Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship ...between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).
Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.
Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.
These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids ...with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.
Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the ...relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration.
Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature.
Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen.
The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we ...identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15 (-/-) mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15 (-/-) liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
1 Department of Anatomy & Physiology, Kansas State University, Manhattan, Kansas; and 2 NuGEN Technologies, Inc., San Carlos, California
Submitted 25 May 2004
; accepted in final form 17 December ...2004
Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 µg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
gene expression microarray analysis; microdissection; nucleic acid amplification techniques
Address for reprint requests and other correspondence: P. Wangemann, Dept. of Anatomy & Physiology, College of Veterinary Medicine, Kansas State Univ., 1600 Denison Ave., Coles Hall 205, Manhattan, KS 66506 (E-mail: wange{at}vet.k-state.edu )
Six cases of esophageal squamous cell carcinoma were identified in six captive adult Pacific (Phoca vitulina richardsii; n = 2) and Atlantic (Phoca vitulina concolor; n = 4) harbor seals. These seals ...presented with intermittent dysphagia, regurgitation, inappetence, and abnormal posturing. Common clinical pathology findings in these seals included azotemia, hyperproteinemia, hyperglobulinemia, and leukocytosis. Gastrointestinal endoscopy commonly revealed an ulcerated mass near the gastroesophageal junction. Each seal was euthanized (n = 3) due to poor prognosis, subsequently died while undergoing an anesthetic procedure (n = 2), or found dead (n = 1). The diagnosis of squamous cell carcinoma was confirmed via biopsy of esophageal mucosa during endoscopy or histopathologic examination of affected tissues after necropsy. On the basis of clinical and postmortem findings, esophageal squamous cell carcinoma should be considered as a differential diagnosis in aged harbor seals exhibiting clinical signs of regurgitation, decreased appetite or anorexia, vomiting, and/or abnormal posturing.
Background: Primary Hyperoxaluria Type 1 (PH1) is an inborn error of metabolism caused by mutations in the AGXT gene, which encodes for the hepatocyte-specific enzyme alanine: glyoxylate ...aminotransferase (AGT). AGT catalyzes the conversion of glyoxylate to glycine in the peroxisome and prevents the build-up of oxalate which occurs in PH1. This causes nephrocalcinosis, systemic oxalosis, and end-stage renal disease. Liver transplant is currently the only curative treatment available. Although a mouse model has previously been generated, the severity of the reported disease phenotype varies, and a better understanding of the genotype-phenotype relationship in both the mouse model and human disease is needed. Methods: We developed an Agxt-/- mouse model using CRISPR/Cas9-mediated gene editing. We performed a natural history study and ethylene glycol (EG) challenge to evaluate the phenotype of this mouse. Results: Agxt-/- mice had elevated plasma glycolate, urine glycolate, and urine oxalate levels compared to Agxt+/+ mice. A small subset of Agxt-/- mice developed minimal nephrocalcinosis (1/8 at 12 weeks, 1/8 at 26 weeks, 0/8 at 39 weeks, and 3/7 at 52 weeks of age). When challenged with 0.7% or 1.2% EG in drinking water for 3 weeks, 2/10 Agxt-/- mice developed nephrocalcinosis. Agxt2mRNA and protein expression were unchanged between Agxt-/- and Agxt+/+ mice. Hydroxy acid oxidase 1(Hao1) messenger ribonucleic acid (mRNA) levels were unchanged, but the corresponding glycolate oxidase protein was increased in Agxt-/- mice. Conclusion: We have created an Agxt-/- mouse model which resembles much of the clinical phenotype of PH1 patients and will be a useful tool in developing novel therapies for this devastating disease. JBCGenetics 2019; 2(1.000): 28-39
Pendred syndrome, characterized by childhood deafness and postpuberty goiter, is caused by mutations of SLC26A4, which codes for the anion exchanger pendrin. The goal of the present study was to ...determine how loss of pendrin leads to hair cell degeneration and deafness. We evaluated pendrin function by ratiometric microfluorometry, hearing by auditory brain stem recordings, and expression of K(+) and Ca(2+) channels by confocal immunohistochemistry. Cochlear pH and Ca(2+) concentrations and endocochlear potential (EP) were measured with double-barreled ion-selective microelectrodes. Pendrin in the cochlea was characterized as a formate-permeable and DIDS-sensitive anion exchanger that is likely to mediate HCO(3)(-) secretion into endolymph. Hence endolymph in Slc26a4(+/-) mice was more alkaline than perilymph, and the loss of pendrin in Slc26a4(-/-) mice led to an acidification of endolymph. The stria vascularis of Slc26a4(-/-) mice expressed the K(+) channel Kcnj10 and generated a small endocochlear potential before the normal onset of hearing at postnatal day 12. This small potential and the expression of Kcnj10 were lost during further development, and Slc26a4(-/-) mice did not acquire hearing. Endolymphatic acidification may be responsible for inhibition of Ca(2+) reabsorption from endolymph via the acid-sensitive epithelial Ca(2+) channels Trpv5 and Trpv6. Hence the endolymphatic Ca(2+) concentration was found elevated in Slc26a4(-/-) mice. This elevation may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells. Degeneration of the hair cells closes a window of opportunity to restore the normal development of hearing in Slc26a4(-/-) mice and possibly human patients suffering from Pendred syndrome.
Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) ...modification, which has a shorter 3′–2′ internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2′-O-methyl or 2′-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2′-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4′-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.
Pendred syndrome, characterized by childhood deafness and postpuberty goiter, is caused by mutations of SLC26A4, which codes for the anion exchanger pendrin. The goal of the present study was to ...determine how loss of pendrin leads to hair cell degeneration and deafness. We evaluated pendrin function by ratiometric microfluorometry, hearing by auditory brain stem recordings, and expression of K+ and Ca... channels by confocal immunohistochemistry. Cochlear pH and Ca... concentrations and endocochlear potential (EP) were measured with double-barreled ion-selective microelectrodes. Pendrin in the cochlea was characterized as a formate-permeable and DIDS-sensitive anion exchanger that is likely to mediate HCO...-secretion into endolymph. Hence endolymph in ... mice was more alkaline than perilymph, and the loss of pendrin in ... mice led to an acidification of endolymph. The stria vascularis of ... mice expressed the K+ channel Kcnj10 and generated a small endocochlear potential before the normal onset of hearing at postnatal day 12. This small potential and the expression of Kcnj10 were lost during further development, and ... mice did not acquire hearing. Endolymphatic acidification may be responsible for inhibition of Ca... reabsorption from endolymph via the acid-sensitive epithelial Ca... channels Trpv5 and Trpv6. Hence the endolymphatic Ca... concentration was found elevated in ... mice. This elevation may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells. Degeneration of the hair cells closes a window of opportunity to restore the normal development of hearing in ... mice and possibly human patients suffering from Pendred syndrome. (ProQuest-CSA LLC: ... denotes formulae/symbols omitted.)
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