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1.
J Vestib Res ; 24(4): 281-8, 2014.
Article in English | MEDLINE | ID: mdl-25095772

ABSTRACT

BACKGROUND: Seasickness is a risk aboard a ship. Histamine is postulated as a causative agent, inversely related to the intake of vitamin C. Persons with mastocytosis experienced improvement of nausea after the intake of vitamin C. OBJECTIVE: To determine whether vitamin C suppresses nausea in 70 volunteers who spent 20 minutes in a life raft, exposed to one-meter-high waves in an indoor pool. METHOD: Double-blind placebo-controlled crossover study. Two grams of vitamin C or placebo was taken one hour before exposure. Blood samples were taken one hour before and after exposure to determine histamine, diamine oxidase, tryptase, and vitamin C levels. Symptom scores were noted on a visual analog scale. On the second day the test persons were asked which day they had felt better. RESULTS: Seven persons without symptoms were excluded from the analysis. Test persons had less severe symptoms after the intake of vitamin C (p < 0.01). Scores on the visual analog scale were in favor of vitamin C, but the difference was not significant. Twenty-three of 63 persons wished to leave the raft earlier: 17 after the intake of placebo and 6 after the intake of vitamin C (p < 0.03). Women (p < 0.02) and men below 27 years of age (p < 0.02) had less pronounced symptoms after the intake of vitamin C. Histamine (p < 0.01) and DAO levels were increased after the intake of vitamin C (p < 0.001) and after placebo (n.s.). The fact that the second test day was rated less stressful by most volunteers is indicative of habituation. CONCLUSIONS: Some of the data show that vitamin C is effective in suppressing symptoms of seasickness, particularly in women and men younger than 27 years of age, and is devoid of side effects. Histamine levels were initially increased after the test persons had been exposed to waves.


Subject(s)
Ascorbic Acid/administration & dosage , Histamine/blood , Motion Sickness/blood , Motion Sickness/drug therapy , Administration, Oral , Adult , Age Factors , Cross-Over Studies , Double-Blind Method , Female , Humans , Male , Middle Aged , Placebos , Sex Factors , Young Adult
2.
Mol Cell Neurosci ; 46(2): 507-15, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21145973

ABSTRACT

Intravitreal injections of recombinant ciliary neurotrophic factor (rCNTF) protect adult rat retinal ganglion cells (RGCs) after injury and stimulate regeneration, an effect enhanced by co-injection with a cAMP analogue (CPT-cAMP). This effect is partly mediated by PKA and associated signaling pathways, but CPT-cAMP also moderates upregulation of suppressor of cytokine signaling (SOCS) pathways after rCNTF injection, which will also enhance the responsiveness of RGCs to this and perhaps other cytokines. We now report that intravitreal injections of CPT-cAMP do not potentiate RGC axonal regeneration when CNTF is expressed via an adeno-associated viral vector (rAAV2), and concomitantly we show that increases in retinal SOCS mRNA expression are less when CNTF is delivered using the vector. We also directly tested the impact of elevated SOCS3 expression on the survival and regeneration of injured adult RGCs by injecting a bicistronic rAAV2-SOCS3-GFP vector into the vitreous of eyes in rats with a peripheral nerve graft sutured onto the cut optic nerve. Overexpression of SOCS3 resulted in an overall reduction in axonal regrowth and almost complete regeneration failure of RGCs transduced with the rAAV2-SOCS3-GFP vector. Furthermore, rAAV2-mediated expression of SOCS3 abolished the normally neurotrophic effects elicited by intravitreal rCNTF injections. In summary, CNTF delivery to the retina using viral vectors may be more effective than bolus rCNTF injections because the gene therapy approach has a less pronounced effect on neuron-intrinsic SOCS repressor pathways. Our new gain of function data using rAAV2-SOCS3-GFP demonstrate the negative impact of enhanced SOCS3 expression on the regenerative potential of mature CNS neurons.


