Biomedical subjects

F Eckstein

Publications and source records attributed to F Eckstein.

At least 145 records · Page 8Linked to original sources

PubMed1998

[Noninvasive analysis of cartilage volume and cartilage thickness in the human elbow joint using MRI].

The aim of the study was to non-invasively analyse the cartilage volume and thickness in the human elbow joint with magnetic resonance imaging. 12 fresh frozen specimens (ages 20 to 69 yrs.) were investigated using a 1.5 T magnet, and a water-excitation FLASH-3D sequence at a resolution of 1 x 0.25 x 0.25 mm3. After linear interpolation to 0.125 x 0.125 mm2 in the image plane, the cartilages were segmented interactively with a Snake algorithm. Following three-dimensional reconstruction, the cartilage volumes were determined, and the mean and maximal cartilage thickness computed by Euclidean distance transformation. The total cartilage volume of the human elbow joint amounted to between 3.90 and 7.17 ml (mean 5.5 ml +/- 20%). The humerus occupied 49 to 60%, the radius 15 to 27% and the ulna 20 to 29% of the total volume. The mean cartilage thickness ranged from an average of 0.9 (proximal part of the ulna) to 1.4 mm (capitulum humeri), and the maximal thickness from 2.3 mm (proximal part of the ulna) to 2.9 mm (distal part of the ulna). The ulnar cartilage showed a more inhomogeneous distribution than that of the humerus and radius. The interindividual variability of the cartilage thickness was less than that of the volume. There was no significant relationship of the volume with age (r = 0.11) or body weight (r = 0.51). However, based on the joint size (r = 0.71-medio-lateral extension of the articular surfaces) about 50% of the variability of the total cartilage volume could be predicted. The technique presented is suitable for designing computer models to investigate the load transmission and functional adaptation of diarthrodial joints, and for diagnosing and monitoring joint disease.

Adult↗

PubMed1998

[Sex-specific analysis of cartilage volume in the knee joint--a quantitative MRI-based study].

The objective of the present study was to determine differences in the normal knee joint cartilage volume of males and females, the analysis of the percentage distribution of the cartilage tissue onto the various joint surfaces, and the determination of the relationship between the cartilage volume, the body weight, and the tibial head diameter. We examined the knee joints of nine healthy men and nine women with a low level of physical activity. The cartilage volume was assessed with magnetic resonance imaging, applying a fat-suppressed gradient-echo sequence with a resolution of 2 x 0.31 x 0.31 mm3 and 3D image reconstruction. In the men, the absolute volumes of the femur and tibia, but not those of the patella, were significantly higher than in the women. The differences between the sexes were considerably lower after normalisation to the body weight and the tibial head diameter and were no more statistically significant. The interindividual variability was reduced after normalisation to these two parameters, the body weight being more effective. We did not observe sex-specific differences in the percentage of the total cartilage volume taken up by the various joint surfaces. Our results suggest that, in young individuals without cartilage lesions, there exist sex-specific differences of the cartilage volume in the knee joint. However, these can be explained in terms of general differences in body constitution (body weight and bone size), without further significant influences of the sex. The knowledge of the normal, sex-specific cartilage volume is relevant when attempting to estimate the amount of tissue loss at the time at which symptoms occur in a patient with degenerative joint disease.

Adult↗

PubMed1997

Inter-domain cross-linking and molecular modelling of the hairpin ribozyme.

The hairpin ribozyme is a small catalytic RNA composed of two helical domains containing a small and a large internal loop and, thus, constitutes a valuable paradigm for the study of RNA structure and catalysis. We have carried out molecular modelling of the hairpin ribozyme to learn how the two domains (A and B) might fold and approach each other. To help distinguish alternative inter-domain orientations, we have chemically synthesized hairpin ribozymes containing 2'-2' disulphide linkages of known spacing (12 or 16 A) between defined ribose residues in the internal loop regions of each domain. The abilities of cross-linked ribozymes to carry out RNA cleavage under single turnover conditions were compared to the corresponding disulphide-reduced, untethered ribozymes. Ribozymes were classed in three categories according to whether their cleavage rates were marginally, moderately, or strongly affected by cross-linking. This rank order of activity guided the docking of the two domains in the molecular modelling process. The proposed three-dimensional model of the hairpin ribozyme incorporates three different crystallographically determined structural motifs: in domain A, the 5'-GAR-3'-motif of the hammerhead ribozyme, in domain B, the J4/5 motif of group I ribozymes, and connecting the two domains, a "ribose zipper", another group I ribozyme feature, formed between the hydroxyl groups of residues A10, G11 of domain A and C25, A24 of domain B. This latter feature might be key to the selection and precise orientation of the inter-domain docking necessary for the specific phosphodiester cleavage. The model provides an important basis for further studies of hairpin ribozyme structure and function.

