The compact and expanded denatured conformations of apomyoglobin in the methanol-water solvent. (49/15056)

We have performed a detailed study of methanol-induced conformational transitions of horse heart apomyoglobin (apoMb) to investigate the existence of the compact and expanded denatured states. A combination of far- and near-ultraviolet circular dichroism, NMR spectroscopy, and small-angle X-ray scattering (SAXS) was used, allowing a phase diagram to be constructed as a function of pH and the methanol concentration. The phase diagram contains four conformational states, the native (N), acid-denatured (U(A)), compact denatured (I(M)), and expanded helical denatured (H) states, and indicates that the compact denatured state (I(M)) is stable under relatively mild denaturing conditions, whereas the expanded denatured states (U(A) and H) are realized under extreme conditions of pH (strong electric repulsion) or alcohol concentration (weak hydrophobic interaction). The results of this study, together with many previous studies in the literature, indicate the general existence of the compact denatured states not only in the salt-pH plane but also in the alcohol-pH plane. Furthermore, to determine the general feature of the H conformation we used several proteins including ubiquitin, ribonuclease A, alpha-lactalbumin, beta-lactoglobulin, and Streptomyces subtilisin inhibitor (SSI) in addition to apoMb. SAXS studies of these proteins in 60% methanol showed that the H states of these all proteins have expanded and nonglobular conformations. The qualitative agreement of the experimental data with computer-simulated Kratky profiles also supports this structural feature of the H state.  (+info)

Tissue sources and blood flow limitations of osmotic water transport across the peritoneum. (50/15056)

Despite the daily use of hypertonic solutions to remove fluid from patients throughout the world who are undergoing peritoneal dialysis, the tissue sources of this water flow are unknown. To study this phenomenon in specific tissues, small plastic chambers were affixed to parietal and visceral surfaces of the peritoneum and were filled with either an isotonic or hypertonic solution. The volume changes over 60 to 90 min were determined and divided by the chamber area to yield the volume flux. The hypertonic solution produced a positive flux into the chamber of 0.6 to 1.1 microl/min per cm2 in all tissues tested. In contrast, the isotonic solution resulted in a net loss or an insignificant change in the chamber volume. Additional experiments tested the influence of blood flow on the hypertonic water flux during periods of control, reduced (50 to 80%), or postmortem (no) blood flow, as determined by laser Doppler flowmetry. With the exception of the liver, small but insignificant changes in the flux into the chamber were observed during the period of reduced flow; all water fluxes were markedly depressed during the postmortem period. It is concluded that both parietal and visceral tissues are sources of osmotically induced water flow into the cavity. Except for the liver, marked blood flow reductions have small but insignificant effects on osmotic water transport.  (+info)

Size of lipid microdroplets effects results of hepatic arterial chemotherapy with an anticancer agent in water-in-oil-in-water emulsion to hepatocellular carcinoma. (51/15056)

We have initially prepared a new drug delivery system for hepatocellular carcinoma (HCC). Using sonication and a detergent, iodinated poppy seed oil (IPSO) can be mixed with an aqueous solution of epirubicin to make a water-in-oil emulsion. The water-in-oil emulsion is further passed through a microporous glass membrane and split into saline to make a long-term inseparable water-in-oil-in-water emulsion (W/O/W) that consists of IPSO microdroplets. To investigate the effect of the size of IPSO microdroplets on the efficacy of injection chemotherapy with W/O/W in patients with HCC, 32 HCC patients were randomly assigned and treated with W/O/W of small IPSO microdroplets (30 micrometers in diameter) containing 60 mg of epirubicin (n = 16, group A) or W/O/W of large IPSO microdroplets (70 micrometers) containing the same amounts of epirubicin (n = 16, group B). Effects were assessed by measuring the percentage of decline of the alpha-fetoprotein (AFP) level in a week from the AFP level immediately before the treatment. The decline was significantly larger in group B (50.5 +/- 19.8, mean +/- S.D.) compared with group A (18.9 +/- 33.1; p <.005). The size of IPSO microdroplets injected into the hepatic artery determines the decrease of serum AFP levels of the patients with HCC.  (+info)

