Jordan Doumanov

EYS is a major gene for rod‐cone dystrophies in France

Autosomal‐recessive retinitis pigmentosa (arRP) was recently associated with mutations in a novel gene EYS, spanning over 2 Mb, making it the largest known gene expressed in the human eye. The purpose of this study was to establish the prevalence and nature of EYS mutations in a clinically well‐characterized cohort of 239 sporadic and arRP French cases. Direct sequencing of EYS was performed in 186 subjects for whom known mutations had previously been excluded by applying microarray technology. We mostly identified novel mutations in EYS in a total of 29 patients: Fifteen of the mutations were predicted to create premature stop codons and two represent exonic deletions. In addition, twenty missense, silent or splice‐site mutations were detected. Patients revealed homozygous or compound heterozygous mutations and in some cases, only a single mutation. Most patients showed classical signs of RP with relatively preserved central vision and visual field until late in the course of the disorder. One patient showed predominance of the disease in the inferior part of the retina suggesting potential phenotypic variability. With a prevalence of 12% or more we provide evidence that EYS is a major gene for RP in France and probably elsewhere.

Disease-Causing Mutations in BEST1 Gene Are Associated with Altered Sorting of Bestrophin-1 Protein

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery.

Best1 Shot Through the Eye—Structure, Functions and Clinical Implications of Bestrophin-1 Protein

ABSTRACT Best1 protein (also known as bestrophin-1) is a highly conservative protein, member of the bestrophin family of anion channels. It is a product of the BEST1 gene and is expressed predominantly on the basolateral plasma membrane of retinal pigment epithelium cells in the human retina. The exact functions of the protein are still under discussion, but its role as an anion channel, regulator of a Ca2+ homeostasis and development of the eye have been proposed. Mutations in the protein are associated with several ocular diseases, named Bestrophinopathies (Best vitelliform macular dystrophy, autosomal dominant vitreoretinochoroidopathy, autosomal recessive bestrophinopathy and adult-onset vitelliform macular degeneration). In this review we present general information about the structure and functions of human Best1 protein and summarize the role of identified Best1 mutations in the development of different pathological conditions.

Effects of Ca2+ ions on bestrophin-1 surface films

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), β-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Effects of vipoxin and its components on HepG2 cells.

Snake venom Phospholipases A2 (svPLA2) are among the main toxic venom components with a great impact on different tissues and organs based on their catalytic specificity and a variety of pharmacological effects, whose mechanism is still under debate. The main toxic component, isolated from the venom of Vipera ammodytes meridionalis, is the heterodimeric postsynaptic ionic complex vipoxin, composed of a basic and toxic PLA2 enzyme subunit (GIIA secreted PLA2) and an acidic, enzymatically inactive and nontoxic subunit - vipoxin acidic component (VAC). This study demonstrates for the first time that vipoxin and its individual subunits affect integrity and viability of HepG2 cells displaying differences in their pharmacological activities. Under the experimental conditions, the individual PLA2 subunit induces cytotoxicity, cytoskeletal rearrangements and triggers early apoptosis in a concentration-dependent manner related to its enzymatic activity. Vipoxin and VAC do not affect cell viability but manifest high degree of genotoxicity, whereas DNA damage induced by PLA2 subunit could be defined as moderate and not associated with its catalytic activity. Our results suggest that the interactions between vipoxin subunits play an important role in HepG2 cell response and most likely affect the observed distinction between cyto- and genotoxicity.

Interaction of Bestrophin-1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in surface films.

Human bestrophin-1 (hBest1) is a transmembrane channel protein, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Although it is clear that hBest1s interactions with lipids are crucial for its function such studies were not performed as the protein was not purified. Here we describe an effective purification of hBest1 from Madin-Darby Canine Kidney (MDCK) cells via simple gel-filtration and affinity chromatographic steps, which makes possible to probe the protein interplay with lipids. The interaction of the purified hBest1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied in Langmuir monolayers. The surface pressure (π)-area (A) isotherms and compression/expansion isocycles of POPC monolayer were recorded in absence and presence of hBest1 in the subphase. The π(A) isotherms were analyzed in terms of surface compressional modulus and via two-dimensional virial equation of state. The dilatational rheological properties of the surface films and their surface potential were also measured. The morphology of the films was observed by Brewster angle microscopy. The inclusion of the protein in the film subphase does not lead to in-depth penetration of hBest1 but interaction takes place in the headgroup region of the monolayer. The hBest1/POPC interaction resulted in formation of more condensed films, which rheological properties and lateral structure differed significantly from the pure POPC monolayers. Our study sheds light on the still unclear question how hBest1 gets in touch with biomembrane phospholipids of eukaryotic cells that might be of key importance for the proper structure and function of RPE biomembranes.

