tissues, we developed a simple method for retaining FPs after resin embedding. This method is based on low-temperature fixation and dehydration, followed by embedding in London Resin White, and avoids the need for cryosections. We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall probes. Using this method we have been able to image green fluorescent protein-labeled plasmodesmata to a depth of more than 40 μm beneath the resin surface. Using correlative light and electron microscopy of the phloem, we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections. Sections were amenable to antibody labeling, and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy) on the same cells. These correlative imaging methods should find several uses in plant cell biology.The localization of fluorescent proteins (FPs) in cells and tissues has become one of the major tools in cell biology (Tsien, 1998; Shaner et al., 2005). Advances in confocal microscopy have meant that many proteins can be tagged with appropriate fluorescent markers and tracked as they move within and between cells (Chapman et al., 2005). Additional approaches involving photobleaching and photoactivation of FPs have opened up new avenues for exploring protein dynamics and turnover within cells (Lippincott-Schwartz et al., 2003). However, not all cells are amenable to live-cell imaging, which in plants is usually restricted to surface cells such as the leaf epidermis. An example is the phloem. The delicate nature of sieve elements and companion cells, which are under substantial hydrostatic pressure, has made studies of the fine structure of these cells particularly difficult (Knoblauch and van Bel, 1998). Despite this, significant advances have been made in imaging the phloem through inventive use of imaging protocols that allow living sieve elements to be observed as they translocate assimilates (for review, see Knoblauch and Oparka, 2012). However, determining the precise localization of the plethora of proteins located within the sieve element (SE)-companion cell (CC) complex remains a technical challenge. The phloem is the conduit for long-distance movement of macromolecules in plants, including viral genomes. For several viruses, the entry into the SE-CC complex is a crucial step that determines the capacity for long-distance movement. Identifying the cell types within the phloem that restrict the movement of some viruses is technically challenging due to the small size of phloem cells and their location deep within plant organs (Nelson and van Bel, 1998).The problems associated with imaging proteins in phloem tissues prompted us to explore methods for retaining the fluorescence of tagged proteins within tissues not normally amenable to confocal imaging. Previously, we used superresolution imaging techniques on fixed phloem tissues sectioned on a Vibroslice, providing information on the association between a viral movement protein (MP) and plasmodesmata (PD) within the SE-CC complex (Fitzgibbon et al., 2010). However, we wished to explore the same cells using correlative light and electron microscopy (CLEM), necessitating the development of methods that would allow sequential imaging of cells using fluorescence microscopy and transmission electron microscopy (TEM). To this end, we developed a protocol that retains fluorescent proteins through aldehyde fixation and resin embedding.In the last 10 years there has been significant interest in imaging fluorescent proteins in semithin sections (for review, see Cortese et al., 2009). Luby-Phelps and colleagues (2003) first described a method for retaining GFP fluorescence after fixation and resin embedding, but their method has not seen widespread application. The advent of superresolution imaging techniques (for review, see Bell and Oparka, 2011) has stimulated considerable interest in this field as the retention of fluorescence in thin sections means that cells can be imaged using techniques such as photoactivation light microscopy and stochastic optical reconstruction microscopy, allowing a lateral resolution of less than 10 nm to be achieved (Subach et al., 2009; Xu et al., 2012). A number of studies have described CLEM on the same cells (Luby-Phelps et al., 2003; Betzig et al., 2006; Watanabe et al., 2011). Advances in this field were reviewed recently (Jahn et al., 2012; see contributions in Muller-Reichert and Verkade, 2012). For example, Pfeiffer et al. (2003) were able to image SEs and CCs using high-pressure freezing, followed by freeze substitution in acetone and resin embedding. They then used thick optical sections of the tissue to locate cells of interest, and these were subsequently imaged using TEM. However, there have been few attempts to retain FPs in resin-embedded plant tissues. Thompson and Wolniak (2008) described the retention of mCitrine fused to an SE-plasma membrane protein in glycol methacrylate sections. The fluorescent signal was stable using wide-field microscopy but bleached rapidly under the confocal microscope.To date, cryosections have been the preferred choice for CLEM in mammalian tissues (Watanabe et al., 2011). Recently, Lee et al. (2011) chemically fixed Arabidopsis (Arabidopsis thaliana) seedlings, cut 50-μm sections, and examined these with a confocal microscope. After confocal mapping the sections were embedded in resin and thin sectioned. These authors were able to locate the same PD pit fields using confocal and TEM, providing important information on the localization of a novel PD protein. As general rule, cryosectioning is a time-consuming process, and subcellular details may be obscured in cryosections because of poor tissue contrast (Watanabe et al., 2011). A major problem with imaging FPs in resin sections has been that GFP and its derivatives are quenched by the acidic, oxidizing conditions required for fixation, dehydration, and embedding of delicate specimens (Tsien, 1998; Keene et al., 2008). Recently, however, Watanabe et al. (2011) explored the retention of FPs in Caenorhabditis elegans cells after fixation by different aldehydes and embedding media. These authors tested a range of resins and found that Citrine and tandem dimer Eos (tdEos) could be retained in methacrylate plastic sections. This material was difficult to cut thinly (<70 nm) compared to epoxy-based resins, but the authors obtained valuable correlative images using stimulated emission depletion microscopy and photoactivation light microscopy followed by low-voltage scanning electron microscopy.Because the retention of fluorescent proteins may differ between plant and animal cells, we explored a number of approaches for retaining fluorescent proteins in resin. Using low-temperature conditions (<8°C) during fixation and dehydration, we could retain strong fluorescence prior to tissue embedding. We also explored different embedding media and found that tissue could be effectively polymerized in London Resin (LR) White while retaining sufficient fluorescence for confocal imaging. Using water-dipping lenses, we were able to detect fluorescent proteins in optical sections up to 40 μm below the surface of the block face. Ultrathin sections from the same blocks showed good structural preservation and allowed CLEM. Subsequently, we cut 1- to 2-μm sections and examined these using confocal microscopy and three-dimensional-structured illumination microscopy (3D-SIM). Sections could be counterstained with a number of conventional fluorophores and antibodies, allowing colocalization studies. These simple methods allow successive imaging of FPs with the light and electron microscope, combining the strengths of both imaging platforms. We believe this approach will have significant utility for tissues that are recalcitrant to conventional confocal imaging.     

