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Biomedical subjects

M H Koch

Publications and source records attributed to M H Koch.

At least 73 records · Page 4Linked to original sources
PubMed1995

Phase behavior of tyloxapol/Triton X100/water mixtures.

The influence of admixture of Triton X100 to the polymer tyloxapol and the phase behavior of the mixtures in contact with water was investigated by viscometry, polarization microscopy, and X-ray scattering. The viscosity of tyloxapol/Triton X100 mixtures is lower than that of pure tyloxapol. With increasing Triton X100 content, the phase behavior of the surfactant mixtures in contact with water evolves from the complex behavior of tyloxapol to the simpler one of Triton X100. In contact with water, mixtures rich in tyloxapol form hexagonal, cubic, and lamellar lyotropic liquid crystalline phases over a wide range of concentration and temperature, whereas Triton X100/water mixtures form only a hexagonal phase in a limited concentration and temperature range. The polymeric nature of tyloxapol stabilizes the liquid crystalline structures, and the size of the supramolecular structures in the water-containing surfactant systems is independent of the tyloxapol/Triton X100 mixing ratio but depends highly on water content. The ability of tyloxapol to form stable lyotropic liquid crystalline phases at body temperature, which has been proposed as a basis for the development of novel peroral sustained-release systems, is not significantly impaired by the addition of appropriate amounts of Triton X100. Admixture of Triton X100 to tyloxapol may thus provide a way to circumvent processing problems during the preparation of pharmaceutical formulations based on the highly viscous tyloxapol.

Chemistry, Pharmaceutical↗
PubMed1995

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties. These differences come to the fore when cholesterol is totally replaced by the spin-label: 3-doxyl-5a-cholestane is significantly less soluble in bile salt and mixed bile salt micelles than cholesterol and, in contrast with cholesterol, it interacts only very weakly, if at all,with phosphatidylcholine. The potential of the ESR method for detecting cholesterol crystal growth in human bile is discussed.

Bile Acids and Salts↗
PubMed1995

Ultrastructural and biochemical modifications of collagen from tissue of Morbus Dupuytren patients.

Small angle X-ray diffraction and biochemical analyses were carried out on normal palmar aponeurosis and on tissue from patients suffering from Dupuytren contractures (MD). Pathological tissue exhibits a higher overall content of collagen III. Type I collagen extracted from pathological tissue has a melting point of 0.8 degrees C higher than that of normal collagen. The only chemical differences compared to normal collagen I are 50% overhydroxylation of lysyl residues and a reduced amount of diglycosylated hydroxylysine residues. Analysis of the electron density distribution inside the collagen repeating period of MD-samples reveals disordered molecular packing in MD samples compared to in normal collagen. The disorder, which is higher in the gap region, is considerably reduced upon stretching.

Carbohydrate Sequence↗
PubMed1994

Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models.

Models of the 50S ribosomal subunit from electron microscopy on isolated particles and on ordered sheets display significantly different features. A model of the shape of the native Escherichia coli 50S subunit in solution and of its RNA-rich core at 4-nm resolution has been produced by using methods for joint interpretation of x-ray and neutron small-angle scattering data obtained by contrast variation. The good agreement between the shape of the entire 50S subunit and the electron microscopic models of isolated particles and between the RNA-rich core and the model obtained from ordered sheets leads to the conclusion that the latter, which is based on the subjective contouring of density maps, is heavily biased toward the RNA.

Escherichia coli↗
PubMed1994

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. I. X-ray synchrotron radiation study.

The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50 S ribosomal subunit of Escherichia coli in solution is described. The experimental X-ray data from contrast variation with sucrose are analysed in terms of the basic functions in real space and the scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins are obtained. From these curves models of the shape of the 50 S subunit and its RNA-rich core are evaluated. These two shapes are positioned so that their difference, which approximates the volume occupied by the proteins, produces a scattering curve which is in good agreement with the scattering from the protein moiety.

Escherichia coli↗
PubMed1994

Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering. II. Neutron scattering study.

The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety. The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm. The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles. The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA.

