The hammerhead ribozyme.
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Base Sequence↗
Biomedical subjects
F Eckstein
Publications and source records attributed to F Eckstein.
On the quality control of antisense oligonucleotides.
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Humans↗
Structure, strain and function of the transverse acetabular ligament.
To assess the morphology and function of the transverse acetabular ligament (TAL), its internal structure and insertion sites were analyzed, and its strain was determined during hip joint loading. Fifteen specimens were embedded in methyl methacrylate and cut into 500-micron sections parallel to the inlet of the acetabulum. Polarized light microscopy and contact radiography revealed important differences between the ventral and dorsal attachment of the TAL. Ventrally, the fibers insert close to the labrum, at a rather extensive site consisting of fibrous cartilage. Dorsally, the ligament arises from a wide attachment at the bony region of the posterior horn, where it is continuous with the periosteum and the joint capsule. In a second part of the study, six specimens (four fixed, two fresh) were investigated, using a testing machine to simulate hip joint loading. The strains of the TAL and of two sites of the acetabular labrum were measured with strain gauges. Loads of 10-2,800 N were applied and the degree of rotation of the femoral head was varied. At 400% body weight, the TAL encountered tensile strains of up to 3.7%, but the labrum only of up to 0.5%. The contact areas of the hip joints were determined with a polyether casting material. At 50% body weight they were found to lie in the anterior and posterior aspects of the lunate surface. These results indicate that, due to a natural incongruity, the acetabular notch is widened during loading and the TAL submitted to tension.
Acetabulum↗
Formation of a free radical of the sulfenylimine type in the mouse ribonucleotide reductase reaction with 2'-azido-2'-deoxycytidine 5'-diphosphate.
Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identical subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibitor 2'-azido-2'-deoxycytidine 5'-diphosphate. The detailed hyperfine structure of the EPR spectrum of the transient radical is somewhat different for the mouse and previously studied E. coli enzymes. When the positive allosteric effector ATP was replaced by the negative effector dATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W103Y and D266A, where the mutations have been shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.
Animals↗
Ribozyme-mediated RNA degradation in nuclei suspension.
Ribozymes containing 2'-fluoro- and 2'-amino-modified pyrimidine nucleosides in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In order to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with proteinase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.
Animals↗
Morphomechanics of the humero-ulnar joint: I. Joint space width and contact areas as a function of load and flexion angle.
BACKGROUND: Previous studies have shown that the trochlear notch is deeper than necessary for an exact fit with the humerus. However, humero-ulnar joint space width and contact areas have so far not been quantified for variations in the load and angle of flexion. METHODS: Six fresh cadaveric specimens were investigated at 30 degrees, 60 degrees, 90 degrees, and 120 degrees of flexion and at loads of 25 and 500 N, simulating resisted elbow extension. The joint space width and contact were determined, using polyether casting material. RESULTS: At 25 N all joints made contact in the ventral and dorsal aspects of the articular surfaces, whereas in the depth of the trochlear notch the joint space was on average between 0.3 and 2.8 mm wide, with some variation between individuals. At 500 N the joint space width was considerably reduced and the contract areas expanded towards the depth of the notch. The size of the dorsal contact areas was significantly smaller at 30 degrees and that of the ventral ones at 120 degrees, their ventro-dorsal ratio decreasing considerably from 30 degrees to 120 degrees (p < 0.01). CONCLUSION: These results indicate that the size of the contact areas depends to a slight extent on the joint position, but that at all loads and flexion angles a bicentric contact and an important central joint space width emerge because of the concave incongruity of the joint. These data may be used for numerical calculations, analysing the effects of incongruity on the joint stress and on the functional adaptation of the subarticular tissues.
Adult↗
Morphomechanics of the humero-ulnar joint: II. Concave incongruity determines the distribution of load and subchondral mineralization.
BACKGROUND: A deeper joint socket (concave incongruity) is found at most angles of flexion of the humero-ulnar joint and maintained over a wide range of physiological loading. It is, however, unclear how far this incongruity affects the distribution of load and subchondral mineralization of this joint as compared with a congruous configuration. METHODS: Two nonlinear, axisymmetrical finite element models with two cartilage layers were constructed, one congruous and one incongruous, with a joint space of realistic magnitude. The distribution of subchondral mineralization was determined by computed tomography osteoabsorptiometry in the same six specimens that were investigated in the first part of the study, and compared with the biomechanical data obtained there and the predictions of the models. RESULTS: In the congruous case, the center of the socket is highly loaded, whereas the periphery does not experience mechanical stimulation. A central bone density maximum is predicted. With concave incongruity the position of the contact areas shifts from the joint margin towards the center as the load increases, and the peak stresses are considerably lower. A bicentric ventro-dorsal distribution pattern of subchondral mineralization is predicted, and this is actually found in the six specimens. CONCLUSIONS: Concave incongruity is shown to determine load transmission and subchondral mineralization of the humero-ulnar joint. It is suggested that this shape leads to a more even distribution of stress, provides intermittent stimulation of the cartilaginous tissue, and has beneficial effects on the metabolism, nutrition, and lubrication of the articular cartilage during cyclic loading.
