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1.
Invest Ophthalmol Vis Sci ; 52(6): 3174-80, 2011 May 16.
Article in English | MEDLINE | ID: mdl-21330663

ABSTRACT

PURPOSE: Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS. METHOD: Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmer's tear test (STT) was used to measure tear production. RESULTS: SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed. CONCLUSIONS: SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.


Subject(s)
Dog Diseases/drug therapy , Keratoconjunctivitis Sicca/veterinary , Lymphocyte Function-Associated Antigen-1/drug effects , Ophthalmic Solutions/pharmacology , Administration, Topical , Animals , Cell Adhesion/drug effects , Cytokines/metabolism , Dog Diseases/diagnosis , Dogs , Dose-Response Relationship, Drug , Female , Humans , Intercellular Adhesion Molecule-1/metabolism , Jurkat Cells/metabolism , Keratoconjunctivitis Sicca/drug therapy , Leukocytes, Mononuclear/drug effects , Lymphocyte Activation/drug effects , Male , Ophthalmic Solutions/pharmacokinetics
3.
Invest Ophthalmol Vis Sci ; 51(10): 5198-204, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20445119

ABSTRACT

PURPOSE: To determine the pharmacokinetics of SAR 1118, a small-molecule antagonist of leukocyte function-associated antigen (LFA)-1, after administration of ophthalmic drops in normal rats, and to determine its pharmacologic activity by assessing the inhibition of retinal leukostasis and vascular leakiness in a streptozotocin (STZ)-induced diabetic retinopathy model. METHODS: The ocular pharmacokinetics of SAR 1118 were studied in rats after a single topical dose of (14)C-SAR 1118 (1 mg/eye; 40 µCi; 15.5 µL). SAR 1118 concentration time profiles in plasma and ocular tissues were quantified by liquid scintillation counting (LSC). The pharmacologic activity of SAR 1118 eye drops administered thrice daily for 2 months at 1% (0.3 mg/eye/d) and 5% (1.5 mg/eye/d) was assessed in an STZ-induced diabetic rat model by determining retinal leukostasis and blood-retinal barrier breakdown. Diabetic rats treated with periocularly administered celecoxib microparticles served as the positive control, and vehicle-treated rats served as the negative control. RESULTS: A single dose of 6.5% (14)C-radiolabeled SAR 1118 ophthalmic drops delivered retinal drug levels greater than 1 µM in less than 30 minutes and sustained levels greater than 100 nM for 8 hours. SAR 1118 eye drops significantly reduced leukostasis and blood-retinal barrier breakdown in a dose-dependent manner. CONCLUSIONS: SAR 1118 ophthalmic drops administered thrice daily deliver therapeutic levels of SAR 1118 in the retina and can alleviate the retinal complications associated with diabetes.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetic Retinopathy/drug therapy , Lymphocyte Function-Associated Antigen-1/drug effects , Ophthalmic Solutions/administration & dosage , Retina/drug effects , Administration, Topical , Animals , Blood-Retinal Barrier/drug effects , Capillary Permeability/drug effects , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/metabolism , Diabetic Retinopathy/pathology , Dose-Response Relationship, Drug , Leukostasis/prevention & control , Male , Microscopy, Confocal , Ophthalmic Solutions/pharmacokinetics , Rats , Rats, Inbred BN , Rats, Sprague-Dawley , Retina/metabolism , Treatment Outcome
4.
Curr Opin Neurobiol ; 15(4): 431-6, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16023852

ABSTRACT

Neurons of the visual, auditory and vestibular systems that signal through graded changes in membrane potential rely upon synaptic ribbons for the exquisite control of neurotransmitter release. Although clearly important for tonic neurotransmission, the precise role of synaptic ribbons remains elusive. In recent years, several genetic, biochemical, electrophysiological and optical approaches have begun to shed light on the functions of these enigmatic organelles.