Escherichia coli↗
PubMed1993

Influence of the supramolecular structure of free lipid A on its biological activity.

The three-dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram-negative species were investigated at 40 degrees C, high water content (80-90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X-ray diffraction and Fourier-transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono- and bi-phosphoryl, as well as those from the non-enterobacterial strains Rhodobacter capsulatus 37b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37 degrees C of the non-enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine-inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non-lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti-complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37 degrees C vary non-systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus. We propose to extend the term 'endotoxic conformation', which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to 'endotoxic supramolecular conformation' which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity.

Carbohydrate Sequence↗
PubMed1993

Age-dependent structural changes in intact human lenses detected by synchrotron radiation X-ray scattering. Correlation with Maillard reaction protein fluorescence.

To understand alterations in the spatial organization of the crystallins, the major determinant of lens transparency, the x-ray scattering by intact normal human lenses from individuals 6-82 years of age was measured using synchrotron radiation. The angular dependence of the integrated scattering intensity is consistent with short-range order within the crystallin assemblies. A significant change in the scattering patterns of the lenses occurs after 55 years of age, in parallel with an increase of the fluorescence of the urea-insoluble crystallin fraction. This correlation suggests a gradual derangement of the short-range order as a result of cross-linking of the crystallin subunits by advanced Maillard reaction products that are generated by the continuous reaction of sugars, such as glucose or fructose, with proteins.

Adolescent↗
PubMed1993

Cloning and expression of Saccharomyces cerevisiae copper-metallothionein gene in Escherichia coli and characterization of the recombinant protein.

The gene sequences for intact and truncated forms of copper-binding metallothionein from Saccharomyces cerevisiae were cloned and overexpressed in Escherichia coli BL21(DE3)pLysE cells. In contrast to several other genes, the intact and truncated metallothionein genes are amplified in the polymerase chain reaction when Mg2+ is replaced by Co2+. The recombinant truncated protein binds copper in vivo and in vitro. A ratio of 8 Cu/12 cysteines was determined from atomic absorption, X-ray fluorescence and amino acid analysis. Extended X-ray absorption spectroscopy indicates that all Cu is in Cu(I) form and coordinated to three S atoms.

Base Sequence↗
PubMed1993

The influence of the effectors of yeast pyruvate decarboxylase (PDC) on the conformation of the dimers and tetramers and their pH-dependent equilibrium.

The influence of effectors of yeast pyruvate decarboxylase, phosphate, pyruvamide, thiamin diphosphate and Mg++, on the pH-dependent equilibrium between dimers and tetramers was studied by synchrotron radiation X-ray solution scattering. Thiamin diphosphate and phosphate shift the equilibrium to higher pH values without altering the structure of the oligomers. Pyruvamide, a substrate analogue activator, induces a significant change in the structure of the tetramer. By eliminating radiation damage by addition of dithioerythrol to the buffers, the scattering curves could be measured accurately over a large angular range. They were expanded in terms of spherical harmonics to obtain the shapes of the dimers and tetramers with higher resolution than was hitherto possible. This also allowed us to position the dimers, which are centrosymmetric at low resolution, in the tetramers which have 222 symmetry. The results indicate that addition of pyruvamide results in a less compact tetramer owing to structural changes in the dimers and to their displacements.

Biophysical Phenomena↗
PubMed1992

Synchrotron radiation solution X-ray scattering study of the pH dependence of the quaternary structure of yeast pyruvate decarboxylase.

The pH dependence of the quaternary structure of pyruvate decarboxylase from yeast was studied in the range 6.2 less than pH less than 8.4. There is an equilibrium with a midpoint around pH 7.5 between tetramers and dimers, and the catalytic activity of the enzyme depends on the volume fraction of tetramer. This equilibrium may provide an additional regulating mechanism besides substrate activation since accumulation of pyruvate would lead to a reduction in pH and hence an increase of the concentration of the catalytically active tetramer. Radiation damage during the X-ray scattering experiments results in a shift of this equilibrium and in the formation of octamers. These effects could be circumvented and analyzed using experimental and data processing methods which can be readily applied to other radiation-sensitive systems. The low-resolution shapes of the dimers and tetramers were determined from the scattering curves using spherical harmonics. The results indicate that a conformational change must occur in the dimers upon formation of the tetramers, in agreement with earlier circular dichroism measurements.

