Electronic Microscopy Unit

Madrid, Spain

Electronic Microscopy Unit

Madrid, Spain

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Foelix R.,Electronic Microscopy Unit | Erb B.,Kilbigstr. 18 | Rast B.,Grutweg 8
Arthropod Structure and Development | Year: 2013

Several studies on tarantulas have claimed that their tarsi could secrete fine silk threads which would provide additional safety lines for maintaining a secure foot-hold on smooth vertical surfaces. This interpretation was seriously questioned by behavioral experiments, and more recently morphological evidence indicated that the alleged spigots (" ribbed hairs" ) were not secretory but most likely sensory hairs (chemoreceptors). However, since fine structural studies were lacking, the sensory nature was not proven convincingly. By using transmission electron microscopy we here present clear evidence that these " ribbed hairs" contain many dendrites inside the hair lumen - as is the case in the well-known contact chemoreceptors of spiders and insects. For comparison, we also studied the fine structure of regular silk spigots on the spinnerets and found them distinctly different from sensory hairs. Finally, histological studies of a tarantula tarsus did not reveal any silk glands, which, by contrast, are easily found within the spinnerets. In conclusion, the alleged presence of silk spigots on tarantula feet is refuted. © 2013 Elsevier Ltd.

Toribio R.,Autonomous University of Madrid | Toribio R.,Technical University of Madrid | Diaz-Lopez I.,Autonomous University of Madrid | Boskovic J.,Electronic Microscopy Unit | Ventoso I.,Autonomous University of Madrid
Nucleic Acids Research | Year: 2016

During translation initiation, eukaryotic initiation factor 2 (eIF2) delivers the Met-tRNA to the 40S ribosomal subunit to locate the initiation codon (AUGi) of mRNA during the scanning process. Stress-induced eIF2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. However, some viral mRNAs can initiate translation in the presence of phosphorylated eIF2 via stable RNA stem-loop structures (DLP; Downstream LooP) located in their coding sequence (CDS), which promote 43S preinitiation complex stalling on the initiation codon. We show here that during the scanning process, DLPs of Alphavirus mRNA become trapped in ES6S region (680-914 nt) of 18S rRNA that are projected from the solvent side of 40S subunit. This trapping can lock the progress of the 40S subunit on the mRNA in a way that places the upstream initiator AUGi on the P site of 40S subunit, obviating the participation of eIF2. Notably, the DLP structure is released from 18S rRNA upon 60S ribosomal subunit joining, suggesting conformational changes in ES6Ss during the initiation process. These novel findings illustrate how viral mRNA is threaded into the 40S subunit during the scanning process, exploiting the topology of the 40S subunit solvent side to enhance its translation in vertebrate hosts. © 2016 The Author(s).

Foelix R.,Electronic Microscopy Unit | Erb B.,Kilbigstrasse 15 | Michalik P.,University of Greifswald
Journal of Arachnology | Year: 2010

Adult male Liphistius have dense hair pads on the ventral side of their tarsi. At first glance they appear like the adhesive scopulae, which are well known from mygalomorph spiders. However, a fine structural analysis of these scopulate hairs shows that they lack the brush-like structure with tiny "endfeet" that is typical for such adhesive hairs. Instead, the smooth hair shaft exhibits a small pore ventrally, about 810 m from the blunt tip. A thin cuticular canal extends from that pore through the middle of the hair shaft and terminates about 30 m above the hair base. Transmission electron microscopy reveals that this central canal contains about 16 delicate dendrites. The morphology of these scopulate hairs thus corresponds closely to contact chemoreceptors known from other spiders. Since these scopulate hairs occur only in adult males, they are likely involved in the perception of female pheromones. © 2010 The American Arachnological Society.

Olm M.A.K.,Child Institute | Kogler Jr. J.E.,University of Sao Paulo | Macchione M.,University of Sao Paulo | Shoemark A.,Electronic Microscopy Unit | And 2 more authors.
Journal of Applied Physiology | Year: 2011

