Direct observation of cell wall glucans in whole cells of Saccharomyces cerevisiae by magic-angle spinning 13C-nmr

Intact cells of Saccharomyces cerevisiae were examined as an aqueous paste by ¹³C-nmr spectroscopy with direct polarization and magic-angle spinning. The spectra obtained were highly resolved, showing numerous resonances in the 60-105 ppm range that were assigned to carbons of a liquid-like domain of the cell wall glucan. Assignments were confirmed by running the spectrum of S. cerevisiae in which the cell wall glucans were labeled with [¹³C] by feeding the cell [¹³C ] galactose. The spectra indicate that the glucan in the cell wall of intact S. cerevisiae assumes a helical conformation and suggest that strain 17A fed with galactose preferentially incorporates the resulting glucose into β(1 → 3)-linkages. © 1994 John Wiley & Sons, Inc.     

A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae

Peptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N-end-rule pathway of the ubiquitin-dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N-end-rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N-end-rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.     

Ups and Downs of Viagra: Revisiting Ototoxicity in the Mouse Model

Sildenafil citrate (Viagra), a phosphodiesterase 5 inhibitor (PDE5i), is a commonly prescribed drug for erectile dysfunction. Since the introduction of Viagra in 1997, several case reports have linked Viagra to sudden sensorineural hearing loss. However, these studies are not well controlled for confounding factors, such as age and noise-induced hearing loss and none of these reports are based on prospective double-blind studies. Further, animal studies report contradictory data. For example, one study (2008) reported hearing loss in rats after long-term and high-dose exposure to sildenafil citrate. The other study (2012) showed vardenafil, another formulation of PDE5i, to be protective against noise-induced hearing loss in mice and rats. Whether or not clinically relevant doses of sildenafil citrate cause hearing loss in normal subjects (animals or humans) is controversial. One possibility is that PDE5i exacerbates age-related susceptibility to hearing loss in adults. Therefore, we tested sildenafil citrate in C57BL/6J, a strain of mice that displays increased susceptibility to age-related hearing loss, and compared the results to those obtained from the FVB/N, a strain of mice with no predisposition to hearing loss. Six-week-old mice were injected with the maximum tolerated dose of sildenafil citrate (10 mg/kg/day) or saline for 30 days. Auditory brainstem responses (ABRs) were recorded pre- and post injection time points to assess hearing loss. Entry of sildenafil citrate in the mouse cochlea was confirmed by qRT-PCR analysis of a downstream target of the cGMP-PKG cascade. ABR data indicated no statistically significant difference in hearing between treated and untreated mice in both backgrounds. Results show that the maximum tolerated dose of sildenafil citrate administered daily for 4 weeks does not affect hearing in the mouse. Our study gives no indication that Viagra will negatively impact hearing and it emphasizes the need to revisit the issue of Viagra related ototoxicity in humans.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain: POSSIBLE ROLE OF CLARIN-1 IN ORGANIZING THE ACTIN CYTOSKELETON*