High-resolution A-DNA crystal structures of d(AGGGGCCCCT). An A-DNA model of poly(dG) x poly(dC). (52/15056)

A-DNA conformation is favored by guanine-rich sequences, such as (dG)n x (dC)n, or under low-humidity conditions. Earlier A-DNA crystal structures revealed some conformational variations which may be the result of sequence-dependent effects and/or crystal packing forces. Here we report the high-resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P212121 or the P6122 space group) to gain insights into the conformation and dynamics of the (dG)n x (dC)n sequence. The P212121 form has been analyzed using data to 1.1 A resolution by the anisotropic temperature factor refinement procedure of the SHELX97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A-DNA structure. The backbone torsional angles fall in a narrow range, except for the alpha/gamma angles which have two distinct combinations (gauche-/gauche+ or trans/trans). An A-DNA model of poly(dG) x poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P212121 form. In the crystal structure of the P6122 space group, the central eight base pairs of the decamer adopt A-DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A-DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.  (+info)

Interaction of the GM2-activator protein with phospholipid-ganglioside bilayer membranes and with monolayers at the air-water interface. (53/15056)

Differential scanning calorimetry (DSC) and film balance measurements were performed to study the interactions of the GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glc1 -->1'Cer (GM2)-activator protein with phospholipid/ganglioside vesicles and monolayers. The nonglycosylated form of the GM2-activator protein, added to unilamellar lipid vesicles of different composition, causes differential effects on the gel to liquid-crystalline phase transition peaks. The phase transition temperature (Tm) of pure dimyristoylglycerophosphocholine (DMPC) bilayer is slightly decreased. When lipids which specifically bind the GM2-activator protein are incorporated into the vesicles (e.g. a sulfatide or gangliosides) a shoulder in the thermograms at higher temperatures is observed, indicating an increase of the stability of the gel phase in relation to the liquid-crystalline phase. We also studied the surface activity of a glycosylated and a nonglycosylated GM2-activator protein at the air-water interface. The glycosylated form showed a slightly lower surface activity than the GM2-activator protein without oligosaccharide moiety. When the GM2-activator protein is added to the sub-phase of a surface covered with a lipid monolayer, it can only insert into the monolayer and reach the air-water interface below a monolayer pressure of 25 mN.m-1, depending on the lipid composition, and not when the monolayers are at the bilayer equivalence pressure of 30-35 mN.m-1. Particularly for Galbeta1-->3GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta 1-->4Glc1-->1'Cer (GM1) and GM2 containing films, the critical pressures (picrit) when no additional increase in surface pressure is observed after addition of the protein into the subphase, are much lower. This leads to the conclusion that binding of the GM2 activator protein to the ganglioside headgroups prevents the protein from reaching the air-water interface. The protein is then located preferentially at the lipid-water interface and cannot penetrate into the chain region.  (+info)

Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts. (54/15056)