Computer Simulation↗

PubMed1997

The newt ribozyme is part of a riboprotein complex.

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a "hammerhead" domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Animals↗

PubMed1997

Mechanism of ribose 2'-group discrimination by an RNA polymerase.

The mechanism by which T7 RNA polymerase (RNAP) discriminates between rNTP and dNTP substrates has been characterized. During transcript elongation T7 RNAP uses rNTPs 70-80-fold more efficiently than dNTPs. Discrimination of the hydrogen-bonding character of the ribose 2'-substituent contributes a largely Km-mediated factor of approximately 20 to this preference for rNTPs. Discrimination of 2'-substituent H-bonding character appears to be made through a hydrogen bond to the hydroxyl group of tyrosine 639. This hydrogen bond makes little net contribution to either rNTP ground or transition state binding energy apparently because it is balanced by the energy of desolvation of the tyrosine hydroxyl. This mechanism may reflect a strategy to facilitate translocation by minimizing contributions from polymerase-NMP moiety interactions to NTP binding energy so as to minimize the affinity of the NTP binding site for the 3'-NMP of the product nucleic acid.

Bacteriophage T7↗

PubMed1997

Intermolecular cleavage by the newt ribozyme.

To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop II. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and III to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondary-structure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and III did. These results suggest that the sequence of the stems I and III impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.

Animals↗

PubMed1997

Isolation of hammerhead ribozymes with altered core sequences by in vitro selection.

The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A9U mutation was observed while in the other two mutations at A9U and U7A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 microM(-1) min(-1), respectively, as compared to 163 microM(-1) min(-1) for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure.

Base Composition↗

PubMed1997

The structure, function and application of the hammerhead ribozyme.

The hammerhead ribozyme is one of the smallest ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the hammerhead, its three-dimensional structure and the proposed mechanism, in addition to ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the ribozyme. The processes involved in designing ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.

Animals↗

PubMed1997

The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells.

The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-1tat-directed asymmetric hammerhead ribozyme alphaYRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of successively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for efficient association with a 645 nt target RNA transcript in vitro(for all constructs kass ranged between 0.3 and 1.8x104/M/s). The cleavage rate constants (kcleav) were independent of the length of the antisense flank and ranged between 0.8 and 1.2x10-4/s. However, the length of the antisense arms, as well as the mode of delivery and the subcellular location of the ribozymes, had a dramatic effect on efficacy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozymes were co-microinjected into the nucleus of human cells, a minimum length of 51 nt in the antisense arm was necessary for antisense- and ribozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorter antisense arms were almost ineffective. Conversely, short chain ribozymes, including those with chemical modifications, were superior to long chain ribozymes when co-microinjected into the cytoplasm. When transfected, all ribozymes showed an antisense effect as well as an additional ribozyme-mediated increase in inhibition. Consequences for the design and application of ribozymes are discussed.

Base Sequence↗

PubMed1997

Thickness distribution of the subchondral mineralization zone of the trochlear notch and its correlation with the cartilage thickness: an expression of functional adaptation to mechanical stress acting on the humeroulnar joint?

BACKGROUND: The thickness of cortical bone and the density of cancellous bone have been shown to reflect local mechanical stress. However, little is known whether this also applies to the thickness of the subchondral mineralization zone (SMZ). Since the humeroulnar joint may be regarded as a model of bicentric load transmission, we examined the thickness distribution of the SMZ of the trochlear notch. METHODS: Fourteen trochlear notches were examined. Eight joint surfaces of these ulnae were completely divided, 4 incompletely subdivided, and 2 remained undivided. After embedding, sagittal sections were prepared. The thickness distributions of SMZ and cartilage were measured, graphically reconstructed, and the correlation between the two calculated for each joint. RESULTS: The thickness of the SMZ lies between 120 and 1,400 microns. Regular patterns were observed with ventral and dorsal maxima, with close correlations between the type of joint and the thickness distribution of the SMZ. The cartilage thickness varied between 350 and 2,000 microns. The pattern of cartilage thickness differed significantly from that of the SMZ. The mean correlation coefficient between SMZ and cartilage thickness was 0.3. CONCLUSIONS: The SMZ thickness depends upon the type of joint and appears to be an expression of the local loading history of the SMZ. The bicentric transmission of force at the humeroulnar joint is reflected in the bicentric distribution of SMZ thickness. A possible explanation for the different distributions of SMZ and cartilage could be the differing types of local mechanical stress acting on bone and cartilage, and/or the different reactions of osteocytes and chondrocytes.

Adaptation, Physiological↗

PubMed1997

Computer simulation of subchondral bone adaptation to mechanical loading in an incongruous joint.

BACKGROUND: We hypothesized that the typically bicentric distribution of subchondral bone density (i.e., two maxima) in incongruous joints with deeper sockets could be predicted by a computer simulation employing a concavely incongruous finite-element model and current bone remodeling theory. Additional objectives were to assess the uniqueness of the solution with respect to assumed model parameters and initial conditions and to determine the relationship between contact areas, subchondral bone stress, and subchondral bone density patterns. METHODS: An idealized model of the humeroulnar joint was constructed with a quantitatively realistic representation of its natural incongruity. A currently accepted remodeling theory was implemented with a finite-element code using a node-based approach. RESULTS: The simulation predicted a dense subchondral bone plate after application of 3,000 daily load cycles for 300 days. The pronounced bicentric distribution of subchondral mineralization emerged. The solution was virtually independent of the initial density distribution and other assumed model parameters. Furthermore, the model predicted high tensile stresses in the subchondral bone, when the joint socket was spread apart during loading. Therefore, the locations of maximal strain energy density in the subchondral bone did not correspond with areas of joint contact. CONCLUSIONS: The results of the bone remodeling simulations are consistent with patterns of subchondral bone density determined experimentally. Furthermore, the solutions exhibited a high degree of uniqueness, were rather insensitive to changes in cartilage stiffness, moderately sensitive to number of applied loading cycles, and highly sensitive to loading magnitude. Tensile stresses seem to play a dominant role in subchondral bone remodeling due to bending in the subchondral bone plate. Thus, we conclude that, in the case of an incongruous joints with deeper sockets, the density of the subchondral bone cannot be regarded as a direct measure of the adjacent articular pressure.

Adaptation, Physiological↗

PubMed1997

Exogenous application of ribozymes for inhibiting gene expression.

Sequence-specific inhibition of gene expression is an attractive concept for the development of a new generation of therapeutics. Two alternatives can be envisaged for the introduction of ribozymes into cells: endogenous or exogenous delivery. In the latter, the ribozyme is prepared by chemical synthesis or transcription and delivered to the cell either unaided or with the help of liposomes. A problem with this approach is the abundance of RNases in the serum, and thus the stabilization of the ribozyme is necessary but without the impairment of catalytic efficiency. This has been achieved by several groups by 2'-modification of the pyrimidine nucleosides and the introduction of a few phosphorothioates at the termini. The selection of ribozyme-accessible sites on the target and the attachment of cholesterol and peptides to the ribozymes will be discussed. Examples of the application of these modified ribozymes in cell cultures will be presented, including the inhibition of expression of the multiple drug resistance gene, after unaided as well as liposome-aided delivery, and studies of animal models demonstrating the potential of this particular application strategy.

Animals↗

PubMed1997

Repeatability of patellar cartilage thickness patterns in the living, using a fat-suppressed magnetic resonance imaging sequence with short acquisition time and three-dimensional data processing.

