Kirilka Mladenova

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.

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.

Polyplex Particles Based on Comb‐Like Polyethylenimine/Poly(2‐ethyl‐2‐oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure

The present contribution is focused on feasibility of using comb-like copolymers of polyethylenimine with poly(2-ethyl-2-oxazoline) (LPEI-comb-PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well-defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub-100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.

Effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimers, Parkinsons disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

Transepithelial resistance in human bestrophin-1 stably transfected Madin-Darby canine kidney cells.

Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin–Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first–fourth day) both cell lines showed the same or similar values of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.