Polymeric vehicles for transport and delivery of DNA via cationic micelle template method

This work describes the preparation of polymeric non-viral system for transport and delivery of nucleic acids. An amphiphilic poly(2-(dimethylamino)ethyl methacrylate)-block-poly(ε-caprolactone)-block-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA20-b-PCL70-b-PDMAEMA20) triblock copolymer was synthesized and used for formation of cationic micelles and subsequent complexation with DNA. Next, a protective polymer coating on the complex surface and removal of copolymer from the particle interior were conducted. In this way, polymer nanocapsules containing DNA molecules were obtained. The synthesized polymer, cationic micelles, complexes, and polymer capsules were investigated by proton nuclear magnetic resonance, gel permeation chromatography, dynamic and electrophoretic light scattering, and transmission electron microscopy. In vitro cytotoxicity assessment of the different systems revealed very good tolerance to human cells.

Interactions of pharmacologically active snake venom sPLA2 with different cell lines

Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity – hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits – a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential.

Changes in the functional characteristics of tumor and normal cells after treatment with extracts of white dead-nettle.

Lamium album L. is a perennial herb widely used in folk medicine. It possesses a wide spectrum of therapeutic activities (anti-inflammatory, astringent, antiseptic, antibiotic, antispasmodic, antioxidant and anti-proliferative). Preservation of medicinal plant could be done by in vitro propagation to avoid depletion from their natural habitat. It is important to know whether extracts from L. album plants grown in vitro possess similar properties as extracts from plants grown in vivo. For these reasons, it is important to examine changes in the composition of secondary metabolites during in vitro cultivation of the plant and how they affect the biological activity. We used A549 human cancer cell line and normal kidney epithelial cells MDCKII (Madin–Darby canine kidney cells II) as controls in assessing the anti-cancer effect of plant extracts. To elucidate changes in some key functional characteristics, adhesion test, MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide), transepithelial resistance (TER), immunofluorescence staining and trypan blue exclusion test were performed. Methanol and chloroform extracts of in vivo and in vitro propagated plants affected differently cancerous and non-cancerous cells. The most pronounced differences were observed in the morphological analysis and in the cell adhesive properties. We also detected suppressed epithelial transmembrane electrical resistance of MDCK II cells, by treatment with plant extracts, compared to non-treated MDCK II cells. A549 cells did not polarize under the same conditions. Altered organization of actin filaments in both cell types were noticed suggesting that extracts from L. album L. change TER and actin filaments, and somehow may block cell mechanisms, leading to the polarization of MDCK II cells.

Investigation of IL-6 Effects on SP-A Expression in A549 Lung Cell Line

ABSTRACT Interleukin-6 (IL-6) is a multifunctional cytokine that is important in inflammatory processes, cell differentiation and growth. Pulmonary surfactant is a protein-lipid complex, which reduce surface tension at the alveolar air-liquid interface, thereby preventing lung collapse. Surfactant protein A (SP-A) is the most abundant of four surfactant proteins (SP-A, SP-B, SP-C and SP-D). It binds to alveolar macrophages, stimulates their chemotaxis and serves as an opsonin in the phagocytosis. Low SP-A levels are associated with increased inflammation in the lung. In the absence of infection SP-A inhibited IL-6 production. In infected macrophages, SP-A augmented IL-6 production. In humans, SP-A levels are altered by a number of disease states. In some of them there is increased levels of SP-A in the bronchoalveolar lavage fluid. Here we investigate presence/absence of correlation between increased levels of IL-6 and production of SP-A from A549 lung cell line. Our results suggest that incubation of A549 cells with IL-6 for different time changes significantly the expression levels of SP-A.