A fast, reproducible, and noninvasive method is required for quantifying cartilage thickness clinically and for studying the deformation of articular cartilage during and after mechanical loading in vivo. The objective of the current investigation was to test the repeatability of regional distribution patterns of patellar cartilage thickness in the living on the basis of a fat-suppressed magnetic resonance imaging sequence with a short acquisition time and three-dimensional digital data processing. The knees of eight healthy volunteers were transversally imaged with a fat-suppressed FLASH-3D (fast low angle shot) sequence (acquisition time: 4 minutes and 10 seconds). In each case, the joint was newly positioned before each of the six replicate measurements was taken. The patellar cartilage was reconstructed three-dimensionally, and the distribution of cartilage thickness was determined with a three-dimensional minimal-distance algorithm. Whereas the cartilage volume ranged from 3,198 to 7,149 mm3, the mean coefficient of variation for the 6-fold volume measurement was 1.35%. On average, 75.1% (+/- 4.1%) of all test pixels could be attributed to the same cartilage thickness interval (0.5 mm) by image analysis; 14.8% (+/- 2.4%) deviated by one interval; 6.6% (+/- 1.5%), by two intervals; and 3.5% (+/- 1.8%), by more than two intervals. We conclude that, on the basis of a magnetic resonance imaging sequence with an acquisition time of less than 5 minutes, the quantitative distribution of cartilage thickness can be determined with high precision in vivo.

Adult↗

PubMed1997

Direct comparison of contact areas, contact stress and subchondral mineralization in human hip joint specimens.

X-ray densitometric and CT osteoabsorptiometric findings suggest that in the human hip subchondral mineralization patterns change from bicentric to monocentric as a function of age. It has been hypothesized that these changes indicate an alteration in the geometric configuration of the joint from incongruous to congruous, possibly associated with the onset of osteoarthrosis. The purpose of this study was therefore to directly compare contact rates, contact stress and subchondral mineralization in the hip joint. Twelve specimens without cartilage lesions (ages 34-86 years) were investigated. Simulating the mid-stance phase, the contact areas were determined by polyether casting and the contact stress with Fuji film. The distribution of subchondral mineralization was assessed non-invasively with CT osteoabsorptiometry. At small loads the load-bearing areas were located at the periphery of the lunate surface. In some joints they were found in the acetabular roof and expanded, with higher loads, to the center of the lunate surface and the anterior and posterior horns. In other joints, the contact areas were recorded at lower loads in the anterior and posterior horns, and only at higher forces they merged in the acetabular roof. The maximal contact stress ranged from 8 t 9 MPa at 300% body weight. Maxima of subchondral mineralization were recorded in the acetabular roof, in the anterior and posterior horns, or in all three locations. There was no clear correlation between the distribution of contact and pressure, and the pattern of subchondral bone density. Incongruity is shown to strongly affect the distribution of contact and pressure in the human hip joint. However, the pattern of subchondral mineralization cannot be readily explained in terms of the contact areas and contact stress during mid-stance. Incongruity may give rise to tensile stresses in the subchondral bone, and the construction of the pelvis as a whole may play an important role in subchondral bone loss loads and adaptation.

Adult↗

PubMed1997

Mechanical implications of humero-ulnar incongruity--finite element analysis and experiment.

Previous studies show that the humero-ulnar joint is physiologically incongruous [Eckstein et al. (1995a) Anat. Rec. 243, 318-326] and exhibits a bicentric (ventro-dorsal) distribution of subchondral mineralization [Eckstein et al. (1995b) J. Orthop. Res. 13, 286-278]. We therefore asked: (1) Does humero-ulnar incongruity bring about a bicentric distribution of contact pressure? (2) Do tensile stresses occur in the subchondral bone of the trochlear notch that are in the same order of magnitude as the compressive stresses? (3) Do ventral and dorsal maxima of subchondral bone density correlate with a bicentric distribution of strain energy density? To that end, a two-dimensional finite element model was designed. The shape and material properties of the bones were based on CT and the boundary conditions selected to agree with resisted elbow extension at 90 degrees of flexion. The incongruity and contact areas were determined experimentally from casts, and the pressure distribution with Fuji Prescale film. In the model and the experiment contact stresses above 2 MPa were recorded in the ventral and dorsal parts of the joint, and values below 0.5 MPa in the depth of the notch. In the model, tensile stresses of 2.9 MPa were observed in the subchondral bone of the ulna, but not in the humerus. The subchondral strain energy density yielded a bicentric pattern in a model with homogeneous subchondral bone properties. It is shown that humero-ulnar incongruity brings about a bicentric distribution of contact pressure, a tensile stress in the notch that is in the same order of magnitude as the compressive stress, and a distribution of strain energy density that correlates with subchondral density patterns.

