Diaminoterephthalate turn-on fluorescence probes for thiols--tagging of recoverin and tracking of its conformational change.

Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these "NiWa Blue" dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal-calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca(2+), irradiation at 280 nm of a tryptophan residue in close proximity to Cys-bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca(2+), the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys-bound NiWa Blue was significantly weakened.

Two cases of cancer-associated retinopathy combined with small-cell lung cancer.

Cancer-associated retinopathy is a rare paraneoplastic syndrome that is often associated with small-cell lung cancer. It is caused by an autoantibody to the 23 kDa photoreceptor protein, recoverin. A small number of reports have described effective treatment for the disease. We report two cases of cancer-associated retinopathy with small-cell lung cancer whose visual symptom preceded the diagnosis of cancer. Their visual acuity and visual field were slightly improved after steroid and anticancer therapy. Steroid therapy was effective, although the period from visual symptom onset to therapy was comparative longer. When cancer-associated retinopathy is suspected, a comparatively large quantity of steroids and anticancer treatment should be combined immediately.

Reverse micelle encapsulation of membrane-anchored proteins for solution NMR studies.

Perhaps 5%-10% of proteins bind to membranes via a covalently attached lipid. Posttranslational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca(2+) signaling pathway in vision, and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol-4,5-bisphosphate. Both proteins have been successfully encapsulated in the lipid-extruded state and high-resolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.

Proteomic analysis of the membrane palmitoylated protein-4 (MPP4)-associated protein complex in the retina.

Membrane palmitoylated protein-4 (MPP4) is a retina-specific scaffolding protein of the membrane-associated guanylate kinase family that has been implicated in organizing presynaptic protein complexes in the photoreceptor ribbon synapse. To isolate the components of this complex we applied a proteomic approach based on immunoaffinity chromatography with a monoclonal anti-MPP4 antibody followed by two-dimensional electrophoresis and mass spectrometry. Among the identified molecules were previously reported proteins of the MPP4 scaffolding complex including adaptor proteins Veli3 and Psd95. Here we demonstrate a selective association between MPP4 and the Psd95-beta isoform that is mediated by interaction of their N-terminal L27 domains. In addition, we have identified recoverin and Hsc70 as novel associated proteins of the MPP4 multiprotein complex in the retina. This study demonstrates the utility of anti-MPP4 antibody precipitation for the elucidation of the MPP4-associated protein complex, which is essential in understanding its precise role in signal transmission at the photoreceptor synapse.

Evaluation of twenty-one human adenovirus types and one infectivity-enhanced adenovirus for the treatment of malignant melanoma.

Advanced melanoma is associated with poor prognosis warranting the development of new therapeutics, such as oncolytic adenoviruses for immunovirotherapy. Since this approach critically depends on efficient transduction of targeted tumor cells, we screened a panel of 22 different adenovirus types for their internalization efficiency in melanoma cells. We demonstrated that the virions of Ad35, Ad38, and Ad3 have significantly higher internalization efficiency in melanoma cells than Ad5, so far the only adenovirus type used in clinical trials for melanoma. Therefore, we developed a conditionally replication-competent Ad5-based vector with the Ad35 fiber shaft and knob domains (Ad5/35) and compared its therapeutic efficacy with the homologous vector carrying the native Ad5 fiber. To further enhance virotherapy, we combined the oncolytic adenovirus vectors with intratumoral expression of measles virus fusogenic membrane glycoproteins H and F (MV-H/F) and dacarbazine chemotherapy. In a human melanoma xenograft model, established from a short-term culture of primary melanoma cells, we demonstrated that the Ad5/35-based therapy had a significantly greater anti-neoplastic effect than the homologous Ad5-based therapy. Furthermore, the combination of virotherapy, intratumoral expression of MV-H/F, and chemotherapy was clearly superior to single- or double-agent therapy. In conclusion, Ad35-based vectors are promising for the treatment of melanoma.

Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space.

Stem and progenitor cells can be combined with polymer substrates to generate tissue equivalents in culture. The replacement of retinal tissue lost to disease or trauma using retinal progenitor cells (RPCs) delivered on polymer scaffolds and transplanted into the sub-retinal space of the damaged retina is a promising therapeutic strategy. Micromachining-based, ultra-thin PMMA poly(methyl methacrylate) scaffolds may provide a suitable cytoarchitectural environment for tissue engineering and transplantation to the diseased eye. Here, adhesion of RPCs to polymer, as well as migration and differentiation in the host retina were compared for PMMA scaffolds (6 microm thickness) with either smooth or porous (11 microm diameter) surface topography. RPCs were cultured under identical conditions on smooth or porous laminin-coated polymer scaffolds and transplanted into the subretinal space of C57BL/6 mice. RPCs could be cultured on both scaffolds with similar results, although transplantation with non-porous scaffolds showed limited RPC retention. Porous scaffolds demonstrated enhanced RPC adherence during transplantation and allowed for greater process outgrowth and cell migration into the host retinal layers. Integrated cells expressed the mature neuronal marker neurofilament-200 (nf-200), the glial marker glial fibrillary acidic protein (GFAP) and the retinal-specific marker recoverin. No host foreign body response was seen. In conclusion, ultra-thin film PMMA scaffolds micromachined to contain through pores retain adherent RPCs to a considerably greater extent than unmachined versions during the transplantation process and can serve as a biocompatible substrate for cell delivery in vivo.

[Cancer associated retinopathy (CAR). Two clinical cases and review of the literature]. / Cancer associated retinopathy (CAR). Présentation de deux cas cliniques et revue de la littérature.

The cancer associated retinopathy (CAR) is a paraneoplasic retinopathy in which an antigen-antibody reaction, due to retinal antigens, also expressed in tumours, leads to degeneration of retinal photoreceptor cells. We observed in CHL-Luxembourg, 2 clinical cases of non-Hodgkin's lymphoma with severe prognosis in whom we described the presence of anti-recoverin antibodies. The CAR is most frequently associated with small cell lung and ovarian carcinomas. Clinical symptoms (phosphenes, progressive loss of eyesight) sometimes, occur before the diagnostics of primary cancer. Retinal degeneration may be assessed by electroretinogram, visual field, fundus oculi. A crossed reactivity between tumour and retinal antigens may initiate an antigen-antibody reaction, that implicates optic lesions. Different antigenic proteins have been evidenced, the most frequent being the recoverin. This protein plays a role in the adaptation to light and darkness. It is expressed in more than 50% of different types of neoplastic cells and would play a role in tumour proliferation. The antigen-antibody reaction leads to death by apoptosis of photoreceptor and bipolar retinal cells. These antirecoverin antibodies are also observed in other retinal degenerative diseases. The diagnosis is confirmed by titration of antibodies in the serum by Western Blot, Elisa and immunohistochemical methods. However, this diagnosis is by exclusion (vs. brain metastasis, drug toxicity, demyelinating diseases, autoimmune non paraneoplastic retinopathies). Corticosteroids are the only therapy that can bring some benefit. There is no value in starting a therapy if the retinal degeneration has reached an advanced stage. Note that the CAR must be distinguished from the Melanoma Associated Retinopathy (MAR) which is a similar paraneoplastic syndrome, but with rapid evolution of its symptoms and different etio-pathogenesis.

CNTF induces dose-dependent alterations in retinal morphology in normal and rcd-1 canine retina.

Ciliary neurotrophic factor (CNTF) provides morphologic preservation of rods in several animal models of retinitis pigmentosa (RP). However, CNTF may alter photoreceptor morphology and rod photoreceptor differentiation in vitro, as well as affecting normal retinal electrophysiology. In addition, the capacity of CNTF to support other cell types affected secondarily in RP (cones and ganglion cells) is unclear. The purposes of this study were to examine the effects of CNTF upon a canine model of RP, the rod-cone degeneration (rcd-1) dog. Archival tissue from a previous study assessing the capacity of CNTF to rescue photoreceptors in rcd-1 dogs was used. One eye was treated for 7 weeks before being explanted. The contralateral eye was untreated. A total of 23 rcd-1 dogs and seven control dogs (four untreated and three CNTF-treated) were used. Morphometric data describing outer and inner nuclear layer thickness, inner retinal thickness, cones and ganglion cells were collected at nine evenly spaced points along each retina and analysed using a mixed effects model. Immunohistochemistry was performed on a subset of 11 dogs for expression of rhodopsin, human cone arrestin (hCAR) and recoverin. CNTF protected the outer nuclear layer and increased inner retinal thickness in a dose-dependent manner (both were maximal at CNTF doses of 1-6 ng day-1). Significant cone loss or reduction of inner nuclear layer width in rcd-1 did not occur in this model, therefore we were unable to assess the protective effect of CNTF upon these parameters. CNTF did not afford significant ganglion cell protection. CNTF induced morphologic changes in rods and ganglion cells, as well as reducing expression of hCAR and rhodopsin, but not recoverin. The dose of CNTF which provided optimal outer nuclear layer protection also resulted in several other effects, including altered ganglion cell morphology, increased thickness of the entire retina, and reduced expression of some phototransduction proteins. These changes were more marked in rcd-1 retinas than in wild-type retinas. This implies that the consequences of CNTF treatment may be substantially influenced by the cellular context into which it is introduced.