Altered cellular localisation and expression, together with unconventional protein trafficking, of prion protein, PrP, in type 1 diabetes.

Aims/hypothesis: Normal cellular prion protein (PrP) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrP is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrP (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases.

Monogenic Diabetes and Integrated Stress Response Genes Display Altered Gene Expression in Type 1 Diabetes.

Type 1 diabetes (T1D) has a multifactorial autoimmune etiology, involving environmental prompts and polygenic predisposition. We hypothesized that pancreata from individuals with and at risk for T1D would exhibit dysregulated expression of genes associated with monogenic forms of diabetes caused by nonredundant single-gene mutations. Using a "monogenetic transcriptomic strategy," we measured the expression of these genes in human T1D, autoantibody-positive (autoantibody+), and control pancreas tissues with real-time quantitative PCR in accordance with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.

Temporal Analysis of Amylase Expression in Control, Autoantibody-Positive, and Type 1 Diabetes Pancreatic Tissues.

Within the human pancreas, exocrine and endocrine cells control secretion of digestive enzymes and production of hormones to maintain metabolic homeostasis, respectively. While the vast majority of type 1 diabetes research efforts have focused on endocrine function and autoimmunity, recent studies identified a series of unique features (e.g.

A Map of Human Type 1 Diabetes Progression by Imaging Mass Cytometry.

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing β cells. A comprehensive picture of the changes during T1D development is lacking due to limited sample availability, inability to sample longitudinally, and the paucity of technologies enabling comprehensive tissue profiling. Here, we analyzed 1,581 islets from 12 human donors, including eight with T1D, using imaging mass cytometry (IMC).

C-terminal proline deletions in KCNC3 cause delayed channel inactivation and an adult-onset progressive SCA13 with spasticity.

Mutations in the potassium channel gene KCNC3 (Kv3.3) cause the autosomal dominant neurological disease, spinocerebellar ataxia 13 (SCA13). In this study, we expand the genotype-phenotype repertoire of SCA13 by describing the novel KCNC3 deletion p.

Persistence of Pancreatic Insulin mRNA Expression and Proinsulin Protein in Type 1 Diabetes Pancreata.

The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β cells has recently been questioned as small amounts of C-peptide are detectable in patients with long-standing disease. We analyzed protein and gene expression levels for proinsulin, insulin, C-peptide, and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+), and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, proinsulin and INS mRNA were detected in the majority of T1D pancreata.

A KCNC3 mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking.

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.

Endothelin-1-mediated vasoconstriction alters cerebral gene expression in iron homeostasis and eicosanoid metabolism.

Endothelins are potent vasoconstrictors and signaling molecules. Their effects are broad, impacting processes ranging from neurovascular and cardiovascular health to cell migration and survival. In stroke, traumatic brain injury or subarachnoid hemorrhage, endothelin-1 (ET-1) is induced resulting in cerebral vasospasm, ischemia, reperfusion and the activation of various pathways.

KCNC3(R420H), a K(+) channel mutation causative in spinocerebellar ataxia 13 displays aberrant intracellular trafficking.

Spinocerebellar ataxia 13 (SCA13) is an autosomal dominant disease resulting from mutations in KCNC3 (Kv3.3), a voltage-gated potassium channel. The KCNC3(R420H) mutation was first identified as causative for SCA13 in a four-generation Filipino kindred with over 20 affected individuals.

A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer, eRNA transcription and the innate immune response.

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli.

Characterization of a mechanism to inhibit ovarian follicle activation.

Objective: To demonstrate that a small molecule can induce the transcription factor Foxo3 in the ovary and lead to inhibition of follicle activation.

Design: Cell culture, organ culture, and animal studies.

Setting: University-based laboratory.

Mutation in the kv3.3 voltage-gated potassium channel causing spinocerebellar ataxia 13 disrupts sound-localization mechanisms.

Normal sound localization requires precise comparisons of sound timing and pressure levels between the two ears. The primary localization cues are interaural time differences, ITD, and interaural level differences, ILD. Voltage-gated potassium channels, including Kv3.

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression.

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions.

Effect of allergy and inflammation on eicosanoid gene expression in CFTR deficiency.

Background: Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2-15% of patients. We hypothesized that sensitization/challenge of CFTR(-/-) mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF.

Methods: FABP-hCFTR(+/-)-CFTR(-/-) mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1β requires a distal enhancer element with a unique role for C/EBPβ.

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1).

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells.

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway.

cPLA(2)α gene activation by IL-1β is dependent on an upstream kinase pathway, enzymatic activation and downstream 15-lipoxygenase activity: a positive feedback loop.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences.

Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis.

Superoxide dismutase (SOD) enzymes are critical in controlling levels of reactive oxygen species (ROS) that are linked to aging, cancer, and neurodegenerative disease. Superoxide (O(2)(*-)) produced during respiration is removed by the product of the SOD2 gene, the homotetrameric manganese superoxide dismutase (MnSOD). Here, we examine the structural and catalytic roles of the highly conserved active-site residue Tyr34, based upon structure-function studies of MnSOD enzymes with mutations at this site.

Distinct functions of CCAAT enhancer-binding protein isoforms in the regulation of manganese superoxide dismutase during interleukin-1beta stimulation.

The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1beta is mediated through a complex intronic enhancer element.

The kinetic mechanism of manganese-containing superoxide dismutase from Deinococcus radiodurans: a specialized enzyme for the elimination of high superoxide concentrations.

Deinococcus radiodurans (Drad), a bacterium with an extraordinary capacity to tolerate high levels of ionizing radiation, produces only a manganese-containing superoxide dismutase (MnSOD). As MnSOD has been shown to remove superoxide radical with varying efficiency depending upon its cellular origin, a comparison of the Drad MnSOD efficiency with that of both human and Escherichia coli MnSODs was undertaken. Pulse radiolysis studies demonstrate that, under identical ratios of enzyme to superoxide radical, the dismutation efficiencies scaled as Drad MnSOD > E.

Metabolic regulation of manganese superoxide dismutase expression via essential amino acid deprivation.

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase.

Engineering and characterization of human manganese superoxide dismutase mutants with high activity and low product inhibition.

Human manganese superoxide dismutase is a mitochondrial metalloenzyme that is involved in protecting aerobic organisms against superoxide toxicity, and has been implicated in slowing tumor growth. Unfortunately, this enzyme exhibits strong product inhibition, which limits its potential biomedical applications. Previous efforts to alleviate human manganese superoxide dismutase product inhibition utilized rational protein design and site-directed mutagenesis.

Structural mobility in human manganese superoxide dismutase.

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine.

Enhanced IgE allergic response to Aspergillus fumigatus in CFTR-/- mice.

To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections.