Biomedical subjects

E F Eikenberry

Publications and source records attributed to E F Eikenberry.

At least 19 recordsLinked to original sources

PubMed2006

The PILATUS 1M detector.

The PILATUS 1M detector is a hybrid pixel array detector with over one million pixels that operate in single photon counting mode. The detector, designed for macromolecular crystallography, is the largest pixel array detector currently in use at a synchrotron. It is a modular system consisting of 18 multichip modules covering an area of 21 cm x 24 cm. The design of the components as well as the manufacturing of the detector including the bump-bonding was performed at the Paul Scherrer Institute (PSI). The use of a single photon counting detector for protein crystallography requires detailed studies of the charge collection properties of the silicon sensor. The 18 modules are read out in parallel, leading to a full frame readout-time of 6.7 ms. This allows crystallographic data to be acquired in fine-varphi-slicing mode with continuous rotation of the sample. The detector was tested in several experiments at the protein crystallography beamline X06SA at the Swiss Light Source at PSI. Data were collected both in conventional oscillation mode using the shutter, as well as in a fine-varphi-slicing mode. After applying all the necessary corrections to data from a thaumatin crystal, the processing of the conventional data led to satisfactory merging R-factors of the order of 8.5%. This allows, for the first time, determination of a refined electron density map of a macromolecular biological crystal using a silicon pixel detector.

Crystallography, X-Ray↗

PubMed2005

Improved data acquisition in grazing-incidence X-ray scattering experiments using a pixel detector.

The use of an area detector in grazing-incidence X-ray experiments lends many advantages in terms of both speed and reliability. Here a discussion is given of the procedures established using the PILATUS pixel detector developed at the Swiss Light Source for optimizing data acquisition and analysis of surface diffraction data at the Materials Science beamline, especially with regard to reflectivity measurements, crystal truncation and fractional order rods, and grazing-incidence diffraction experiments.

Journal Article↗

PubMed2000

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils.

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.

Animals↗

PubMed1998

A Pixel-Array Detector for Time-Resolved X-ray Diffraction.

An integrating pixel-array detector for recording time-resolved X-ray diffraction measurements on microsecond timescales has been designed and tested as a 4 x 4 pixel prototype. Operational characteristics and radiation tolerance are discussed. A 100 x 92 array with 151.2 micro m square pixels is currently under construction.

Journal Article↗

PubMed1996

Correlation between lipid plane curvature and lipid chain order.

The 1-palmitoyl-2-oleoyl-phosphatidylethanolamine: 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE:POPC) system has been investigated by measuring, in the inverted hexagonal (HII) phase, the intercylinder spacings (using x-ray diffraction) and orientational order of the acyl chains (using 2H nuclear magnetic resonance). The presence of 20 wt% dodecane leads to the formation of a HII phase for the composition range from 0 to 39 mol% of POPC in POPE, as ascertained by x-ray diffraction and 2H nuclear magnetic resonance. The addition of the alkane induces a small decrease in chain order, consistent with less stretched chains. An increase in temperature or in POPE proportion leads to a reduction in the intercylinder spacing, primarily due to a decrease in the water core radius. A temperature increase also leads to a reduction in the orientational order of the lipid acyl chains, whereas the POPE proportion has little effect on chain order. A correlation is proposed to relate the radius of curvature of the cylinders in the inverted hexagonal phase to the chain order of the lipids adopting the HII phase. A simple geometrical model is proposed, taking into account the area occupied by the polar headgroup at the interface and the orientational order of the acyl chains reflecting the contribution of the apolar core. From these parameters, intercylinder spacings are calculated that agree well with the values determined experimentally by x-ray diffraction, for the variations of both temperature and POPE:POPC proportion. This model suggests that temperature increases the curvature of lipid layers, mainly by increasing the area subtended by the hydrophobic core through chain conformation disorder, whereas POPC content affects primarily the headgroup interface contribution. The frustration of lipid layer curvature is also shown to be reflected in the acyl chain order measured in the L alpha phase, in the absence of dodecane; for a given temperature, increased order is observed when the curling tendencies of the lipid plane are more pronounced.

Biophysical Phenomena↗

PubMed1995

High-resolution macromolecular structure determination using CCD detectors and synchrotron radiation.

BACKGROUND: Synchrotron radiation sources have made impressive contributions to macromolecular crystallography. The delay in development of appropriate X-ray detectors has, however, been a significant limitation to their efficient use. New technologies, based on charge-coupled devices (CCDs), provide capabilities for faster, more accurate, automated data collection. RESULTS: A CCD-based X-ray detector has been developed for use in macromolecular crystallography and has been in operation for about one and a half years at the Cornell High Energy Synchrotron Source. It has been used for a variety of crystallographic projects, including a number of high-resolution structural studies. The statistical quality of the data, the detector's ease and efficiency of use, and the growing number of structural results illustrate the practical utility of this new detector system. CONCLUSIONS: The new detector has enhanced capabilities for measuring diffraction patterns from crystals of macromolecules, especially at high resolution, when the X-ray intensities are weak. The survey of results described here ranges from virus crystallography to weakly diffracting small-molecule structure determination and demonstrates the potential of CCD detectors when combined with synchrotron radiation sources.

Amino Acid Sequence↗

PubMed1994

Collagen fibril structure in lamprey.

X-ray diffraction and electron microscopy are used to compare the molecular and higher order structure of collagen fibrils in three tissues of the lamprey: the dermis, perinotochord and notochord sheath. These lamprey tissues are known to contain five distinct genetic types of fibrillar collagen. The modes of axial and lateral packing of collagen molecules in fibrils of the lamprey tissues demonstrate the three major motifs seen in higher vertebrate D-periodic collagen fibrils. In particular, lamprey dermis was found to have a decreased D period resulting from a molecular tilt that is seen in skins of higher vertebrates. Our results suggest the molecular-packing motifs for collagen fibrils were in place at the dawn of vertebrate evolution and have been conserved since. In contrast, the diameters of fibrils and their spatial orientation in lamprey tissues do not in general, correspond to features found in mammalian tissues. Only for lamprey notochord is there a strong similarity, with fibril diameters and organization closely resembling those seen in type II tissues of higher vertebrates. This suggests that for all tissues except those with type II collagen, higher level organization of fibrils evolved along with the diversification of vertebrates.

Animals↗

PubMed1991

Nonbilayer phases of membrane lipids.

Numerous liquid crystalline biomembrane lipids are known to exhibit non-lamellar phases characterized by curvature of their component lipid monolayers. An understanding of the phase stability of these systems begins with analysis of the energy of bending the monolayers, the interactions which lead to the bending energy, and the geometrical constraints which lead to competing energy terms which arise when the monolayers are bent and packed onto lattices with different structures. Diffraction and other techniques suitable for probing lipid phase structure are described. A phenomenological model is reviewed which successfully explains many of the qualitative features of lipid mesomorphic phase behavior. A key result of this model is that lipid bilayer compositions which are close to the non-lamellar phase boundaries of their phase diagrams are characterized by a frustrated elastic stress which may modulate the activity of imbedded membrane proteins and which may provide a rationale for the prevalence of non-lamellar-tending lipid species in biomembrane bilayers. Areas in need of future research are discussed.

Kinetics↗

PubMed1991

Assessment of cell proliferation on porous microcarriers by means of image analysis.

Spherical porous microcarriers (PMCs) made from collagen-glycosaminoglycan crosslinked copolymers have exhibited considerable promise as growth surfaces for the proliferation of anchorage-dependent mammalian cell lines and have demonstrated the ability to entrap anchorage-independent cells. However, quantification of cell growth on PMCs has proved difficult. A method of measuring the proliferation of PMCs, based on image analysis, is presented. Using CV1 and CHO cell lines, samples of PMCs were removed from culture at various times, fixed, embedded and sectioned. The 2 microns sections were stained, photographed and digitized in three colors. A computer program was developed to evaluate digitized PMC cross-sections and to classify pixels as conforming to either background, cytoplasmic, matrix or nuclear parameters, based on a set of classification rules determined by statistical analysis. Growth curves were generated by relating the number of pixels occupied by cellular material to the total number of pixels in the PMC cross-section. The PMCs were found to foster cell proliferation, with cell densities approaching 100% occupancy.

Animals↗

PubMed1988

Isolation and partial characterization of collagen chains dimerized by sugar-derived cross-links.

Incubation of tail tendon from a young rat in solutions containing D-ribose resulted in attachment of the monosaccharide to collagen and subsequent cross-link formation at a rate much faster than found for glucose. The collagen rapidly became resistant to solubilization and showed increasing fluorescence. Ribose bound to all major CNBr peptides of collagen, with some preference for the alpha 2-CB3,5 peptide and the triple-helical region of alpha 1-CB6, and was incorporated into higher molecular weight material. Extensive pepsin digestion permitted isolation of dimers of alpha chains cross-linked in triple-helical regions as a result of incubation with ribose. The dimers were identified as beta 11, beta 12, and beta 22 components, and the limited degree of heterogeneity of these components indicated that cross-linking occurred at several sites, some of which must be intermolecular. Isolated beta components were strongly fluorescent with a spectrum similar to that of collagen in aged tissues. Fluorescent dimers with similar characteristics were found in pepsin digests of tail tendons from older rats.

Animals↗

PubMed1988

Glycation induces expansion of the molecular packing of collagen.

Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.

Animals↗

PubMed1988

Fibril-forming collagens in lamprey.

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.

Amino Acids↗

PubMed1987

Ultrastructural organization of skin: classification on the basis of mechanical role.

In our previous developmental study on skin we reported that correlations existed between the collagen fibril diameter distribution, glycosaminoglycan content and composition, and mechanical role. In this work we present new but related electron microscope data on the ultrastructure of skins from flounder, marlin, cod, frog, toad, caiman, viper, pigeon, whale and opossum. It is shown that mammalian and avian body skins generally contain relatively sharp unimodal distributions of fibril diameter consistent with a "passive" mechanical role. In contrast most reptilian and fish skins have broad, right-skewed (or bimodal) distributions of fibril diameters compatible with the exotendinous attributes required of an "active" skin. A study of the changing form of the collagen fibril diameter distribution with depth in the dermis is also reported.

Alligators and Crocodiles↗

PubMed1986

Type II collagen of lamprey.

The major collagen in lamprey notochord is type II, as determined by its amino acid composition and solubility properties. This collagen has a distribution of charged residues indistinguishable from higher vertebrate Type II collagens as judged by its SLS banding pattern. Lamprey type II collagen has a higher thermal stability than lamprey skin collagen, in contrast to the identical melting temperatures for these types in mammals. A minor collagen in lamprey notochord has solubility properties, amino acid composition, and electrophoretic mobility similar to that of 1 alpha, 2 alpha, 3 alpha collagen in human cartilage.

Amino Acids↗

PubMed1985

Characterization of the tissue form of type V collagen from chick bone.

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.

Animals↗

PubMed1985

High post-translational modification levels in type II procollagen are not a consequence of slow triple-helix formation.

The kinetics of triple helix formation of newly synthesized type II procollagen in chick embryo sternal cartilage cells were determined. The data obtained were in agreement with a model for triple helix formation in cellulo proposed recently (Bruckner et al., 1981). The time required to produce fully triple helical type II procollagen in cartilage cells was only 30% longer than for type I procollagen in tendon cells. Therefore, the several-fold increase in post-translational modification observed for type II collagen as compared to type I collagen cannot be attributed solely to slow helix formation but rather is a result of higher levels of enzymic activity of post-translational modification present in cartilage cells.

Animals↗

PubMed1984

Crystalline fibril structure of type II collagen in lamprey notochord sheath.

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I. The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1 alpha, 2 alpha and 3 alpha chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.

Animals↗

PubMed1984

Formation of the triple helix of type I procollagen in cellulo. Temperature-dependent kinetics support a model based on cis in equilibrium trans isomerization of peptide bonds.

The kinetics of triple-helix formation in type I procollagen at 37 degrees C in cellulo have been found to agree with predictions from the following model: triple-helix formation is initiated after completion of the synthesis of the procollagen polypeptide chains and after the chains associate to form interchain disulfide bonds within the C-propeptide; triple-helix formation propagates from this single nucleation site toward the N terminus of the molecule, interrupted by the random occurrence of peptide bonds in the cis configuration; cis-trans isomerization controls the rate of triple-helix formation. This model predicts that the activation energy of the rate-limiting process should be strongly positive. However, studies of triple-helix formation in vitro using thermally denatured material have shown only a low, or even negative, dependence of the rate on temperature in the physiological range. Here we report the temperature dependence of the rate of triple-helix formation in cellulo and a novel procedure for analyzing the resulting data to give an estimate of the Arrhenius activation energy of the rate-controlling process. It was found that this rate showed a strong, positive dependence on temperature, as expected, and that the activation energy was in satisfactory agreement with independent direct determinations of this parameter for cis-trans isomerizations. These findings lend further support to the model of triple-helix formation described above.

Animals↗