Biomedical subjects

M Demura

Publications and source records attributed to M Demura.

27 records · Page 2Linked to original sources

PubMed1994

Structural analysis of highly oriented poly(p-phenylene-terephthalamide) by 15N solid-state nuclear magnetic resonance.

The structure of an aromatic polyamide, poly(p-phenylene-terephthalamide) (PPTA), was studied in the solid state using 15N nuclear magnetic resonance (NMR) spectroscopy. Spectra of uniaxially aligned molecules placed with the axis of alignment both parallel with and perpendicular to the applied magnetic field were analyzed to yield the orientations of specific molecular bonds with respect to the fiber axis. The 15N chemical shift tensor was characterized by simulating powder pattern spectra of both PPTA and a model compound, benzanilide. Chemical shift and dipolar coupled chemical shift line shapes were calculated through Euler angle transformations from the principal axis system (PAS) reference frame to the fiber axis system (FAS) frame. The orientations of NH and NC' bonds in PPTA are determined as well as the orientational distribution of the PPTA fiber axis. The structural parameters determined for PPTA are compared with those obtained by X-ray diffraction.

Magnetic Resonance Spectroscopy↗

PubMed1993

A method for studying the structure of uniaxially aligned biopolymers using solid state 15N-nmr: application to Bombyx mori silk fibroin fibers.

Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction. In the present work, a new method is developed in which solid state 15N-nmr spectra obtained from uniaxially aligned molecules placed with the axis of alignment both parallel and perpendicular to the magnetic field are analyzed to yield the orientations of specific molecular bonds. Analytical expressions are derived that utilize spectral features read from 15N chemical shift anisotropy line shapes to calculate a discrete number of possible orientations for a specific site. The 15N-1H dipolar interaction is employed to further narrow the number of unique orientations possible for a given site. With this method, a neighborhood of possible orientations is quickly determined, and full line-shape simulations within this region of allowed space can be performed to refine the limits of orientation. This technique demonstrates the use of a single type of isotopic label to determine the orientation of a specific molecular group such as a peptide plane within a protein. Results from the application of this method to the Bombyx mori silk fibroin protein provide structural detail that is consistent with currently accepted structural models based on fiber diffraction studies.

Animals↗

PubMed1992

A method for the calculation of protein alpha-CH chemical shifts.

The chemical shifts of C(alpha)H protons have been calculated for 9 proteins, based on coordinates taken from high-resolution crystal structures. Chemical shifts were calculated using ring-current shifts, shifts arising from magnetic anisotropies of bonds, and shifts arising from the polarizing effect of polar atoms on the C(alpha)H bond. The parameters used were refined iteratively to give the best fit to (experimental-random coil) shifts over the set of 9 proteins. A further small correction was made to the averaged Gly C(alpha)H shift. The calculated shifts match observed shifts with correlation coefficients varying between 0.45 and 0.86, with a standard deviation of about 0.3 ppm. The differences between calculated and observed shifts have been studied in detail, including an analysis of different crystal structures of the same protein, and indicate that most of the differences can be accounted for by small differences between the structure in solution and in the crystal. Calculations using NMR-derived structures give a poor fit. The calculations reproduce the experimentally observed differences between chemical shifts for C(alpha)H in alpha-helix and beta-sheet. Most of the differentiation in secondary-structure-dependent shifts arises from electric field effects, although magnetic anisotropy also makes a large contribution to the net shift. Applications of the calculations to assignment (including stereospecific assignment) and structure determination are discussed.

Carbon↗

PubMed1992

Characterization of low-temperature-plasma treated silk fibroin fabrics by ESCA and the use of the fabrics as an enzyme-immobilization support.

Bombyx mori silk fibroin fabrics were treated with low-temperature-plasma using various gases. Alkaline phosphatase was immobilized onto the fabrics using the CNBr method. The enzyme activity was much improved by plasma treatment, especially when both O2 and CF4 were used. Using the ESCA technique it was found that the activities of the immobilized enzyme were strongly correlated with the C1s peak intensities of the atomic group -C-O-CF3. The apparent Michaelis constant for the enzyme decreased with increasing amounts of -C-O-CF3.

Alkaline Phosphatase↗

PubMed1990

Detection of adenovirus type41 in stool samples by a latex agglutination method.

