Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their ...bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.
The attachment of lysine 48 (Lys48)-linked polyubiquitin chains to proteins is a universal signal for degradation by the proteasome. Here, we report that long Lys48-linked chains are resistant to ...many deubiquitinating enzymes (DUBs). Representative enzymes from this group, Ubp15 from yeast and its human ortholog USP7, rapidly remove mono- and diubiquitin from substrates but are slow to remove longer Lys48-linked chains. This resistance is lost if the structure of Lys48-linked chains is disrupted by mutation of ubiquitin or if chains are linked through Lys63. In contrast to Ubp15 and USP7, Ubp12 readily cleaves the ends of long chains, regardless of chain structure. We propose that the resistance to many DUBs of long, substrate-attached Lys48-linked chains helps ensure that proteins are maintained free from ubiquitin until a threshold of ubiquitin ligase activity enables degradation.
Deubiquitinating enzymes (DUBs) remove polyubiquitin chains from proteins.
Protein-attached monoubiquitin is rapidly removed by many DUBs, but polyubiquitin chains linked through lysine 48 are resistant to cleavage.
Intramolecular chain interactions limit DUB activity toward polyubiquitin chains.
High activity toward monoubiquitin allows many DUBs to minimize inappropriate ubiquitin signaling.
The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread ...of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive "LAMPshade" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.
Genetic Effects on Age-Dependent Onset and Islet Cell Autoantibody Markers in Type 1 Diabetes
Jinko Graham 1 ,
William A. Hagopian 2 ,
Ingrid Kockum 3 ,
Lou Sheng Li 3 ,
Carani B. Sanjeevi 3 ,
Robert ...M. Lowe 4 ,
Jonathan B. Schaefer 4 ,
Marjan Zarghami 4 ,
Heather L. Day 4 ,
Mona Landin-Olsson 5 ,
Jerry P. Palmer 4 ,
Marta Janer-Villanueva 6 ,
Leroy Hood 6 ,
Göran Sundkvist 7 ,
Åke Lernmark 4 ,
Norman Breslow 8 ,
Gisela Dahlquist 9 ,
for the Swedish Childhood Diabetes Study Group ,
Göran Blohmé 10 and
for the Diabetes Incidence in Sweden Study Group
1 Department of Statistics and Actuarial Science, Simon Fraser University, Burnaby, Canada
2 Pacific Northwest Research Institute, Seattle, Washington
3 Karolinska Institute, Department of Molecular Medicine, Stockholm, Sweden
4 Department of Medicine, University of Washington, Seattle, Washington
5 Department of Medicine, University of Lund, Lund, Sweden
6 Institute for Systems Biology, Seattle, Washington
7 Department of Endocrinology, University Hospital Malmö, University of Lund, Lund, Sweden
8 Department of Biostatistics, University of Washington, Seattle, Washington
9 Department of Paediatrics, Umeå University, Umeå, Sweden
10 Diabetes Center, Södersjukhuset, Stockholm, Sweden
Abstract
Age-dependent associations between type 1 diabetes risk genes HLA, INS VNTR, and CTLA-4 and autoantibodies to GAD65 (GADAs),
ICA512/IA-2, insulin, and islet cells were determined by logistic regression analysis in 971 incident patients with type 1
diabetes and 702 control subjects aged 0–34 years. GADAs were associated with HLA-DQ2 in young but not in older patients ( P = 0.009). Autoantibodies to insulin were negatively associated with age ( P < 0.0001) but positively associated with DQ8 ( P = 0.03) and with INS VNTR ( P = 0.04), supporting possible immune tolerance induction. ICA512/IA-2 were negatively associated with age ( P < 0.0001) and with DQ2 ( P < 0.0001) but positively associated with DQ8 ( P = 0.04). Males were more likely than females to be negative for GADA ( P < 0.0001), autoantibodies to islet cells ( P = 0.04), and all four autoantibody markers ( P = 0.004). The CTLA-4 3′ end microsatellite marker was not associated with any of the autoantibodies. We conclude that age
and genetic factors such as HLA-DQ and INS VNTR need to be combined with islet autoantibody markers when evaluating the risk
for type 1 diabetes development.