Subject(s)
Axons/metabolism , Ciliary Neurotrophic Factor/administration & dosage , Genetic Therapy/methods , Nerve Regeneration/physiology , Retinal Ganglion Cells/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , Adenoviridae/genetics , Animals , Ciliary Neurotrophic Factor/genetics , Ciliary Neurotrophic Factor/metabolism , Cyclic AMP/administration & dosage , Cyclic AMP/analogs & derivatives , Female , Gene Expression , Genetic Vectors/genetics , Immunohistochemistry , Intravitreal Injections , Microscopy, Confocal , Nerve Regeneration/drug effects , Neuroprotective Agents/administration & dosage , Optic Nerve Injuries/physiopathology , Optic Nerve Injuries/therapy , Polymerase Chain Reaction , RNA, Messenger/analysis , Rats , Rats, Wistar , Recombinant Proteins/administration & dosage , Retinal Ganglion Cells/drug effects , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/drug effects , Transduction, Genetic
3.
Exp Neurol ; 223(1): 207-20, 2010 May.
Article in English | MEDLINE | ID: mdl-19646436

ABSTRACT

Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves.


Subject(s)
Gene Expression Regulation/physiology , Motor Neurons/pathology , Nerve Growth Factors/metabolism , Nerve Regeneration/physiology , Radiculopathy , Replantation/methods , Animals , Cell Survival/physiology , Choline O-Acetyltransferase/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay/methods , Female , Hindlimb/pathology , Lumbosacral Plexus/pathology , Lumbosacral Plexus/physiopathology , Muscle, Skeletal/physiopathology , Nerve Growth Factors/genetics , Radiculopathy/pathology , Radiculopathy/physiopathology , Radiculopathy/surgery , Rats , Rats, Wistar , Sciatic Nerve/metabolism , Sciatic Nerve/pathology , Time Factors
4.
Gene Ther ; 16(4): 521-32, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19092858

ABSTRACT

Recombinant adeno-associated virus (rAAV) vectors are increasingly being used as tools for gene therapy, and clinical trials have begun in patients with genetically linked retinal disorders. Intravitreal injection is optimal for the transduction of retinal ganglion cells (RGCs), although complete selectivity has not been achieved. There may also be advantages in using intravitreal approaches for the transduction of photoreceptors. Here we compared the cellular tropism and transduction efficiency of rAAV2/1, -2/2, -2/3, -2/4, -2/5, -2/6 and -2/8 in adult rat retina after intravitreal injection. Each vector encoded green fluorescent protein (GFP), and the number, laminar distribution and morphology of transduced GFP(+) cells were determined using fluorescent microscopy. Assessment of transduced cell phenotype was based on cell morphology and immunohistochemistry. rAAV2/2 and rAAV2/6 transduced the greatest number of cells, whereas rAAV2/5 and rAAV2/8 were least efficient. Most vectors primarily transduced RGCs; however, rAAV2/6 had a more diverse tropism profile, with 46% identified as amacrine or bipolar cells, 23% as RGCs and 22% as Müller cells. Müller cells were also frequently transduced by rAAV2/4. The highest photoreceptor transduction was seen after intravitreal rAAV2/3 injection. These data facilitate the design and selection of rAAV vectors to target specific retinal cells, potentially leading to an improved gene therapy for various human retinal pathologies.


Subject(s)
Dependovirus/genetics , Genetic Vectors/administration & dosage , Retina/metabolism , Transduction, Genetic , Animals , Dependovirus/classification , Dependovirus/physiology , Female , Injections, Intraocular , Microscopy, Confocal , Rats , Rats, Wistar , Retina/virology , Serotyping , Viral Tropism , Vitreous Body
5.
J Virol ; 75(2): 961-70, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11134309

ABSTRACT

We have studied the infection pathway of Autographa californica multinuclear polyhedrosis virus (baculovirus) in mammalian cells. By titration with a baculovirus containing a green fluorescent protein cassette, we found that several, but not all, mammalian cell types can be infected efficiently. In contrast to previous suggestions, our data show that the asialoglycoprotein receptor is not required for efficient infection. We demonstrate for the first time that this baculovirus can infect nondividing mammalian cells, which implies that the baculovirus is able to transport its genome across the nuclear membrane of mammalian cells. Our data further show that the virus enters via endocytosis, followed by an acid-induced fusion event, which releases the nucleocapsid into the cytoplasm. Cytochalasin D strongly reduces the infection efficiency but not the delivery of nucleocapsids to the cytoplasm, suggesting involvement of actin filaments in cytoplasmic transport of the capsids. Electron microscopic analysis shows the cigar-shaped nucleocapsids located at nuclear pores of nondividing cells. Under these conditions, we observed the viral genome, major capsid protein, and electron-dense capsids inside the nucleus. This suggests that the nucleocapsid is transported through the nuclear pore. This mode of transport seems different from viruses with large spherical capsids, such as herpes simplex virus and adenovirus, which are disassembled before nuclear transport of the genome. The implications for the application of baculovirus or its capsid proteins in gene therapy are discussed.