We have developed a simple agglutination (LA) method for the detection of enteric adenovirus (EAd) in stool samples from infants with acute gastroenteritis. Ad type 41 (Ad41) was detected with high sensitivity and specificity by a slide agglutination test using latex particles coated with antiAd41 antibody (LA-antiAd41). The agglutination of LA-antiAd41 with Ad41 on a glass slide was evident macroscopically within 2 min. The sensitivity of the LA method was four times higher than that of the EM method.

Adenoviruses, Human↗

PubMed1987

Mitochondrial membrane potential estimated with the correction of probe binding.

Lipophilic ions are widely used as the probe for estimation of the membrane potential. It is suggested that the correction of the probe binding to the membrane and/or intracellular constituents is a problem to be solved in order to evaluate the membrane potential accurately. Previously, we proposed a method for the correction of the probe binding (Demura, M., Kamo, N. and Kobatake, Y. (1985) Biochim. Biophys. Acta 820, 207-215). In this paper, the method was applied to the determination of the membrane potential of intact mitochondria. The probes used constitute a homologous series of (Phe)3-P+-(CH2)n-CH3 (n = 0-4) and tetraphenylphosphonium (TPP+). Binding of these probes to de-energized mitochondria followed the Langmuir isotherm. However, values of parameters determined at high (50-800 microM) and low (under 20 microM) probe concentrations were different, suggesting the existence at least two, high- and low-affinity, binding sites. With extrapolation to the 'state of no binding', the membrane potential of intact mitochondria was estimated to be -147 mV (interior-negative) when they were energized by 5 mM succinate in medium consisting of 125 mM KCl, 10 mM MgCl2, 5 mM phosphate, 0.4 mM EDTA and 50 mM Tris-HCl (pH 7.5) at 25 degrees C. Parameters appearing in the equation for the correction of probe binding were determined with the use of this value of the membrane potential. The validity of the equation and the value of the parameters were revealed by the fact that after the correction, all probes used gave approximately the same value under the same conditions. We expanded the method so as to include the langmuir adsorption isotherm. When the modified equation is used, the estimated membrane potentials were less dependent on a probe concentration less than 10 microM.

Animals↗

PubMed1987

Binding of lipophilic cations to the liposomal membrane: thermodynamic analysis.

Lipophilic ions are widely used as probes for measuring membrane potentials. Since binding of the probes to the membrane interferes with the accurate estimation of the membrane potential, it is necessary to clarify the characteristics of probe binding to membranes. The present paper deals with the binding of lipophilic cations to liposomes. The results can be summarized as follows: (1) The binding of triphenylmethylphosphonium, its homologues and tetraphenylphosphonium to liposomes of dipalmitoylphosphatidylcholine followed the Langmuir adsorption isotherm. (2) Spin-labeled lipophilic cations were synthesized and the binding to liposomes of egg phosphatidylcholine was examined. The binding also followed the Langmuir adsorption isotherm. The dissociation constant (the concentration giving half-maximal binding), K, was independent of the temperature, indicating that the binding is entropy-driven. (3) The binding was influenced by the fluidity of the membrane. Except in the case of triphenylmethylphosphonium (TPMP+), K and A (maximum amounts of binding) increased above the transition temperature. In other words, above the phase transition temperature the binding affinity is decreased, while maximum amounts of binding are increased for all phosphoniums used except TPMP+.

Adsorption↗

PubMed1987

Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5.

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.

Adenoviruses, Human↗

PubMedDate not supplied

Structural analysis of silk with 13C NMR chemical shift contour plots.

The polymorphic structures of silk fibroins in the solid state were examined on the basis of a quantitative relationship between the 13C chemical shift and local structure in proteins. To determine this relationship, 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues, and the C alpha chemical shift plot for Gly residues were prepared using atomic co-ordinates from the Protein Data Bank and 13C NMR chemical shift data in aqueous solution reported for 40 proteins. The 13C CP/MAS NMR chemical shifts of Ala, Ser and Gly residues of Bombyx mori silk fibroin in silk I and silk II forms were used along with 13C CP/MAS NMR chemical shifts of Ala residues of Samia cynthia ricini silk fibroin in beta-sheet and alpha-helix forms for the structure analyses of silk fibroins. The allowed regions in the 13C chemical shift contour plots for C alpha and C beta carbons of Ala and Ser residues for the structures in silk fibroins, i.e. Silk II, Silk I and alpha-helix, were determined using their 13C isotropic NMR chemical shifts in the solid state. There are two area of the phi,psi map which satisfy the observed Silk I chemical shift data for both the C alpha and C beta carbons of Ala and Ser residues in the 13C chemical shift contour plots.

Alanine↗