Footnotes
Address correspondence and reprint requests to Åke Lernmark, University of Washington Department of Medicine, R.H. Williams
Laboratory, 1959 N.E. Pacific St., Box 357710 Seattle, WA 98195-7710. E-mail: ake{at}u.washington.edu .
Received for publication 15 November 2001 and accepted in revised form 24 January 2002.
*Authors are listed in the acknowledgments .
GADA, GAD65 autoantibody; IA-2A, ICA512/IA-2 autoantibody; IAA, insulin autoantibody; ICA islet cell autoantibody; IRD, infrared
dye; JDF, Juvenile Diabetes Foundation; VNTR, variable number tandem repeat, WHO, World Health Organization.
DIABETES
Porphyria cutanea tarda (PCT) is characterized by a skin blistering eruption that develops in sun exposed areas of the skin. It is the most common cutaneous porphyria world-wide, and classically ...associated with hepatic injury but also estrogen use, cigarette smoking, and HIV. In any case of photodistributed persistent blistering skin condition, PCT must be high on the differential. This case of a carpenter diagnosed with PCT not only illustrates a classic case but also the opportunity to achieve significant response to therapy in a motivated patient particularly with improved access to direct-acting antivirals (DAA) for hepatitis C treatment.
Agents that increase intracellular cAMP inhibit the activation and function of T cells and can lead to cell death. Recently, it has been postulated that cAMP inhibits T cell function in large part by ...acting as a brake on the T cell receptor and costimulatory receptor pathways. Therefore, for full activation of the T cell to occur, this inhibitory influence must be removed. One likely mechanism for accomplishing this is by up-regulation and/or activation of specific cyclic nucleotide phosphodiesterases (PDEs), and such a mechanism for one phosphodiesterase, PDE7A1, has been reported. In this paper, we extend this mechanism to another isozyme variant of the same PDE family, PDE7A3. We also report the full-length sequence of human PDE8A1 and show that it also is induced in response to a combination of T cell receptor and costimulatory receptor pathway activation. However, the time course for induction of PDE8A1 is slower than that of PDE7A1. The basal level measured and, therefore, the apparent fold induction of PDE7A1 mRNA and protein depend in large part on the method of isolation of the T cells. On the other hand, regardless of the isolation method, the basal levels of PDE7A3 and PDE8A1 are very low and fold activation is much higher. Constitutively expressed PDE8A1 and PDE7A3 also have been isolated from a human T cell line, Hut78.
The Jun family of activator protein 1 (AP-1) transcription factors (c-Jun, JunB, and JunD) is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and ...hypertrophy. The role of individual AP-1 transcription factors in the stressed heart is not clear. In the present study we analyzed the role of JunD in survival, hypertrophy, and angiogenesis in the pressure-overloaded mouse heart after thoracic aortic constriction.
Mice lacking JunD (knockout KO) showed increased mortality and enhanced cardiomyocyte apoptosis and fibrosis associated with increased levels of hypoxia-induced factor-1alpha, vascular endothelial growth factor (VEGF), p53, and Bax protein and reduced levels of Bcl-2 protein after 7 days of severe pressure overload compared with wild-type (WT) siblings. Cardiomyocyte hypertrophy in surviving KO mice was enhanced compared with that in WT mice. Chronic moderate pressure overload for 12 weeks caused enhanced left ventricular hypertrophy in KO mice, and survival and interstitial fibrosis were comparable with WT mice. Cardiac function, 12 weeks after operation, was comparable among shams and pressure-overloaded mice of both genotypes. In addition, KO mice exposed to chronic pressure overload showed higher cardiac capillary density associated with increased protein levels of VEGF.
Thus, JunD limits cardiomyocyte hypertrophy and protects the pressure-overloaded heart from cardiac apoptosis. These beneficial effects of JunD, however, are associated with antiangiogenic properties.