Ciliary beat frequency (CBF) measurements provide valuable information for diagnosing of primary ciliary dyskinesia (PCD). We developed a system for measuring CBF, used it in association with electron microscopy to diagnose PCD, and then analyzed characteristics of PCD patients.1 The CBF measurement system was based on power spectra measured through digital imaging. Twenty-four patients suspected of having PCD (age 1-19 yr) were selected from a group of 75 children and adolescents with pneumopathies of unknown causes. Ten healthy, nonsmoking volunteers (age ≥17 yr) served as a control group. Nasal brush samples were collected, and CBF and electron microscopy were performed. PCD was diagnosed in 12 patients: 5 had radial spoke defects, 3 showed absent central microtubule pairs with transposition, 2 had outer dynein arm defects, 1 had a shortened outer dynein arm, and 1 had a normal ultrastructure. Previous studies have reported that the most common cilia defects are in the dynein arm. As expected, the mean CBF was higher in the control group (P < 0.001) and patients with normal ultrastructure (P < 0.002), than in those diagnosed with cilia ultrastructural defects (i.e., PCD patients). An obstructive ventilatory pattern was observed in 70% of the PCD patients who underwent pulmonary function tests. All PCD patients presented bronchial wall thickening on chest computed tomography scans. The protocol and diagnostic techniques employed allowed us to diagnose PCD in 16% of patients in this study. Copyright © 2011 the American Physiological Society.

Saluja R.,Pharmacology Division | Jyoti A.,Pharmacology Division | Chatterjee M.,Pharmacology Division | Habib S.,CSIR - Central Electrochemical Research Institute | And 5 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2011

Nitric oxide synthase (NOS) expression and catalytic status in human peripheral blood mononuclear cells (PBMCs) is debatable, while its sub-cellular distribution remains unascertained. The present study characterizes NOS transcripts by real time PCR, NOS protein by immunoprecipitation (IP)/Western blot (WB), nitric oxide (NO) generation by DAF-2DA and NOS sub-cellular distribution by immunogold electron microscopy in resting PBMCs, monocytes and lymphocytes obtained from healthy donors. We observed constitutive expression of full length NOS isoforms (nNOS, iNOS and eNOS) in PBMCs: with the highest expression of iNOS in comparison to nNOS and eNOS. Isolated monocytes expressed more eNOS transcript and protein as compared to nNOS and iNOS. Lymphocytes however had more iNOS transcripts and protein than nNOS and eNOS. NOS was catalytically active in PBMCs, monocytes as well as in lymphocytes as evident by NO generation in the presence of substrate and cofactors, which was significantly reduced in the presence of NOS inhibitor. Immunogold electron microscopy and morphometric analysis revealed the distinct pattern of NOS distribution in monocytes and lymphocytes and also exhibited differences in the nuclear-cytoplasmic ratio. nNOS localization was much more in the cytosol than in the nucleus among both monocytes and lymphocytes. Interestingly, iNOS distribution was comparable in both cytosol and nucleus among monocytes, but in lymphocytes iNOS was predominantly localized to the cytosol. The present study exhibits constitutive presence of all the NOS isoforms in PBMCs and reports the distinct pattern of NOS distribution among monocytes and lymphocytes. © 2011 Elsevier B.V.

Corsini M.,University of Brescia | Moroni E.,University of Brescia | Ravelli C.,University of Brescia | Andres G.,Electronic Microscopy Unit | And 5 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2014

OBJECTIVE - Angiogenesis and inflammation are closely related processes. Gremlin is a novel noncanonical vascular endothelial growth factor receptor-2 (VEGFR2) ligand that induces a proangiogenic response in endothelial cells (ECs). Here, we investigated the role of the cyclic adenosine monophosphate-response element (CRE)-binding protein (CREB) in mediating the proinflammatory and proangiogenic responses of ECs to gremlin. APPROACH AND RESULTS - Gremlin induces a proinflammatory response in ECs, leading to reactive oxygen species and cyclic adenosine monophosphate production and the upregulation of proinflammatory molecules involved in leukocyte extravasation, including chemokine (C-C motif) ligand-2 (Ccl2) and Ccl7, chemokine (C-X-C motif) ligand-1 (Cxcl1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). Accordingly, gremlin induces the VEGFR2-dependent phosphorylation, nuclear translocation, and transactivating activity of CREB in ECs. CREB activation mediates the early phases of the angiogenic response to gremlin, including stimulation of EC motility and permeability, and leads to monocyte/macrophage adhesion to ECs and their extravasation. All these effects are inhibited by EC transfection with a dominant-negative CREB mutant or with a CREB-binding protein-CREB interaction inhibitor that competes for CREB/CRE binding. Also, both recombinant gremlin and gremlin-expressing tumor cells induce proinflammatory/proangiogenic responses in vivo that are suppressed by the anti-inflammatory drug hydrocortisone. Similar effects were induced by the canonical VEGFR2 ligand VEGF-A165. CONCLUSIONS - Together, the results underline the tight cross-talk between angiogenesis and inflammation and demonstrate a crucial role of CREB activation in the modulation of the VEGFR2-mediated proinflammatory/proangiogenic response of ECs to gremlin. © 2013 American Heart Association, Inc.