Adult↗

PubMed1997

Non-invasive determination of cartilage thickness throughout joint surfaces using magnetic resonance imaging.

Data on articular cartilage thickness in the living are important for the design for computer models, aimed at preoperatively assessing the effect of surgical procedures on joint contact and load transmission, and for the calculation of cartilage material properties from its deformational behavior as determined during arthroscopy. A non-invasive method for measuring cartilage thickness in living subjects is, however, not available. A technique based on magnetic resonance imaging has therefore been tested for assessing articular cartilage thickness throughout joint surfaces. The accuracy is determined by comparing cartilage thickness maps obtained from three patellar specimens with a fat-suppressed three-dimensional gradient-echo sequence (resolution 2 x 0.31 x 0.31 mm) to those obtained with CT arthrography, A-mode ultrasound and anatomical sections. The distribution patterns are quantitatively compared using image analysis. The highest agreement was obtained for MRI and the sections (50% identical pixels), but all techniques yielded very similar results. On average, MR slightly underestimated the cartilage thickness compared with CT and the sections, and overestimated it compared with ultrasound. No evidence of differences in the degree of similarity could be detected in areas of thin and thick cartilage. We conclude that, if the resolution and accuracy of the method presented are considered acceptable, MRI is available for repeatable determination of topographical maps of articular cartilage thickness in living subjects. These data can be used to determine joint contact and stress in computer models and to calculate cartilage material properties in vivo.

Adult↗

PubMed1997

A non-invasive technique for 3-dimensional assessment of articular cartilage thickness based on MRI. Part 2: Validation using CT arthrography.

Established methods for the measurement of articular cartilage thickness are invasive and cannot be sequentially applied in living subjects. In the present study, the distribution of cartilage thickness throughout entire joint surfaces was determined from MR images obtained with a fat-suppressed gradient-echo sequence at a resolution of 0.31 x 0.31 x 2.00 mm3, and compared to that derived from CT arthrography. A minimal distance algorithm was employed to produce 3D cartilage thickness maps of seven cadaveric human knee joints. The mean amount of deviation of the cartilage volumes was 5.6% (+/- 4.6), statistical analysis showing that there was high agreement between the two methods (r = 0.995, slope = 1.037, y-intercept = -90.5 mm3). The 3D thickness maps yielded a striking agreement between the two methods, the maximum values generally yielding a deviation of none or one thickness interval of 0.5 mm. This investigation shows that accurate 3D assessment of articular cartilage thickness can be performed with MRI, this technique having the advantage that it is suitable for investigating living subjects.

Adult↗

PubMed1997

A non-invasive technique for 3-dimensional assessment of articular cartilage thickness based on MRI. Part 1: Development of a computational method.

Articular cartilage thickness is of relevance in various fields in diagnostics and biomedical research. In view of recent improvements of MR cartilage imaging a computational method has been developed for three-dimensional determination of cartilage thickness from tomographic datasets. A correction algorithm that compensates for the error implied in the voxel based distance measurements is implemented. Four different thickness definitions have been applied to two numerical test structures in order to judge their usability in the medical realm. The results for each of the thickness measurement methods are shown as color-coded thickness maps wrapped round the test objects. An algorithm determining at each point the minimal distance from the articular surface to the bone-cartilage interface is suggested to give the most suitable demonstration of articular cartilage. This algorithm is successfully applied to a 3-dimensional data set of human knee joint cartilage obtained with a fat-suppressed gradient-echo sequence from a healthy volunteer. A non-invasive method for determining cartilage thickness could become a very valuable tool in diagnostic radiology, orthopaedic practice and biomechanics.

Adult↗