Here we report the cloning, expression, and characterization of a cAMP-specific phosphodiesterase (PDE) from Trypanosoma brucei (TbPDE2B). Using a bioinformatic approach, two different expressed ...sequence tag clones were identified and used to isolate the complete sequence of two identical PDE genes arranged in tandem. Each gene consists of 2,793 bases that predict a protein of 930 aa with a molecular mass of 103.2 kDa. Two GAF (for cGMP binding and stimulated PDEs, Anabaena adenylyl cyclases, and Escherichia coli FhlA) domains, similar to those contained in many signaling molecules including mammalian PDE2, PDE5, PDE6, PDE10, and PDE11, were located N-terminal to a consensus PDE catalytic domain. The catalytic domain is homologous to the catalytic domain of all 11 mammalian PDEs, the Dictyostelium discoideum RegA, and a probable PDE from Caenorhabditis elegans. It is most similar to the T. brucei PDE2A (89% identity). TbPDE2B has substrate specificity for cAMP with a Kmof 2.4 µM. cGMP is not hydrolyzed by TbPDE2B nor does this cyclic nucleotide modulate cAMP PDE activity. The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine, papaverine and pentoxifyline are poor inhibitors of TbPDE2B. Similarly, PDE inhibitors selective for the mammalian PDE families 2, 3, 5, and 6 (erythro-9-3-(2-hydroxynonyl)-adenine, enoximone, zaprinast, and sildenafil) were also unable to inhibit this enzyme. However, dipyridamole was a reasonably good inhibitor of this enzyme with an IC50of 27 µM. cAMP plays key roles in cell growth and differentiation in this parasite, and PDEs are responsible for the hydrolysis of this important second messenger. Therefore, parasite PDEs, including this one, have the potential to be attractive targets for selective drug design.
RB from a Bud's Eye View Schaefer, Jonathan B; Breeden, Linda L
Cell,
06/2004, Letnik:
117, Številka:
7
Book Review, Journal Article
Recenzirano
Odprti dostop
RB and related proteins block transcriptional activation of genes critical to initiation of the cell cycle and suppress unwanted cell division. The circuitry controlling this response is generally ...conserved from humans to yeast, but no negative regulator like RB has been found in yeast. In this issue of
Cell, two studies
(Costanzo et al., 2004; de Bruin et al., 2004) reveal that Whi5 appears to play the role of RB in preventing precocious cell cycle entry in budding yeast.
Wild bank voles (Clethrionomys glareolus) may develop diabetes in laboratory captivity. The aim of this study was to test whether bank voles develop type 1 diabetes in association with Ljungan virus. ...Two groups of bank voles were analyzed for diabetes, pancreas histology, autoantibodies to glutamic acid decarboxylase (GAD65), IA-2, and insulin by standardized radioligand-binding assays as well as antibodies to in vitro transcribed and translated Ljungan virus antigens. Group A represented 101 trapped bank voles, which were screened for diabetes when euthanized within 24 hours of capture. Group B represented 67 bank voles, which were trapped and kept in the laboratory for 1 month before being euthanized. Group A bank voles did not have diabetes. Bank voles in group B (22/67; 33%) developed diabetes due to specific lysis of pancreatic islet beta cells. Compared to nondiabetic group B bank voles, diabetic animals had increased levels of GAD65 (P < .0001), IA-2 (P < .0001), and insulin (P = .03) autoantibodies. Affected islets stained positive for Ljungan virus, a novel picorna virus isolated from bank voles. Ljungan virus inoculation of nondiabetic wild bank voles induced beta-cell lysis. Compared to group A bank voles, Ljungan virus antibodies were increased in both nondiabetic (P < .0001) and diabetic (P = .0015) group B bank voles. Levels of Ljungan virus antibodies were also increased in young age at onset of newly diagnosed type 1 diabetes in children (P < .01). These findings support the hypothesis that the development of type 1 diabetes in captured wild bank voles is associated with Ljungan virus. It is speculated that bank voles may have a possible zoonotic role as a reservoir and vector for virus that may contribute to the incidence of type 1 diabetes in humans.
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