Kumar S.,Cincinnati Childrens Research Foundation | Ciraolo G.,Electronic microscopy Unit | Hinge A.,Cincinnati Childrens Research Foundation | Filippi M.-D.,Cincinnati Childrens Research Foundation
Journal of Immunological Methods | Year: 2014

Transmission electron microscopy (TEM) provides ultra-structural details of cells at the sub-organelle level. However, details of the cellular ultrastructure, and the cellular organization and content of various organelles in rare populations, particularly in the suspension, like hematopoietic stem cells (HSCs) remained elusive. This is mainly due to the requirement of millions of cells for TEM studies. Thus, there is a vital requirement of a method that will allow TEM studies with low cell numbers of such rare populations. We describe an alternative and novel approach for TEM studies for rare cell populations. Here we performed a TEM study from 10,000 HSC cells with relative ease. In particular, tiny cell pellets were identified by Evans blue staining after PFA-GA fixation. The cell pellet was pre-embedded in agarose in a small microcentrifuge tube and processed for dehydration, infiltration and embedding. Semi-thin and ultra-thin sections identified clusters of numerous cells per sections with well preserved morphology and ultrastructural details of golgi complex and mitochondria. Together, this method provides an efficient, easy and reproducible process to perform qualitative and quantitative TEM analysis from limited biological samples including cells in suspension. © 2013 Elsevier B.V.

Magellan K.,Rhodes University | Magellan K.,South African Institute For Aquatic Biodiversity | Pinchuck S.,Electronic Microscopy Unit | Swartz E.R.,South African Institute For Aquatic Biodiversity
Journal of Fish Biology | Year: 2014

This study investigated two potential strategies to survive short and longer-term aerial exposure in a galaxiid. This scaleless fish possesses cutaneous pores that dilated in the short-term (15min-3h) but contracted over longer periods (15h) out of water, suggesting that these organs are used to cope with shorter durations of air exposure. Pores on the abdominal surface showed the greatest variation while those on the operculum surface hardly changed. Conversely, thickening of the epithelial layer of secondary gill lamellae showed a slower increase but persisted in an approximately linear fashion over the duration of this study, indicating that this is a strategy that facilitates longer-term aerial exposure. Thus, this species has the capacity to accommodate both short and long-term exposure to air. © 2014 The Fisheries Society of the British Isles.

Schatz D.,Bar - Ilan University | Schatz D.,Weizmann Institute of Science | Nagar E.,Bar - Ilan University | Sendersky E.,Bar - Ilan University | And 9 more authors.
Environmental Microbiology | Year: 2013

Biofilms are consortia of bacteria that are held together by an extracellular matrix. Cyanobacterial biofilms, which are highly ubiquitous and inhabit diverse niches, are often associated with biological fouling and cause severe economic loss. Information on the molecular mechanisms underlying biofilm formation in cyanobacteria is scarce. We identified a mutant of the cyanobacterium Synechococcus elongatus, which unlike the wild type, developed biofilms. This biofilm-forming phenotype is caused by inactivation of homologues of type II secretion /type IV pilus assembly systems and is associated with impairment of protein secretion. The conditioned medium from a wild-type culture represses biofilm formation by the secretion-mutants. This suggested that the planktonic nature of the wild-type strain is a result of a self-suppression mechanism, which depends on the deposition of a factor to the extracellular milieu. We also identified two genes that are essential for biofilm formation. Transcript levels of these genes are elevated in the mutant compared with the wild type, and are initially decreased in mutant cells cultured in conditioned medium of wild-type cells. The particular niche conditions will determine whether the inhibitor will accumulate to effective levels and thus the described mechanism allows switching to a sessile mode of existence. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Foelix R.,Electronic Microscopy Unit | Erb B.,Electronic Microscopy Unit
Journal of Arachnology | Year: 2010

Although venom glands were described for the Mesothelae many years ago (Bristowe & Millot 1933), a more recent monograph (Haupt 2003) denied the existence of such glands in the Mesothelae. Our morphological studies of nine different species of Liphistius demonstrated the presence of venom gland openings on the cheliceral fangs in all of these species. Also, we observed a small venom gland in the anterior portion of the cheliceral basal segment. The possibility that venom glands may be lacking in adult males is discussed. The presence of venom glands in the Mesothelae indicates that this is a plesiomorphic character of all Araneae. © 2010 The American Arachnological Society.

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