A complex of the synthetic tetrasaccharide AGA*IM [GlcN, 6-SO3-alpha(1-4)-GlcA-beta(1-4)-GlcN,3, 6-SO3-alpha(1-4)-IdoA-alphaOMe] and the plasma protein antithrombin has been studied by NMR spectroscopy. 1H and 13C chemical shifts, three-bond proton-proton (3JH-H) and one-bond proton-carbon coupling constants (1JC-H) as well as transferred NOEs and rotating frame Overhauser effects (ROEs) were monitored as a function of the protein : ligand molar ratio and temperature. Considerable changes were observed at both 20 : 1 and 10 : 1 ratios (AGA*IM : antithrombin) in 1H as well as 13C chemical shifts. The largest changes in 1H chemical shifts, and the linewidths, were found for proton resonances (A1, A2, A6, A6', A1*, A2*, A3*, A4*) in GlcN, 6-SO3 and GlcN,3,6-SO3 units, indicating that both glucosamine residues are strongly involved in the binding process. The changes in the linewidths in the IdoA residue were considerably smaller than those in other residues, suggesting that the IdoA unit experienced different internal dynamics during the binding process. This observation was supported by measurements of 3JH-H and 1JC-H. The magnitude of the three-bond proton-proton couplings (3JH1-H2 = 2.51 Hz and 3JH4-H5 = 2.23 Hz) indicate that in the free state an equilibrium exists between 1C4 and 2S0 conformers in the ratio of approximately 75 : 25. The chair form appears the more favourable in the presence of antithrombin, as inferred from the magnitude of the coupling constants. In addition, two-dimensional NOESY and ROESY experiments in the free ligand, as well as transferred NOESY and ROESY spectra of the complex, were measured and interpreted using full relaxation and conformational exchange matrix analysis. The theoretical NOEs were computed using the geometry of the tetrasaccharide found in a Monte Carlo conformational search, and the three-dimensional structures of AGA*IM in both free and bound forms were derived. All monitored NMR variables, 1H and 13C chemical shifts, 1JC-H couplings and transferred NOEs, indicated that the changes in conformation at the glycosidic linkage GlcN, 6-SO3-alpha(1-4)-GlcA were induced by the presence of antithrombin and suggested that the receptor selected a conformer different from that in the free state. Such changes are compatible with the two-step model [Desai, U.R., Petitou, M., Bjork, I. & Olson, S. (1998) J. Biol. Chem. 273, 7478-7487] for the interaction of heparin-derived oligosaccharides with antithrombin, but with a minor extension: in the first step a low-affinity recognition complex between ligand and receptor is formed, accompanied by a conformational change in the tetrasaccharide, possibly creating a complementary three-dimensional structure to fit the protein-binding site. During the second step, as observed in a structurally similar pentasaccharide [Skinner, R., Abrahams, J.-P., Whisstock, J.C., Lesk, A.M., Carrell, R.W. & Wardell, M.R. (1997) J. Mol. Biol. 266, 601-609; Jin, L., Abrahams, J.-P., Skinner, R., Petitou, M., Pike, R. N. & Carrell, R.W. (1997) Proc. Natl Acad. Sci. USA 94, 14683-14688], conformational changes in the binding site of the protein result in a latent conformation.  (+info)

Cell water balance of white button mushrooms (Agaricus bisporus) during its post-harvest lifetime studied by quantitative magnetic resonance imaging. (55/15056)

A combination of quantitative water density and T2 MRI and changes therein observed after infiltration with 'invisible' Gd-DTPA solution was used to study cell water balances, cell water potentials and cell integrity. This method was applied to reveal the evolution and mechanism of redistribution of water in harvested mushrooms. Even when mushrooms did not lose water during the storage period, a redistribution of water was observed from stipe to cap and gills. When the storage condition resulted in a net loss of water, the stipe lost more water than the cap. The water density in the gill increased, probably due to development of spores. Deterioration effects (i.e. leakage of cells, decrease in osmotic water potential) were found in the outer stipe. They were not found in the cap, even at prolonged storage at 293 K and R.H.=70%. The changes in osmotic potential were partly accounted for by changes in the mannitol concentration. Changes in membrane permeability were also indicated. Cells in the cap had a constant low membrane (water) permeability. They developed a decreasing osmotic potential (more negative), whereas the osmotic potential in the outer stipe increased, together with the permeability of cells.  (+info)

Adhesion of adhesive resin to dental precious metal alloys. Part II. The relationship between surface structure of Au-In alloys and adhesive ability with 4-META resin. (56/15056)

Adhesion of 4-META to Au-In alloy was improved by adding In equivalent to .15% of Au content. On the basis of the results of Au-In alloys analyzed by XPS, the present study investigated the reason why adhesion of the Au-In alloy was improved. The O 1s spectrum could be separated into three oxygen chemical states, In2O3, chemisorbed H2O, and physisorbed H2O. The amount of chemisorbed H2O decreased remarkably with increasing amount of In. It is considered that the poor adhesive ability of the pure gold and alloys containing only small amounts of In was due to the chemisorbed H2O molecules and insufficient indium oxide on the alloy surface. It was established that excellent adhesion requires an oxide with chemical affinity for 4-META to cover at least 50% of the alloy surface.  (+info)