Hydrogen-Ion Concentration↗
PubMed1991

Rate and mechanism of the assembly of tropomyosin with actin filaments.

The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM MgCl2, 100 mM KCl, pH 7.5, 25 degrees C) and at the protein concentrations used (2.5-24 microM actin, 0.2-3.4 microM tropomyosin) the half-life time of assembly of tropomyosin with actin filaments was found to be less than 1 s. The results were analyzed quantitatively by a model in which tropomyosin initially binds to isolated sites. Further tropomyosin molecules bind contiguously to bound tropomyosin along the actin filaments. Good agreement between the experimental and theoretical time course of assembly was obtained by assuming a fast preequilibrium between free and isolatedly bound tropomyosin.

Actins↗
PubMed1991

The induction of lamellar stacking by cholesterol in lecithin-bile salt model systems and human bile studied by synchrotron X-radiation.

Small angle X-ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin-bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol, isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1], where cholesterol crystals were expected, we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.

Bile↗
PubMed1991

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers.

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.

Animals↗
PubMed1991

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

Bacteriorhodopsins↗
PubMed1991

Structural analysis of turkey tendon collagen upon removal of the inorganic phase.

Calcified leg flexor tendons in which the inorganic phase content had been lowered by progressive demineralization were studied by small angle X-ray diffraction and thermogravimetry. The X-ray diffraction results agree very well with the data previously obtained on calcified turkey tendon indicating that the method used to decalcify tendons provides good correspondence with the process of calcification. Up to five thermal processes can be detected in the thermogravimetric scans: (1) water release; (2) collagen decomposition; (3 and 4) combustion of the residual organic components; (5) carbonate removal from the apatitic phase. The temperature of collagen decomposition decreases at lower inorganic phase content in agreement with the higher thermal stability of calcified collagen fibrils compared with uncalcified ones. The decrease of collagen thermal stability upon decalification is paralleled by a decrease of the structural order of the collagen fibrils as indicated by small angle X-ray diffraction data. Decalcification down to about 40% wt of inorganic phase does not significantly alter the inorganic blocks that are regularly arranged inside the gap zone of the collagen. Further removal of inorganic phase down to about 15% wt provokes a variation of the intensity distribution of the small angle meridional reflections that can be ascribed to a reduction of the mean height of the inorganic blocks. At inorganic phase contents below 15% wt the gap region is more free to contract upon air drying as a result of the reduction of the mean length of the inorganic blocks.

Animals↗
PubMed1991

Collagen structural organization in uncalcified and calcified human anterior longitudinal ligament.

Collagen structure and collagen-apatite structural relationship has been investigated in human anterior ligament, where the mineral deposition occurs on collagen fibrils morphologically different from those of bone and tendons. Ultrastructural observations made on replicas of cryoprotected and freeze fractured uncalcified samples display a "helicoidal" morphology of the collagen fibrils. X-ray diffraction analysis carried out using conventional and synchrotron radiation sources revealed that the D-axial spacing is 65.0 nm and the electron density distribution inside the repeating period is very similar to those of tendon collagen in the same conditions of hydration. The short D-period can be interpreted as due to a greater angle of molecular crimping and/or molecular tilt compared to that of tendon. Air drying does not cause any appreciable variation in the D-axial period and induces an increase of the gap/overlap ratio that can be ascribed to telopeptide disorder. In spite of the different morphology of the collagen fibrils, the structural relationship between collagen and the mineral phase in calcified ligament is very close to that observed in bone and tendons. The apatitic phase is laid down in blocks along the collagen fibrils with the same axial periodicity, D = 65.0 nm, as that of uncalcified collagen fibrils. The mean height of the mineral blocks, which are 0.45D long, is even higher than in bone and masks any further fluctuation of the electron density due to the organic matrix.

Calcinosis↗