Subject(s)
Exocytosis/physiology , Synapses/metabolism , Synapses/ultrastructure , Animals , Eye Proteins , Humans , Synaptic Vesicles/ultrastructure
5.
Proc Natl Acad Sci U S A ; 101(16): 6068-73, 2004 Apr 20.
Article in English | MEDLINE | ID: mdl-15073335

ABSTRACT

We identify Rpa12p of RNA polymerase I (Pol I) as a termination factor. Combined analyses using transcription run-on, electron microscopy-visualized chromatin spreading and RT-PCR have been applied to the rRNA-encoding genes of Saccharomyces cerevisiae. These confirm that Pol I termination occurs close to the Reb1p-dependent terminator in wild-type strains. However, deletion mutants for the 3' end-processing enzyme Rnt1p or the Rpa12p subunit of Pol I both show Pol I transcription in the spacer. For Deltarpa12, these spacer polymerases are devoid of nascent transcripts, suggesting they are immediately degraded. The homology of Rpa12p to the small subunit Rpb9p of Pol II and Rpc11p of Pol III, both implicated in transcriptional termination, points to a common termination mechanism for all three classes of RNA polymerase.


Subject(s)
RNA Polymerase I/physiology , Saccharomyces cerevisiae Proteins/physiology , Transcription, Genetic/physiology , Base Sequence , DNA Primers , DNA, Ribosomal/genetics , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics
6.
Science ; 300(5623): 1284-8, 2003 May 23.
Article in English | MEDLINE | ID: mdl-12764195

ABSTRACT

The capsaicin receptor (TRPV1), a heat-activated ion channel of the pain pathway, is sensitized by phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis after phospholipase C activation. We identify a site within the C-terminal domain of TRPV1 that is required for PIP2-mediated inhibition of channel gating. Mutations that weaken PIP2-TRPV1 interaction reduce thresholds for chemical or thermal stimuli, whereas TRPV1 channels in which this region is replaced with a lipid-binding domain from PIP2-activated potassium channels remain inhibited by PIP2. The PIP2-interaction domain therefore serves as a critical determinant of thermal threshold and dynamic sensitivity range, tuning TRPV1, and thus the sensory neuron, to appropriately detect heat under normal or pathophysiological conditions.


Subject(s)
Phosphatidylinositol 4,5-Diphosphate/metabolism , Receptors, Drug/chemistry , Receptors, Drug/metabolism , Amino Acid Sequence , Animals , Arsenicals/pharmacology , Binding Sites , Capsaicin/metabolism , Capsaicin/pharmacology , Carrier Proteins , ErbB Receptors/metabolism , Hot Temperature , Humans , Ion Channel Gating , Membrane Proteins , Molecular Sequence Data , Mutation , Oocytes , Patch-Clamp Techniques , Phosphorylation , Potassium Channels, Inwardly Rectifying/chemistry , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Protein Structure, Tertiary , Rats , Receptor, trkA/metabolism , Receptors, Drug/genetics , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Type C Phospholipases/metabolism , Xenopus
7.
Proc Natl Acad Sci U S A ; 99(13): 8796-801, 2002 Jun 25.
Article in English | MEDLINE | ID: mdl-12077310

ABSTRACT

Transcriptional interference between genes and the regulatory elements of simple eukaryotes such as Saccharomyces cerevisiae is an unavoidable consequence of their compressed genetic arrangement. We have shown previously that with the tandem arranged genes GAL10 and GAL7, inefficient transcriptional termination of the upstream gene inhibits initiation of transcription on the downstream gene. We now show that transcriptional interference can occur also with S. cerevisiae RNA polymerase II genes arranged convergently. We demonstrate that when the GAL10 and GAL7 genes are rearranged in a convergent orientation, transcriptional initiation occurs at full levels. However, as soon as the two transcripts begin to overlap, elongation is restricted, resulting in a severe reduction in steady-state mRNA accumulation. This effect is observed only in cis arrangement, arguing against RNA-interference effects acting on the potential generation of antisense transcripts. These data reinforce the necessity of separating adjacent RNA polymerase II transcription units by efficient termination signals.


Subject(s)
Genes, Fungal , Saccharomyces cerevisiae/genetics , Transcription, Genetic , Base Sequence , Blotting, Northern , DNA Primers , Saccharomyces cerevisiae/growth & development
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