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1^−/− mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products.Usher syndrome is the most common cause of human inherited deafness and blindness, accounting for ∼50% of all cases (1). There are three clinical types of Usher syndrome, types I, II, and III (1–3). Usher type I is characterized by profound congenital deafness and vestibular dysfunction, and Usher type II is characterized by moderate to severe deafness. Usher type III is distinguished from types I and II by progressive (non-congenital) deafness together with variable impairment of vestibular function. All Usher types lead to progressive retinal degeneration with a retinitis pigmentosa-like appearance. Five causative genes have been identified for Usher syndrome type I, and three genes for type II (3). The protein products of Usher type I and II genes are functionally heterogeneous, including an unconventional myosin, scaffold proteins, G-protein-coupled receptor, and cadherins. Adding to this heterogeneity, the Usher syndrome type III gene encodes a novel transmembrane protein named clarin-1 (CLRN1)³ (4–6) with an unknown function. The heterogeneity of genes involved in Usher syndrome makes it extremely challenging to elucidate shared and distinctive disease mechanisms.CLRN1 belongs to a superfamily of four-transmembrane proteins that includes the tetraspanin and claudin families. CLRN1 and its paralogues, CLRN2 and CLRN3, form the Clarin family, which is conserved throughout vertebrate species and shows limited sequence homology to the tetraspanins (4). Tetraspanins are considered to be structural proteins that interact laterally with other membrane proteins such as ion channels, integrins, and other tetraspanins (7, 8) to form tetraspanin-enriched microdomains. Tetraspanin-enriched microdomains embody other proteins to allow localized transmission of signals, cell-cell adhesion/fusion, cell-matrix interactions, and/or formation of diffusion barriers against small molecules. Similar to tetraspanins, CLRN1 retains only limited hydrophilic regions exposed to cytoplasmic or extracellular aqueous phases (Fig. 1A) and, apparently, lacks any functional domains. Although CLRN1 is structurally related and similar to tetraspanins, it is currently unknown whether CLRN1 can form specific microdomains. The question also remains as to what one or more functions CLRN1 microdomains serve if indeed they do exist.Open in a separate windowFIGURE 1.CLRN1 is a plasma membrane protein localized at F-actin-enriched protrusions. A, the topology and transmembrane domains shown were predicted with the HMMTOP transmembrane topology prediction server (55). The possible N-linked glycosylation site is indicated. Also shown (red circle) is the previously predicted motif near the CLRN1 C-terminal tail that may serve as a PDZ-binding site (4). B, immunolocalization of Human WT CLRN1. C, immunolocalization of Na/K ATPase in HEK293 cells stably expressing CLRN1. D, merged image of B and C indicates that CLRN1 and Na/K ATPase co-localize. Images B–D are single optical sections of HEK293 cells. E, cell surface biotinylation was performed to separate cell surface proteins (avidin-bound) (AB) from intracellular proteins (flow-through) (FT). Immunoblots of both fractions reveal that most of the CLRN1 protein localized to the plasma membrane. HEK293 cells alone and HEK293 cells expressing CLRN1 were preincubated for 30 min with Sulfo-NHS-SS-Biotin to label cell surface proteins. After cells were harvested, biotin-labeled CLRN1 protein levels were measured by immunoblotting. F, localization of human WT CLRN1 in HEK293 cells stably expressing CLRN1. G, F-actin in HEK293 cells stably expressing CLRN1. F-actins were labeled with phalloidin-Alexa 488. H, merged image of F and G. CLRN1 localized at both microvilli (arrows) and lamellipodia (arrowheads). I–K, CLRN1 localization studied by immunofluorescence confocal microscopy after disruption of F-actin by cytochalasin D treatment. I, CLRN1 localized diffusely on the plasma membrane. J, F-actin localization is shown. K, merged image of I and J. After disruption of F-actin, CLRN1 and F-actin no longer co-localize. Images F–K were generated from multiple optical sections by a maximum intensity projection. Scale bars, 50 μm.CLRN1 is expressed in sensory hair cells (4) where it may interact with other co-existing Usher gene products or cellular machinery essential for the maintenance of these cells. Increasing evidence suggests that products of Usher type I and II genes form large networks of interacting proteins, and that F-actin plays a major role in organizing these networks (reviewed in Refs. 2, 9). The core of these networks is the Usher type IC gene product, Harmonin, which interacts directly with F-actin in vitro and stabilizes F-actin when it is expressed heterologously in HeLa cells (10). Harmonins retain multiple PDZ domains dedicated to interacting with products of Usher type I and type II genes (reviewed in Refs. 2, 9) and also serve as PDZ domain-based scaffolds to anchor Usher proteins to F-actin. A link between Usher gene products and actin-based organelles also has been established in vivo. In Usher syndrome I and II mouse models, the actin-enriched stereocilia are morphologically and functionally defective (11–14). Because the causative gene for Usher type III was identified more recently than those of Usher types I and II, little is known about the pathogenesis of Usher syndrome III. Epistatic interactions between Usher syndrome type IB and Usher syndrome III may suggest linkage among CLRN1, Myosin VIIa, and F-actin (15). Clinically, patients with the N48K CLRN1 mutation have a rod and cone degenerative phenotype similar to Usher type IIA patients (16), suggesting a common pathological pathway for Usher types IIA and III. Despite the genetic and phenotypic characterization in humans, the molecular function of CLRN1 remains elusive, as well as its relationship and interaction with other Usher gene products. Therefore, identifying possible interactive partners of CLRN1 should improve understanding of the function of CLRN1 and the common pathological pathways of progressive hearing and vision loss in the Usher syndromes.Here we investigated whether CLRN1 can form microdomains similar to the tetraspanin-enriched microdomain, and if so, what the function of such microdomains might be. Our studies indicate that CLRN1 forms membranous cholesterol-rich compartments on plasma membranes and interacts with and regulates the machinery involved in actin filament organization. To understand the pathogenesis of Usher syndrome, we asked whether and how the Usher syndrome III causative mutation, N48K, results in dysfunction of the clarin-1-enriched microdomains involved in organizing actin. To determine whether Clrn1 is involved in the regulation of actin cytoskeleton in vivo, we studied the structure of F-actin-enriched stereocilia bundles in Clrn1^−/− mouse. Because actin provides important scaffolds in Usher interactome, the observations described herein provide a novel molecular link between CLRN1 and the identified gene products of Usher types I and II.     

Surgical Induction of Endolymphatic Hydrops by Obliteration of the Endolymphatic Duct

Surgical induction of endolymphatic hydrops (ELH) in the guinea pig by obliteration and obstruction of the endolymphatic duct is a well-accepted animal model of the condition and an important correlate for human Meniere''s disease. In 1965, Robert Kimura and Harold Schuknecht first described an intradural approach for obstruction of the endolymphatic duct (Kimura 1965). Although effective, this technique, which requires penetration of the brain''s protective covering, incurred an undesirable level of morbidity and mortality in the animal subjects. Consequently, Andrews and Bohmer developed an extradural approach, which predictably produces fewer of the complications associated with central nervous system (CNS) penetration.(Andrews and Bohmer 1989) The extradural approach described here first requires a midline incision in the region of the occiput to expose the underlying muscular layer. We operate only on the right side. After appropriate retraction of the overlying tissue, a horizontal incision is made into the musculature of the right occiput to expose the right temporo-occipital suture line. The bone immediately inferio-lateral the suture line (Fig 1) is then drilled with an otologic drill until the sigmoid sinus becomes visible. Medial retraction of the sigmoid sinus reveals the operculum of the endolymphatic duct, which houses the endolymphatic sac. Drilling medial to the operculum into the area of the endolymphatic sac reveals the endolymphatic duct, which is then packed with bone wax to produce obstruction and ultimately ELH. In the following weeks, the animal will demonstrate the progressive, fluctuating hearing loss and histologic evidence of ELH.Open in a separate windowClick here to view.^{(41M, flv)}     

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.