The effect of rimantadine on the structure of model and biological membranes

The action of the antiviral drug rimantadine on the structure of bilayer lipid membranes (BLM) and RBC membranes was investigated. Structural changes in BLM were recorded by ionophore conductivity changes and by changes in the third harmonic of capacity current signal due to lateral compression of BLM in an electric field. It was shown that the adsorption of rimantadine on BLM results in an increase in ionophore mobility in bilayer membranes of dioleolyllecithin (DOL) and common lipids of bovine brain (CL) and in a decrease in those of azolectin (A). Relative changes in the third harmonic signal also depend on the membrane composition and have different signs. The results may be explained by the rimantadine action on the lipid bilayer structure: "rigidification" of A-membranes and "fluidization" of BLM from DOL and CL. Structural reorganization of RBC membranes as investigated by the ability of the cells to enter a micropipette (inner diameter greater than or equal to 3 microns) thereby undergoing deformation. It was shown that rimantadine influences RBC deformability due to drug induced inhomogenous mechanical membrane properties. Also, rimantadine accelerated the process of artificially induced aggregation of erythrocytes. The relation of the effects on artificial and biological membranes, and the structural changes in the lipid phase of membrane are discussed.     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Survival of clonogenic cells of Lewis lung carcinoma cells forming colonies in agar cultures in diffusion chambers during gamma-irradiation and exposure to gamma-neutron californium-252 irradiation

Clonogenic cells forming colonies in agar cultures in diffusion chambers and those isolated from subcutaneously transplanted Lewis lung carcinoma do not differ in their sensitivity to 60Co gamma-rays with respect to tumor growth stages. The dose-survival curves for all studied cells are S-shaped with a small shoulder. A cumulative dose-survival curve for malignant clonogenic cells is characterized by the average value of mean lethal dose D0 = 2.24 Gy and extrapolation number n = 2.0. When exposed to gamma-neutron-radiation (252Cf) malignant clonogenic cells exhibit a nearly exponential dose-survival curve with D0 = 0.56 Gy (with respect to a neutron component). The RBE of gamma-neutron radiation (252Cf) is 2.5.     

Mechanisms of Candida Resistance to Antimycotics and Promising Ways to Overcome It: The Role of Probiotics

Probiotics and Antimicrobial Proteins - Pathogenic Candida and infections caused by those species are now considered as a serious threat to public health. The treatment of candidiasis is...     

A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed.     

C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.