Subject(s)
Cell Nucleus/virology , Endocytosis , Nucleocapsid/metabolism , Nucleopolyhedroviruses/physiology , Animals , Cell Line , Endosomes/virology , Fluorescent Antibody Technique , Genome, Viral , Green Fluorescent Proteins , Humans , Hydrogen-Ion Concentration , In Situ Hybridization, Fluorescence , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Electron , Nuclear Pore/metabolism , Nuclear Pore/virology , Nucleopolyhedroviruses/genetics , Rats , Receptors, Virus/physiology , S Phase/physiology , Tumor Cells, Cultured
6.
Acta Ophthalmol Scand ; 78(4): 411-5, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10990042

ABSTRACT

PURPOSE: Corneal biopsies are important in diagnosing multiple corneal diseases. They were previously performed by way of keratectomy, a method that causes corneal opacity and topographic changes due to scarring. Microbiopsy is a new way to perform corneal biopsies. Before microbiopsy may be performed on human corneas, the safety of this procedure has to be proved concerning clinical development, histological changes and topographic changes after multiple biopsies. METHODS: The healthy right cornea of 24 rabbits was punctured. 12 microtrephinations in 4 different symmetric patterns were performed. The clinical development of the bioptic sites as well as the topographic changes were observed over 5 months. After enucleation, serial sections of the corneas were analysed histologically. RESULTS: Out of 294 performed biopsies, 291 samples could be collected. 4 perforations occurred. The initial epithelial defect closed within 3 days. A pale stromal scar remained. The histological analysis of these scars showed a facette underlined by a dense hypocellular fibrous layer and a typical star-shaped figure consisting of a loose hypercellular stromal tissue. Only dioptric power of corneas with circle-pattern showed a statistically significant decrease. CONCLUSION: Micropuncture is a safe and efficient bioptic procedure. Even 12 micropunctures do not lead to significant changes of dioptric power in most patterns. Further studies are necessary to evaluate the reproducibility of refractive changes by circle patterns and corrections of astigmatisms.


Subject(s)
Biopsy/methods , Cornea/pathology , Animals , Contact Lenses , Corneal Topography , Follow-Up Studies , Male , Microscopy, Electron, Scanning , Punctures , Rabbits , Refraction, Ocular , Wound Healing
7.
Gene Ther ; 7(17): 1505-15, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11001371

ABSTRACT

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.


Subject(s)
Genetic Therapy/methods , Genetic Vectors/metabolism , Integrin beta1/metabolism , Transfection/methods , Yersinia pseudotuberculosis/genetics , Antigen-Antibody Reactions , Fatty Acids, Monounsaturated , Genetic Engineering , Humans , Plasmids/metabolism , Polyethyleneimine , Protein Binding , Quaternary Ammonium Compounds , Tumor Cells, Cultured
8.
Proc Natl Acad Sci U S A ; 95(4): 1466-71, 1998 Feb 17.
Article in English | MEDLINE | ID: mdl-9465038

ABSTRACT

A recently cloned isoform of cGMP-dependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl- secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl- channel. Mutation of the cGK II N-terminal myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membrane-anchoring domain of cGK II was fused to the N terminus of cGK Ibeta, acquired the ability to associate with the membrane and activate the CFTR Cl- channel. The potency order of cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent of 32P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl- channel activation in intact cells.


Subject(s)
Chloride Channels/physiology , Cyclic GMP-Dependent Protein Kinases/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Animals , Cell Compartmentation , Cell Membrane/enzymology , Humans , Ion Channel Gating , Myristates , Phosphorylation , Protein Processing, Post-Translational , Rats , Recombinant Proteins , Transfection
9.
J Biol Chem ; 271(12): 7025-9, 1996 Mar 22.
Article in English | MEDLINE | ID: mdl-8636133

ABSTRACT

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.


Subject(s)
Cyclic GMP-Dependent Protein Kinases/metabolism , Isoenzymes/metabolism , Myristic Acids/metabolism , Animals , Base Sequence , Cell Line , Cell Membrane/enzymology , DNA Primers , Humans , Lipid Metabolism , Molecular Sequence Data , Myristic Acid , Substrate Specificity
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