Absorptiometry, Photon↗
Subchondral bone density in the human elbow assessed by computed tomography osteoabsorptiometry: a reflection of the loading history of the joint surfaces.
The functional adaptation of bone tissue to the mechanical stresses acting on it has been convincingly established. This association should apply as well to the subchondral bone, reflecting the long-term distribution of stress over the joint surfaces. Thirty-six specimens of the human elbow joint were investigated by computed tomography osteoabsorptiometry in order to assess the distribution of the subchondral mineralization. The distal surfaces usually were more highly mineralized than the proximal components of the joint, whereas the humeroulnar and the humeroradial parts exhibited a similar degree of mineralization. The fovea of the radial head always showed a central density maximum, and the trochlear notch usually presented a bicentric distribution pattern, with maxima beneath the ventral and dorsal regions of the articular surface. The different patterns of subchondral mineralization were shown to reflect the loading history of the overlying articular surfaces, which is determined mainly by geometrical factors. The flatter socket of the humeroradial joint leads to central load transmission, but the deeper socket of the humeroulnar joint will, by contrast, give rise to bicentric stress distribution.
Absorptiometry, Photon↗
The thickness of the subchondral plate and its correlation with the thickness of the uncalcified articular cartilage in the human patella.
The regional thickness distributions of the subchondral plate and the unmineralized part of the articular cartilage were morphometrically determined in normal human patellae, and the correlation coefficient for each specimen calculated from the paired measurements. For this purpose the patellae were embedded in methyl methacrylate and cut as serial sections, which were assessed with a Vidas image-analyzing system (Kontron). The values obtained were used to reconstruct the individual and average thickness distributions and to calculate the correlation coefficients for each subject. Both the thickness of the subchondral plate and that of the cartilage revealed regular distributions which, however, followed different patterns. Central regions with maximum values from which the thickness decreased concentrically towards the periphery were found in both. However, the distribution patterns of the unmineralized cartilage and the subchondral plate could be clearly distinguished, both by the position of the maxima and by the arrangement of the isocrassids (contour lines of equal thickness). The thicknesses of the two tissues showed a correlation between 0.38 and 0.82 (mean 0.6). We attribute this to their different reactions to the type of stress acting upon them. It appears that the thickness of the subchondral plate is principally determined by stresses acting over a longer period of time with low frequency, whereas the thickness of the articular cartilage seems to be a response to intermittent dynamic stresses of a higher frequency.
Adult↗
Phosphate analogs for study of DNA polymerases.
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DNA Polymerase I↗
The potential of magnetic resonance imaging (MRI) for quantifying articular cartilage thickness -- a methodological study.
The thickness of patellar articular cartilage was assessed in a cadaveric human knee joint by magnetic resonance imaging. Imaging was conducted at 1.0 T, using three-dimensional gradient-echo sequences. From each of the sequences the total cartilage volume, the size of the articular surface, the mean cartilage thickness and the regional distribution of cartilage thickness were determined by image analysis. These values were then compared with those obtained from anatomical sections. The fat-suppressed FLASH sequence was found to allow the most accurate evaluation of the total volume and the regional distribution of the articular cartilage. Slight underestimation of the cartilage thickness by about 5% may be due to the fact that the calcified layer is not made visible by magnetic resonance imaging. There is, however, a very high degree of similarity between the distribution patterns obtained from the MR images and the anatomical sections. The contrast-to-noise ratios and reproducibility were also highest with the fat-suppressedFLASH sequence. This pulse sequence can therefore be recommended for experimental and clinical use.
Journal Article↗
RNA cleavage by small catalytic RNAs.
Recent studies of the hammerhead ribozyme have provided an insight into its three-dimensional structure. In addition, studies using chemical probes, functional-group modification and mutational analysis, in combination with computer modelling, have led to proposals for the structure of both the hairpin and hepatitis delta virus ribozymes. Such structural elucidations will aid understanding of the mechanism of ribozyme catalysis. The discovery that certain RNA-binding proteins can increase the catalytic efficiency of ribozymes in encouraging for their use in the inhibition of gene expression in vivo.
Animals↗
Structure-function relationships of hammerhead ribozymes: from understanding to applications.