The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.     

How calcium signals in myocytes and pericytes are integrated across in situ microvascular networks and control microvascular tone

The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca²⁺ signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca²⁺ signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca²⁺ signalling and contractility of myocytes and pericytes. Ca²⁺ signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca²⁺ signals arise from Ca²⁺ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca²⁺ release and not via ryanodine receptors or Ca²⁺ entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca²⁺ signals between arteriolar and venular networks dependent on gap junctions and Ca²⁺ entry via L-type Ca²⁺ channels. Endothelial Ca²⁺ signalling inhibits intracellular Ca²⁺ oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca²⁺ signals via EDHF. Increases of Ca²⁺ in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes.     

Migrations of Leptophlebia vespertina L. and L. marginata L. (Ins.: Ephemeroptera) in the estuary of a coastal stream

Investigations carried out in a coastal stream flowing to the Northern Bothnian Sea (Ängerån, 63°35'N, 19°50'E) have shown a high drift rate of mayfly nymphs towards the coastal areas. The nymphal development takes place in the estuaries with low salinity (conductivity between 47 and 9800 μS; salinity between 0 and 4–5%₀). After the emergence (May/June) the adults fly from the coastal areas to lay their eggs in the stream biotope in the Ängerån. There thus appears to be a colonization cycle between the coastal stream and adjacent coastal areas. The migration movements of downstream drift and the compensatory upstream directed flight are interpreted as asurvival strategy of the species concerned.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.