The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Influence of helio-geophysical factors (HGF) on environments, on health state and reliability of man's professional functioning, on processes of society development

The present work reveals the helio-geophysical factors HGF) influence on natural surroundings, human and society, health state and professional reliability of human functioning in technogenic and social systems of extreme risk. The interdisciplinary complex investigations were performed on the social-population, organismal, organ cell and molecular levels. The HGF synergistic influence on a human state and functioning and the society especially within megalopolises' non-equilibrium geo-dynamic zones was studied in details. We analyzed the HGF influence on the causes and development of instabilities in ecology-social processes and the formation of intellectual and social climate. We also worked out some recommendations on stabilizing the society development concerning the geo-space and ecology-humanitarian imperative on the threshold of the current maximum of solar activity in 2000 - 2003.     

Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80(gag) gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.     

Comparative characterization of the two primary pumps, H+-ATPase and Na+-ATPase, in the plasma membrane of the marine alga Tetraselmis viridis

The transport characteristics of the plasma membrane H⁺‐ATPase (PMHA) and Na⁺‐ATPase (PMNA) from marine unicellular green alga Tetraselmis viridis Rouch. were studied using sealed plasma membrane vesicles isolated from this species. The activities of the ATPases were investigated by monitoring the ATP‐dependent pH changes in the vesicle lumen. PMHA operation led to acidification of the vesicle lumen, whereas Na⁺ translocation into plasma membrane vesicles catalysed by PMNA was accompanied by H⁺ efflux, namely the alkalization of the vesicle lumen (Balnokin et al., FEBS Lett 462: 402–406, 1999). The intravesicular acidification and alkalization were detected with the ΔpH probe acridine orange and the pH probe pyranine, respectively. PMHA and PMNA were found to operate in distinct pH regions, maximal activity of PMHA being observed at pH 6.5 and that of PMNA at pH 7.8. Kinetic studies revealed that the ATPases have similar affinities to their primary substrate, MgATP complex (an apparent K_m = 34 ± 6.2 µM for PMHA and 73 ± 8.7 µM for PMNA). At the same time, the ATPases were differently affected by free Mg²⁺ and ATP. Free Mg²⁺ appeared to be a mixed‐type inhibitor for PMNA (K_i′ = 210 µM) but it did not suppress PMHA. Conversely, free ATP markedly suppressed PMHA being a mixed‐type inhibitor (K_i′ = 330 µM), but PMNA was affected by free ATP only slightly. Furthermore, the ATPases substantially differed in their sensitivities to the inhibitors of membrane ATPases, such as orthovanadate, N‐ethylmaleimide and N,N′‐dicyclohexylcarbodiimide. The differences found in the properties of the PMHA and PMNA are discussed in terms of regulation of their activities and their capacity to be involved in cytosolic ion homeostasis in T. viridis cells.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

Atomic force microscopy analysis of icosahedral virus RNA

Single-stranded genomic RNAs from four icosahedral viruses (poliovirus, turnip yellow mosaic virus (TYMV), brome mosaic virus (BMV), and satellite tobacco mosaic virus (STMV)) along with the RNA from the helical tobacco mosaic virus (TMV) were extracted using phenol/chloroform. The RNAs were imaged using atomic force microscopy (AFM) under dynamic conditions in which the RNA was observed to unfold. RNAs from the four icosahedral viruses initially exhibited highly condensed, uniform spherical shapes with diameters consistent with those expected from the interiors of their respective capsids. Upon incubation at 26 degrees C, poliovirus RNA gradually transformed into chains of globular domains having the appearance of thick, irregularly segmented fibers. These ultimately unwound further to reveal segmented portions of the fibers connected by single strands of RNA of 0.5-1 nm thickness. Virtually the same transformations were shown by TYMV and BMV RNA, and with heating, the RNA from STMV. Upon cooling, the chains of domains of poliovirus RNA and STMV RNA condensed and re-formed their original spherical shapes. TMV RNAs initially appeared as single-stranded threads of 0.5-1.0 nm diameter but took on the structure of the multidomain chains upon further incubation at room temperature. These ultimately condensed into short, thick chains of larger domains. Our observations suggest that classical extraction of RNA from icosahedral virions produces little effect on overall conformation. As tertiary structure is lost however, it is evident that secondary structural elements are arranged in a sequential, linear fashion along the polynucleotide chain. At least in the case of poliovirus and STMV, the process of tertiary structure re-formation from the linear chain of secondary structural domains proceeds in the absence of protein. RNA base sequence, therefore, may be sufficient to encode the conformation of the encapsidated RNA even in the absence of coat proteins.     