The hammerhead ribozyme is the smallest member of the naturally occurring family of RNA molecules that are capable of catalysing the site-specific cleavage of RNA. Functional-group modifications have led to an identification of groups that are important for catalysis, and have helped in the understanding of the role of Mg2+, which is required for catalysis. Recent studies on the three-dimensional structure of the hammerhead ribozyme, including X-ray analysis, have contributed significantly towards an understanding of its mode of action. In addition to contributing to our understanding of RNA catalysis, these studies have also stimulated investigations into the possibility of using ribozymes in gene therapy to cleave specific mRNAs.
Animals↗
Probing RNA tertiary structure: interhelical crosslinking of the hammerhead ribozyme.
Distinct structural models for the hammerhead ribozyme derived from single-crystal X-ray diffraction and fluorescence resonance energy transfer (FRET) measurements have been compared. Both models predict the same overall geometry, a wishbone shape with helices II and III nearly colinear and helix I positioned close to helix II. However, the relative orientations of helices I and II are different. To establish whether one of the models represents a kinetically active structure, a new crosslinking procedure was developed in which helices I and II of hammerhead ribozymes were disulfide-crosslinked via the 2' positions of specific sugar residues. Crosslinking residues on helices I and II that are close according to the X-ray structure did not appreciably reduce the catalytic efficiency. In contrast, crosslinking residues closely situated according to the FRET model dramatically reduced the cleavage rate by at least three orders of magnitude. These correlations between catalytic efficiencies and spatial proximities are consistent with the X-ray structure.
Base Sequence↗
[Magnetic resonance tomography in trauma of the knee joint].
Technical improvements of MRI have significantly improved the visualization of joints. New sequences and experience with MRI has lead to increased sensitivity and specificity of internal joint lesions. For the first time, contusions of the subchondral bone which are prognostically significant for the developing of chondral lesions can be identified. To date, no other tests are available to identify such lesions. This review article outlines the current value of MRI for evaluating post-traumatic knee joint injuries.
Bone Marrow↗
[Knee joint cartilage in magnetic resonance tomography. MR chondrovolumetry (MR-CVM) using fat-suppressed FLASH 3D sequence].
UNLABELLED: The objective of this study was to optimize the demonstration of articular cartilage with magnetic resonance tomography (MRT) and to assess its accuracy in determining the articular cartilage volume of the knee joint. METHODS: A fat-suppressed FLASH-3D sequence was optimized on healthy volunteers. A fresh cardaveric knee joint was removed from a 82-year-old man and immediately imaged without being frozen or fixed. MRT was carried out at 1.5 T and 25 mT/m (Vision, Siemens, Erlangen, Germany) with a conventional CP knee coil. Sagittal and transverse sections were acquired perpendicular to the articular surfaces, and then, parallel to these imaging planes, anatomical sections were obtained with a diamond band saw. The volumes of the patellar, tibial and femoral cartilages were determined from both the images and the sections, using an image analysing system. RESULTS: Signal intensities and contrast-to-noise ratios depend on the parameters used. The highest contrast between the cartilage and the periarticular tissues was obtained at a flip angle of 30 degrees (TR = 60 ms, TE = 11 ms). However, a flip angle of 60 degrees was judged to provide optimal subjective image quality. Using these parameters, the deviations between the radiological and the anatomical cartilage volumes were -4.7% in the patella, -3.1% in the tibial plateau and -4.2% in the femur. CONCLUSIONS: The volumes of the knee-joint cartilages may be accurately determined with MRT if an appropriate pulse sequence is chosen. From a clinical point of view the differences between MR images and anatomical sections can be considered negligible. These differences may be explained on the basis that MRT delineates uncalcified cartilage only and that the calcified layer is demonstrated as part of the subchondral bone plate. However, close correlation of the thicknesses of the uncalcified and the calcified layer has been reported in the literature, so the relative distribution of articular cartilage should be accurately reflected in MR images.
Aged↗
Translation of 2'-modified mRNA in vitro and in vivo.
2'-Fluoro- and 2'-amino-2'-deoxynucleoside triphosphates have been used for in vitro transcription of 2'-modified luciferase mRNA. The 2'-modified deoxynucleoside-containing transcripts were tested for the expression of luciferase in X.Laevis oocytes as well as in rabbit reticulocyte lysate. Only 2'-fluoro-2'-deoxy-adenosine-modified mRNA gave rise to luciferase as shown by SDS gel as well as by enzyme activity measurements in vivo as well as in vitro. 2'-Fluoro-2'-deoxy-pyrimidine nucleoside-modified mRNA did not give rise to luciferase activity. However, they directed incorporation of 35S-labeled methionine into peptide fragments in rabbit reticulocyte lysate indicating premature termination of translation. No or only extremely little of such incorporation could be detected with 2'-amino modified transcripts.
Animals↗
A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements.
For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.
Base Composition↗