Folic acid modified gelatine coated quantum dots as potential reagents for in vitro cancer diagnostics

Background

Highly hydrophobic surfaces can have very low surface energy and such low surface energy biological interfaces can be obtained using fluorinated coatings on surfaces. Deposition of biocompatible organic films on solid-state surfaces is attained with techniques like plasma polymerization, biomineralization and chemical vapor deposition. All these require special equipment or harsh chemicals. This paper presents a simple vapor-phase approach to directly coat solid-state surfaces with biocompatible films without any harsh chemical or plasma treatment. Hydrophilic and hydrophobic monomers were used for reaction and deposition of nanolayer films. The monomers were characterized and showed a very consistent coating of 3D micropore structures.

Results

The coating showed nano-textured surface morphology which can aid cell growth and provide rich molecular functionalization. The surface properties of the obtained film were regulated by varying monomer concentrations, reaction time and the vacuum pressure in a simple reaction chamber. Films were characterized by contact angle analysis for surface energy and with profilometer to measure the thickness. Fourier Transform Infrared Spectroscopy (FTIR) analysis revealed the chemical composition of the coated films. Variations in the FTIR results with respect to different concentrations of monomers showed the chemical composition of the resulting films.

Conclusion

The presented approach of vapor-phase coating of solid-state structures is important and applicable in many areas of bio-nano interface development. The exposure of coatings to the solutions of different pH showed the stability of the coatings in chemical surroundings. The organic nanocoating of films can be used in bio-implants and many medical devices.     

Coenzyme Reorientations in the Active Sites of Aspartate Aminotransferase Isoenzymes Studied by Linear Dichroism Method

Abstract

Cytosolic and mitochondrial pig heart aspartate aminotransferases (cAspAT and mAspAT) and chicken heart cAspAT have been oriented in a compressed slab of polyacrylamide gel and their linear dichroism LD spectra have been recorded. The coenzyme's tilt angles in the active sites of chicken cAspAT and pig mAspAT and their quasisubstrate complexes imitating catalytic intermediates have been computed. The computations are based on reduced linear dichroism values (ΔA/A), the known directions of the transition dipole moments in the coenzyme ring and atomic coordinates of the coenzyme obtained by X-ray crystallography. It has been found that formation of the enzyme complex with glutarate and protonation of the internal pyridoxal-lysine aldimine induce reorientations of the coenzyme. As a result of protonation, the coenzyme ring tilts by 27° in cAspAT and 13° in mAspAT. Formation of the external aldimine with 2-mehtylaspartate is accompanied by tilting of the coenzyme ring by 44° in cAspAT and 39° in mAspAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63° in cAspAT and 53° in mAspAT. It is inferred that the basic features of the active site dynamics are similar in the three AspATs studied. The diiferences in the coenzyme tilt angles between cAspAT and mAspAT may be linked to catalytic and structural peculiarities of the isoenzymes.     

Hypertrophic cardiomyopathy-causing Asp175asn and Glu180gly Tpm1 mutations shift tropomyosin strands further towards the open position during the ATPase cycle

To understand the molecular mechanism by which the hypertrophic cardiomyopathy-causing Asp175Asn and Glu180Gly mutations in α-tropomyosin alter contractile regulation, we labeled recombinant wild type and mutant α-tropomyosins with 5-iodoacetamide-fluorescein and incorporated them into the ghost muscle fibers. The orientation and mobility of the probe were studied by polarized fluorimetry at different stages of the ATPase cycle. Multistep alterations in the position and mobility of wild type tropomyosin on the thin filaments during the ATP cycle were observed. Both mutations were found to shift tropomyosin strands further towards the open position and to change the affinity of tropomyosin for actin, with the effect of the Glu180Gly mutation being greater than Asp175Asn, showing an increase in the binding strong cross-bridges to actin during the ATPase cycle. These structural changes to the thin filament are likely to underlie the observed increased Ca²⁺-sensitivity caused by these mutations which initiates the disease remodeling.